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Multi-omic data mining to elucidate molecular adaptation mechanisms of filamentous fungi exposed to space environment

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Multi-omic data mining to elucidate molecular adaptation mechanisms of filamentous fungi exposed to space environment

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Content Multi-omic data mining to elucidate molecular
adaptation mechanisms of filamentous fungi exposed
to space environment
Dissertation by:
Adriana Blachowicz
In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy (Pharmaceutical Sciences)
UNIVERSITY OF SOUTHERN CALIFORNIA
Los Angeles, California
May 2019
© 2019
Adriana Blachowicz
ORCID: https://orcid.org/0000-0001-9027-5670
All rights reserved
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Between stimulus and response
there is a space. In that space is
our power to choose our
response. In our response lies
our growth and our freedom.
Viktor E. Frankl
To my family.
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Acknowledgements
My journey away from Poland began five years ago with a six-month internship
at the NASA Jet Propulsion Laboratory (JPL). This opportunity has been fostered
by the collaborative work of professor A. Paszczynski, from the University of Idaho,
and Dr. K. Venkateswaran, from JPL. Back then, as a young student, I did not
even think of starting a doctoral degree, yet, five years later, here I am ready
to defend my PhD thesis on fungal adaptation to space environment from the
collaboration between professor C. C. C. Wang, from the University of Southern
California (USC), and Dr. K. Venkateswaran, from JPL.
The multiple projects described in this thesis are the natural result of the
productive synergy with professor C. C. C. Wang and Dr. K. Venkateswaran. Their
converging scientific interests, yet different philosophy for details, represented
a constant guiding light, which taught me how to approach problems and plan
their scientific exploration from a holistic perspective, before indulging into the
scientific minutiae. This broadening exercise has been the most fundamental step in
developing my critical and scientific thinking, which arguably is the most important
accomplishment of my PhD, and for which my gratitude goes to professor C. C. C.
Wang and Dr. K. Venkateswaran.
I would like to thankfully mention the professors who kindly accepted to serve
on my defense committee: professors C. Okamoto, I. Haworth, J. Xie, and T.
Williams. This thesis could not have assumed the current shape without their
insights throughout the years of research and, in particular, towards the end of my
academic journey as a student.
I would also like to extend my gratitude to Dr. Y.-M. Chiang and all the
former and current lab members, Dr. W. Sun, Dr. J. Van Dijk, Dr. K. Lin, Y.-E.
Liao, C. Rabot, E. Yuan, P. Lehman, S. J. Lim, J. Wang, N. L. H. Pham and G.
Xu. The meticulous work of all these people contributed making professor C. C.
C. Wang’s laboratory an incredible place to work. I wish the same luck to the
forthcoming students.
I am especially grateful to my “partner in crime”, Dr. Jillian Romsdahl. Our
numerous interactions – on and off campus – have contributed to making this
journey an incredible adventure, culminated in the dear friendships I have been
fortunate to cultivate along the way. In particular, I would like to mention my
closest fellows: Michelle Grau, Melissa Kordahi, Xianhui Chen, and Hsuan-Yao
Wang. They have been an incredible certainty and a true inspiration, and I look
forward to keep nurturing our friendship in the years to come.
Last, but not least, my family, to which I would like to dedicate this thesis
and my entire PhD. The undivided support I received throughout these years has
represented a solid dogma I could base my successes on. You are my World!
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Abstract
Filamentous fungi are dual organisms that can be both useful for mankind and
threatening to human habitats and health. These omnipresent extremotolerant
microorganisms are associated with a range of hostile environments and the human
body. Immense adaptability to a variety of conditions enables fungi to thrive in
what seems like inhospitable niches, including man-made closed habitats. One
such habitat is the International Space Station (ISS), which is a research platform
under strict microbiological scrutiny. Recent advances in the next generation
sequencing technologies have transformed the scientists’ approach to study fungal
adaptation mechanisms. Insights into genome, proteome and metabolome allow for
holistic assessment of changes in the molecular suite of fungi exposed to extreme
conditions. In this work multi-omic approach was applied to study response of
variousfilamentousfungitospaceenvironment. Weinvestigatedboth, unintentional
fungal “hitchhikers” that unknowingly followed people and cargo aboard the ISS
Aspergillus fumigatus (Chapter 2 and 3) and Aspergillus niger (Chapter 6 and 7),
and fungi sent to the ISS in strictly controlled experiments, including several
Chernobyl isolated fungi, ISS-isolated A. niger (Chapter 4 and 5), and Aspergillus
nidulans (Chapter 8).
Aspergillus fumigatus is a ubiquitous in nature saprophytic fungus that may
pose a health hazard to immunocompromised individuals. Upon isolation from
the ISS, during microbial observatory study, two A. fumigatus isolates were
characterized to investigate space conditions-induced phenotype when compared to
well-studied Af293 and clinical isolate CEA10. Initial analyses showed that genomic
diversity of both ISS-isolates was within the genetic variance of 95 environmental
and clinical isolates, however both ISS isolates showed increased virulence in larval
zebrafish model of invasive aspergillosis (Chapter 2). Observed increase in virulence
prompted further molecular analyses of ISS isolates. Proteome characterization
revealed up-regulation of proteins involved in carbohydrate metabolism, secondary
metabolism and stress responses in both ISS isolates when compared to clinical
isolates. Among increased in abundance proteins were TpcK, TpcF and TpcA
involved in trypacidin biosynthesis, Asp-hemolysin, AcuE and PdcA involved in
glyoxylate cycle and ethanol fermentation, respectively (Chapter 3).
A. niger, ubiquitousinbuiltandnaturalenvironmentsfungus, whichhasknown
biotechnological applications was among ISS-isolated and further characterized
strains. Proteomic analysis of ISS-isolated A. niger revealed altered abundance of
proteins involved in carbohydrate metabolism, cell wall modulation, and oxidative
stress response when compared to well-studied ATCC 1015. Similarly to A. fumiga-
tus whole genome analysis revealed genetic variance within the diversity observed
among other species in the A. niger/ welwitschiae/lacticoffeatus clade (Chapter 6).
Further, secondary metabolite (SM) analysis showed significant increase in the
yields of produced compounds, especially antioxidant Pyranonigrin A. Gene tar-
geted deletion was used to determine gene cluster responsible for Pyranonigrin A
production. Lastly, radiation protective potential of Pyranonigrin A against UV-C
was tested (Chapter 7).
To study fungal adaptation to space conditions in controlled experiments
several Chernobyl- and ISS-isolated fungi were selected and characterized prior
to space flight. Assessment of UV-C and simulated Mars conditions resistance
revealed strain-dependent survival. Further, upon exposure to SMC two surviving
strains A. fumigatus and Cladosporium cladosporoides showed induced production
of SM and differential expression of proteins involved in translation, carbohydrate
metabolism and energy conversion processes when compared to unexposed control
strains (Chapter 4). Genomic characterization of ISS-grown A. niger revealed
accumulation of SNPs within specific regions of the genome, while observed
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INDELs were distributed across all chromosomes. Proteome analysis showed
differential expression of proteins involved in carbohydrate metabolism, and stress
response, while SM characterization revealed that SM production is altered by
space conditions (Chapter 5). Lastly, when fungal model organism A. nidulans
and three mutant strains were grown aboard the ISS and compared to ground
counterparts alterations in specific genome regions were observed. Additionally,
proteins involved in carbohydrate metabolism and stress response were differentially
expressed among the ISS-grown strains and alterations in SM yields were revealed
(Chapter 8).
This thesis underscores the significance of multi-omic characterization in
comprehension of the fungal response to distinct environmental conditions. Such
in depth analyses are imperative for elucidating plausible mechanisms of enhanced
virulence of A. fumigatus and may unveil biotechnological applications of fungi
during space explorations. Further, understanding of possible molecular alterations
triggered by irradiation is crucial for the success of long-term manned space flights
to ensure both astronauts’ health and maintenance of the closed habitat.
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TABLE OF CONTENTS
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi
Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii
Chapter 1: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Adaptability and persistence of fungi in various environments . . . . 1
1.2 Fungi in confined spaces on the example of the ISS . . . . . . . . . 2
1.3 Evaluation of fungal adaptation mechanisms . . . . . . . . . . . . 3
Chapter 2: Characterization of Aspergillus fumigatus isolated from air and
surfaces of the International Space Station . . . . . . . . . . . . . . . . 8
2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.3.1 Identification of A. fumigatus samples from the ISS . . . . . 11
2.3.2 Visual characterization and growth rates of ISS strains in vitro 13
2.3.3 ISS strains show no enhanced resistance to chemical stresses
in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3.4 Secondary metabolite analysis among ISS and clinical isolates 15
2.3.5 ISS strains exhibit increased virulence in a vertebrate model
of invasive aspergillosis . . . . . . . . . . . . . . . . . . . . . 21
2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.5 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 27
2.5.1 Isolation and verification of A. fumigatus isolates from the ISS 27
2.5.2 Fungal culture . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.5.3 Physiological analysis . . . . . . . . . . . . . . . . . . . . . 29
2.5.4 Stress tests . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.5.5 Secondary metabolite extraction and analysis . . . . . . . 30
2.5.6 Zebrafish care and maintenance . . . . . . . . . . . . . . . . 31
2.5.7 Larval zebrafish virulence assay . . . . . . . . . . . . . . . . 31
2.5.8 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . 32
Chapter 3: Proteomic characterization of Aspergillus fumigatus isolated from
air and surfaces of the International Space Station . . . . . . . . . . . . 33
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3.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.3.1 Proteome analysis overview . . . . . . . . . . . . . . . . . 35
3.3.2 Secondary metabolism and toxins . . . . . . . . . . . . . . 37
3.3.3 Stress response . . . . . . . . . . . . . . . . . . . . . . . . 40
3.3.4 Carbohydrate metabolic processes . . . . . . . . . . . . . . 40
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 46
3.5.1 Isolation and identification of A. fumigatus . . . . . . . . . 46
3.5.2 Growth conditions . . . . . . . . . . . . . . . . . . . . . . 46
3.5.3 Protein extraction . . . . . . . . . . . . . . . . . . . . . . 46
3.5.4 Tandem mass tag (TMT) labeling . . . . . . . . . . . . . . 47
3.5.5 LC-MS/MS analysis . . . . . . . . . . . . . . . . . . . . . 48
3.5.6 Proteome data processing . . . . . . . . . . . . . . . . . . 48
3.5.7 Secondary metabolie analysis . . . . . . . . . . . . . . . . 49
3.5.8 Sporulation capacity assessment . . . . . . . . . . . . . . . 50
Chapter 4: Proteomic and metabolomic characteristics of extremophilic fungi
under simulated Mars conditions . . . . . . . . . . . . . . . . . . . . . . 51
4.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.3.1 Identification of fungal strains . . . . . . . . . . . . . . . . 54
4.3.2 Survival of desiccated conidial spores under UV-C irradiation 56
4.3.3 Survival of desiccated conidia under simulated Mars condi-
tions (SMC) . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3.4 Secondary metabolite (SM) profiling of Aspergillus fumigatus
and Cladosporium cladosporioides exposed to SMC . . . . 58
4.3.5 Proteome profiling of Aspergillus fumigatus exposed to SMC 58
4.3.6 Proteome profiling of Cladosporium cladosporioides exposed
to SMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.3.7 Increased resistance to UV-C of SMC exposed Aspergillus
fumigatus conidia . . . . . . . . . . . . . . . . . . . . . . . 69
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.5 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 73
4.5.1 Sample collection sites . . . . . . . . . . . . . . . . . . . . 73
4.5.2 Preparation of aluminum coupons with monolayers of dried
fungal conidia . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.5.3 UV-C exposure and recovery . . . . . . . . . . . . . . . . . 74
4.5.4 Simulated Martian conditions (SMC) . . . . . . . . . . . . 74
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4.5.5 Secondary metabolite extraction and analysis . . . . . . . 75
4.5.6 Proteome samples extraction and processing . . . . . . . . 76
4.5.7 Quantitative proteomics analysis . . . . . . . . . . . . . . 77
4.5.8 Genome annotation . . . . . . . . . . . . . . . . . . . . . . 78
Chapter 5: The International Space Station environment triggers molecular
responses in Aspergillus niger . . . . . . . . . . . . . . . . . . . . . . . 79
5.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
5.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.3.1 Genome variation in the ISS-grown JSC-093350089 A. niger. 81
5.3.2 Proteomic characterization of ISS-grown JSC-093350089
A. niger . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5.3.3 Metabolomic characterization of ISS-grown JSC-093350089
A. niger . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
5.5 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 97
5.5.1 Isolation and identification of A. niger . . . . . . . . . . . 97
5.5.2 Growth conditions . . . . . . . . . . . . . . . . . . . . . . 97
5.5.3 Secondary metabolite (SM) extraction and analysis . . . . 98
5.5.4 SM statistical analysis . . . . . . . . . . . . . . . . . . . . 98
5.5.5 Protein extraction . . . . . . . . . . . . . . . . . . . . . . 98
5.5.6 Tandem mass tag (TMT) labeling . . . . . . . . . . . . . . 99
5.5.7 LC-MS/MS analysis . . . . . . . . . . . . . . . . . . . . . 99
5.5.8 Proteome data analysis . . . . . . . . . . . . . . . . . . . . 100
5.5.9 Genomic DNA extraction and whole genome sequencing
(WGS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.5.10 Genetic variation identification . . . . . . . . . . . . . . . . 101
Chapter 6: Characterization of Aspergillus niger isolated from the Interna-
tional Space Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
6.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
6.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
6.3.1 Identification of A. niger sampled from the ISS . . . . . . 106
6.3.2 Visual characterization and growth rates of JSC-093350089
in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
6.3.3 Overview of proteome analysis . . . . . . . . . . . . . . . . 108
6.3.4 Differential abundance of cell wall modulation proteins . . 109
6.3.5 Differential abundance of stress response proteins . . . . . . 111
6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
6.5 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 117
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6.5.1 Isolation and identification of the ISS A. niger isolate . . . 117
6.5.2 Genome sequencing, assembly, and annotation . . . . . . . 117
6.5.3 Phylogenetic analysis . . . . . . . . . . . . . . . . . . . . . 118
6.5.4 Growth conditions . . . . . . . . . . . . . . . . . . . . . . 119
6.5.5 Physiological analysis . . . . . . . . . . . . . . . . . . . . . 119
6.5.6 Protein extraction . . . . . . . . . . . . . . . . . . . . . . 120
6.5.7 Tandem mass tag (TMT) labeling . . . . . . . . . . . . . . 120
6.5.8 LC-MS/MS analysis . . . . . . . . . . . . . . . . . . . . . . 121
6.5.9 Proteome data analysis . . . . . . . . . . . . . . . . . . . . 122
Chapter 7: Metabolomic analysis of Aspergillus niger isolated from the
International Space Station reveals the radiation resistance potential of
pyranonigrin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
7.3.1 Secondary metabolite analysis of JSC-093350089 . . . . . . 126
7.3.2 Analysis of the potential gene clusters responsible for pro-
duction of pyranonigrin A in silico . . . . . . . . . . . . . 127
7.3.3 Development of an efficient gene targeting system in JSC-
093350089 and identification of the PKS-NRPS hybrid re-
sponsible for pyranonigrin A biosynthesis . . . . . . . . . . 129
7.3.4 Identification of pyranonigrin A biosynthesis gene cluster
boundaries . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
7.3.5 Assessment of the UV resistance potential of pyranonigrin A 132
7.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
7.5 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 136
7.5.1 Secondary metabolite extraction and analysis . . . . . . . 136
7.5.2 Strains and molecular manipulations . . . . . . . . . . . . 136
7.5.3 Radiation resistance analysis . . . . . . . . . . . . . . . . . 137
Chapter 8: International Space Station conditions alter genomics, proteomics,
and metabolomics in Aspergillus nidulans . . . . . . . . . . . . . . . . 138
8.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
8.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
8.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
8.3.1 Genome variation among ISS-grown samples . . . . . . . . . 141
8.3.2 Proteomic profiling of ISS-grown A. nidulans . . . . . . . . 143
8.4 Secondary metabolome alterations in ISS-grown A. nidulans . . . 149
8.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
8.6 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 155
8.6.1 Strains, media, and growth conditions . . . . . . . . . . . . 155
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8.6.2 Genomic DNA extraction, library preparation, and genome
sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
8.6.3 Genetic mutation identification . . . . . . . . . . . . . . . 156
8.6.4 Protein extraction . . . . . . . . . . . . . . . . . . . . . . 157
8.6.5 Tandem mass tag labeling . . . . . . . . . . . . . . . . . . 157
8.6.6 LC-MS/MS analysis . . . . . . . . . . . . . . . . . . . . . 158
8.6.7 Quantitative proteomics analysis . . . . . . . . . . . . . . 159
8.6.8 Secondary metabolite extraction and analysis . . . . . . . 160
Chapter 9: Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . 162
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
- xiii -
LIST OF FIGURES
Number Page
2.1 Isolation and phylogenetic characterization of ISS strains . . . . . . 12
2.2 In vitro growth of ISS isolates compared to growth of the clinical
isolates Af293 and CEA10 . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 ISS isolates showed no enhanced resistance to chemical stresses in vitro 16
2.4 Secondary metabolite production of ISS strains . . . . . . . . . . . 18
2.5 Secondary metabolite production of ISS strains, with a focus on an
unknown A. fumigatus compound . . . . . . . . . . . . . . . . . . 19
2.6 High-resolution mass spectrometry of unknown compound. . . . . . 20
2.7 Virulence assessment in a larval zebrafish model of invasive aspergillosis 20
3.1 AspGD GO Slim terms of proteins normalized to Af293 . . . . . . 36
3.2 AspGD GO Slim terms of proteins differentially expressed in space
strains when compared to Af293 and CEA10 . . . . . . . . . . . . 38
3.3 Monomethylsulochrin yields in clinical and ISS-isolated strains . . 39
3.4 Sporulation capacity of clinical and ISS-isolated strains . . . . . . . 40
4.1 UV-C resistance of Chernobyl and ISS-isolated fungal strains . . . 56
4.2 Secondary metabolite production of SMC-exposed ISSFT-021-30 and
IMV 00236-30 when compared to unexposed ISSFT-021 and IMV 00236 59
4.3 AspGD GO Slim terms of differentially expressed proteins in ISSFT-
021-30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.4 Biological process COG categories of differentially expressed proteins
in IMV 00236-30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4.5 UV-C resistance of A. fumigatus ISS-isolated and clinical strains . 69
4.6 UV spectra (200 to 400 nm) of the solar simulator employed in this
study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
5.1 AspGD GO Slim terms of differentially expressed proteins in ISS-
grown JSC-093350089 . . . . . . . . . . . . . . . . . . . . . . . . . 86
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5.2 Secondary metabolite production of ISS-grown JSC-093350089 when
compared to ground controls . . . . . . . . . . . . . . . . . . . . . 92
5.3 Secondary metabolite production of regrown ISS- and ground-grown
JSC-093350089 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.1 PhylogeneticcharacterizationofJSC-093350089displayingitsrelative
placement within the A. niger/welwitschiae/lacticoffeatus clade . . 107
6.2 In vitro growth of JSC-093350089 compared to ATCC 1015 . . . . 108
6.3 Biological process GO Slim categories of differentially expressed
proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
7.1 Secondary metabolite production in JSC-093350089 relative to ATCC
1015 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
7.2 Schematic representation of pyr cluster . . . . . . . . . . . . . . . . 131
7.3 Percent survival following exposure to varying doses of UV-C radia-
tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8.1 Schematic overview of the A. nidulans ISS experiment . . . . . . . . 141
8.2 Overview of proteomic analysis . . . . . . . . . . . . . . . . . . . . 146
8.3 Asperthecin production in ISS-grown LO1362 and CW12001 . . . . 150
8.4 LC-MS profile of SMs identified in A. nidulans . . . . . . . . . . . . 151
8.5 Secondary metabolite quantification when compared to ground controls152
- xv -
LIST OF TABLES
Number Page
3.1 Proteins involved in secondary metabolism and toxin biosynthesis
that revealed increased or decreased abundance . . . . . . . . . . . 39
3.2 Proteins involved in stress response that revealed increased or de-
creased abundance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.3 Proteins involved in carbohydrate metabolism that revealed increased
or decreased abundance . . . . . . . . . . . . . . . . . . . . . . . . 42
4.1 Fungal isolates used in the study and their significance . . . . . . . 55
4.2 Quantitative analysis of the simulated Mars Conditions (SMC) toler-
ance of selected extremotolerant Chernobyl- and ISS-isolated fungi 57
4.3 Differentially expressed proteins involved in translation and ribosome
biogenesis in ISSFT-021-30 subjected to SMC . . . . . . . . . . . . . 61
4.4 Differentially expressed proteins involved in carbohydrate metabolism
in ISSFT-021-30 subjected to SMC . . . . . . . . . . . . . . . . . . 62
4.5 Differentially expressed proteins involved in response to stress in
ISSFT-021-30 subjected to SMC . . . . . . . . . . . . . . . . . . . 63
4.6 Differentially expressed proteins involved in translation, ribosomal
structure and biogenesis in IMV 00236-30 subjected to SMC . . . . 65
4.7 Differentially expressed proteins involved in post-translational modifi-
cation, protein turnover, and chaperones in IMV 00236-30 subjected
to SMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.8 Differentially expressed proteins involved in carbohydrate transport
and metabolism in IMV 00236-30 subjected to SMC . . . . . . . . 67
4.9 Differentially expressed proteins involved in energy production and
conversion in IMV 00236-30 subjected to SMC . . . . . . . . . . . 68
5.1 Summary of genetic variations observed in ISS-grown JSC-093350089
when compared to ground control . . . . . . . . . . . . . . . . . . 82
5.2 Singlenucleotidepolymorphisms(SNPs)inISS-grownJSC-093350089
when compared to ground control . . . . . . . . . . . . . . . . . . 82
5.3 Differentially expressed proteins involved in carbohydrate metabolism 88
- xvi -
5.4 Differentially expressed proteins involved in stress response . . . . 89
5.5 Differentially expressed proteins involved in cellular amino acid
metabolic processes . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.6 Differentially expressed proteins involved in protein catabolic processes 91
6.1 Relative abundance of cell wall modulation proteins . . . . . . . . 112
6.2 Relative abundance of stress response proteins . . . . . . . . . . . 113
7.1 Putative function of genes within the pyranonigrin A biosynthetic
gene cluster and their homologs in Penicillium thymicola. . . . . . 130
8.1 Features of SNPs and INDELs . . . . . . . . . . . . . . . . . . . . 143
8.2 Comparative analysis of nonsynonymous SNPs occurring during
spaceflight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
8.3 Differentially expressed proteins by strain and biological process . . 147
- xvii -
Nomenclature
§§§
AspGD Aspergillus genome database
ATCC American Type Culture Collection
BRIC biological research in canister
CFU colony forming unit
ChEZ Chernobyl exclusion zone
ChNPP Chernobyl nuclear power plant
COG cluster of orthologous genes
COH City of Hope
CZ Czapek medium
DNA deoxyribonucleic acid
FC fold change
FDR false discovery rate
GMM glucose minimal medium
GO gene ontology
HEPA high-efficiency particulate arrestance filter
- xviii -
HPLC-DAD-MS high-performance liquid chromatography–photodiode array
detection–mass spectroscopy
IA invasive aspergillosis
ILMAH inflated Lunar/Mars analogue habitat
IMV Institute for Microbiology and Virology (Academy of Sciences),
Kiyv, Ukraine
INDEL insertion or deletion of bases in the genome of an organism
ISS International Space Station
ISS-MO International Space Station Microbial Observatory
ITS internal transcribed spacer
JGI Joint Genome Institute
JPL Jet Propulsion Laboratoy
JSC Johnson Space Center
KSC Kenedy Space Center
LBNL Lawrence Berkeley National Laboratory
LC-MS liquid chromatography-mass spectrometry
LEA late embryogenesis abundant
NASA National Aeronautics and Space Administration
NER nucleotide excision repair
NGS next generation sequencing
NRPS nonribosomal peptide synthetase
PD potato dextorose medium
PDA potato dextorose agar
PHAB plate habitat
PKS polyketide synthase
- xix -
PR photoreactivation
PVA polyvinyl alcohol
RNA ribonucleic acid
ROS reactive oxygen species
SABL Space Automated Bioproduct Lab
SM secondary metabolite
SMC simulated Mars conditions
SNP single nucleotide polymorphism
TMT tandem mass tags
UV-C ultraviolet C
WGS whole genome sequencing
WT wild type
- xx -
Chapter1
Introduction
1
1.1 Adaptability and persistence of fungi in various envi-
ronments
Microorganisms are omnipresent. Both bacteria and filamentous fungi are
capable of thriving in various abiotic conditions, sometimes colonizing what seem
like inhospitable environments. Fungi are capable of withstanding a wide range of
extreme temperatures [1, 2] and pHs [3, 4, 5], nutrient deprivation, desiccation [6],
irradiation [7] and space conditions [8, 9]. Being ubiquitous fungi have been isolated
from a vast array of environments on Earth, including barren lands, caves [10,
11, 12], deserts [13, 14], ice of the Antarctic [15, 16, 17, 18], and nuclear accident
sites [7].
Apart from occupying environmental niches, many fungi are known human
commensals [19, 20]. Each individual is characterized by a personal microbial
cloud [21] that is dominated by bacteria (99.9%). However, despite their low
abundance in the human microbiota, fungi may play a key role in the overall
maintenance of microbial community structure [20]. Further, the human myco-
biome has an emerging role in health and disease, as some disease-causing fungi
are of endogenous origin [19, 22]. Fungal genera Aspergillus, Penicillium, and
Cladosporium are predominant in nasal and oral cavities [23, 24], whereas human
1
Blachowicz

et al., Methods in Microbiology, 2018
1.2. Fungi in confined spaces on the example of the ISS A. Błachowicz
skin is dominated by genus Malassezia [25]. Various species of fungi have also been
associated with the human gut, lungs and urogenital tract [19, 26]. Since microor-
ganisms play an integral role in many human physiological processes, introducing
them to man-made habitats is inevitable. Additionally, as some subsets of fungi
are difficult to eradicate from man-made indoor environments [27] the presence of
fungi indoors may pose a health hazard for people with impaired immune functions
and be detrimental for habitat maintenance therefore, assessing the impact of
fungal presence in confined spaces is fundamental.
1.2 Fungi in confined spaces on the example of the ISS
One extraordinary example of a man-made closed habitat is the International
Space Station (ISS), which is a research platform in low Earth orbit (LEO)
inhabited by 3 to 10 astronauts at all times [28]. The ISS was built through the
joint effort of USA, European, Japanese, Russian, and Canadian space agencies.
Its environment is characterized by enhanced radiation, microgravity, controlled
temperature, and humidity [29]. The ISS is utilized to carry out experiments on
microorganisms [30, 31], plants [32, 33], animals [34, 35], and humans [36, 37].
Results from these experiments are compared to ground counterparts to elucidate
the impact of space conditions on the physiology of living organisms. Other studies
investigate the molecular phenotype and community dynamics of microbes that
inhabit the ISS as a result of anthropogenic contamination by astronauts and
cargo [8, 38, 39]. Such microbial surveillance is important as the ISS crew changes
periodically and both humans and microbes are affected by space conditions [40,
41, 42, 43]. Further, space agencies have increased their efforts to understand the
host-microbe interactions, as fungi may pose a threat to both habitat maintenance
and human well-being during outer space exploration and life beyond low Earth
orbit. For example, some fungi are technophiles capable of biodegrading polymers
and corroding metals [44], while other fungi are causative agents of a variety of
health conditions, spanning from mild allergies [45] to potentially life-threatening
invasive aspergillosis (IA) in immunocompromised individuals [46]. In order to
facilitate studying the host-microbe interactions and the microbial dynamics of
- 2 -
1.3. Evaluation of fungal adaptation mechanisms A. Błachowicz
confined spaces several simulated closed habitats, including the ILMAH, an inflated
lunar/Mars analogue habitat [47, 48, 49], the Antarctic Concordia Station [50,
51], and the Mars500 facility [52] have been used. Knowledge gathered from these
confined areas is necessary for the development of proper maintenance strategies
and ensuring safe microenvironment.
1.3 Evaluation of fungal adaptation mechanisms
One characteristic of filamentous fungi that confers environmental advantage is
their ability to sense external stimuli and readily adapt to a spectrum of ecological
niches. For example, it has been reported that fungi isolated from highly irradiated
environments, like the Chernobyl nuclear power plant (ChNPP) [7] or “Evolution
Canyon” [53] are highly melanized. Melanins, which are pigments ubiquitous in
nature [54] possess UV protective properties. Additionally, 20% of fungi isolated
from ChNPP accident sites exhibited the previously unknown phenomenon of
radiotrophism, in which the fungus is capable of converting radiation into energy for
growth. Studiesshowedthatmelaninsundergochangesintheirelectronicproperties
following the exposure to ionizing radiation to enhance energy transduction [55,
56]. Melanins along with myriad other secondary metabolites (SMs), widely
produced by fungi, allow their producers to conquer versatile environmental
niches [57]. Often, SMs play ecological fitness roles thanks to broad range of
activities, including antifungal, antimicrobial or antiparasitic. Further, SMs help
with nutrient acquisition, and inter- and intraspecies signaling and symbiosis and
can impact the fungal developmental processes [57, 58, 59].
Apart from alterations in secondary metabolism fungi developed countless
other survival mechanisms, including modulation of protein and enzyme expression.
For example in order to adapt to low-nutrient environment and starvation, fungi
alter carbohydrate metabolism, which leads to utilizing alternative carbon sources
like C
2
compounds and chitin [60, 61, 62]. Further, amino acid starvation may
triggeractivationofmostoftheaminoacidbiosyntheticenzymesviatranscriptional
activator GCN4p (CpcA/Cpc1) in yeast and Aspergilli [63, 64]. Other transcription
factors, including AP-1-like transcription factor Cap1 in Candida albicans, which
- 3 -
1.3. Evaluation of fungal adaptation mechanisms A. Błachowicz
is an orthologue of Saccharomyces cerevisiae Yap1 and Schizosaccharomyce pombe
Pap1 are important for oxidative stress adaptation [65], while Rim101 is involved
in conferring pH resistance of C. albicans [66].
Recent advancements in the next generation sequencing (NGS) technologies
have enabled in-depth investigation of microbial communities. Multi-omic analyses,
such as genomic, transcriptomic, metabolomic, and proteomic, have enhanced our
ability to unveil the molecular state of the cell in response to varying environ-
mental factors. Whole genome sequencing (WGS) is a comprehensive method of
elucidating genetic sequence of any organism. In fact, an exponential increase
in the availability of fungal genomes [67, 68] drives both, discoveries of unknown
secondary metabolite gene clusters and comparative analyses to discern alterations
in the fungal genetic sequence. The latter application may therefore lead to identi-
fication of crucial adaptive responses and alterations however, only when combined
with other omic analyses the active changes in the molecular state of the microor-
ganism can be determined. Proteomic and metabolomic profiles of fungi change
accordingly to environmental conditions therefore provide an excellent snapshot
of a molecular phenotype triggered by studied conditions. Observed differentially
expressed proteins may add to our understanding of altered biological responses, as
unknown functions and protein-protein interactions may be elucidated and further
characterized [69, 70]. Similarly, metabolomic analyses may result in identifica-
tion of unique secondary metabolites conferring environmental advantage under
selected conditions. Such holistic approach translates to significant advances in
understanding of fungal adaptation mechanisms, which can both unravel enormous
biotechnological potential of fungi and the threat they pose for human well-being
during space explorations.
- 4 -
1.3. Evaluation of fungal adaptation mechanisms A. Błachowicz
Chapter 1 is incorporated from Blachowicz, A., Venkateswaran, K., Wang,
C.C.C., (2018). Persistence of Fungi in Atypical, Closed Environments: Cultivation
to Omics, Methods in Microbiology, 45, 67-86, DOI: 10.1016/bs.mim.2018.07.006.
Chapter 2 is incorporated from Knox, B.P., †Blachowicz, A., †Palmer, J.M.,
Romsdahl, J., Huttenlocher, A., Wang, C. C. C., Keller, N. P., and Venkateswaran,
K., (2016). Characterization of Aspergillus fumigatus Isolated From Air and Sur-
facesoftheInternationalSpaceStation, mSphere 1(5):e00227-16. DOI:10.1128/mSphere.00227-
16.
The author conducted secondary metabolite (SM) analysis and drafted correspond-
ing results, materials and methods, and discussion sections.
Chapter 3 is incorporated from Blachowicz, A., Chiang, A.J., Romsdahl,
J., Kalkum, M., Wang, C.C.C., Venkateswaran, K., (2019). Proteomic Char-
acterization of Aspergillus fumigatus Isolates from the Air and Surfaces of the
International Space Station, Fungal Genetics and Biology, 1087-1845(19), DOI:
10.1016/j.fgb.2019.01.001.
Chapter 4 is incorporated from Blachowicz, A., Chiang, A.J., Elsaesser, A.,
Kalkum, M., Ehrenfreund, P., Stajich, J.E., Torok, T., Wang, C.C.C., and
Venkateswaran, K., Proteomic and Metabolomic Characteristics of Extremophilic
Fungi Under Simulated Mars Conditions, which is under review in Frontiers in
Microbiology.
Chapter 5 is incorporated from Blachowicz, A., Romsdahl, J., A., Chiang,
A.J., Masonjones. S., M., Kalkum, M., Stajich J.E., Torok, T., Wang, C.C.C.,
and Venkateswaran K., The International Space Station Environment Triggers
Molecular Responses in Aspergillus niger, which is being prepared for submission
to Microbial Genomics.
Chapter 6 is incorporated from Romsdahl, J., Blachowicz, A., Chiang, A.J.,
Singh, N., Stajich J.E., Kalkum, M., Venkateswaran K., Wang, C.C.C., (2018).
Characterization of Aspergillus niger Isolated from the International Space Station,
mSystems, 3:e00112-18. DOI: 10.1128/mSystems.00112-18
- 5 -
1.3. Evaluation of fungal adaptation mechanisms A. Błachowicz
The author helped with the proteome analysis and preparation of the corresponding
figure.
Chapter 7 is incorporated from Romsdahl, J., Blachowicz, A., Chiang Y.M.,
Venkateswaran K., Wang, C.C.C., Metabolomic Analysis of Aspergillus niger
Isolated from the International Space Station Facilitates Identification of the
Pyranonigrin A Biosynthetic Gene Cluster, which is under review in Fungal
Genetics and Biology.
The author analysed SM profiles of the ISS and ATCC isolates and prepared
the corresponding figure. The author helped with the initial set up of the UV-C
experiments and the figure preparation.
Chapter8isincorporatedfromRomsdahl, J., Blachowicz, A., Chiang, A.J., Chi-
ang, Y.M., Masonjones, S., Yaegashi, J., Countryman, S., Karouia, F., Kalkum, M.,
Stajich J.E., Venkateswaran K., Wang, C.C.C., (2018). International Space Station
Conditions Alter Genomics, Proteomics, and Metabolomics in Aspergillus nidulans,
Applied Microbiology and Biotechnology, 103(3):1363-1377, DOI: 10.1007/s00253-
018-9525-0.
The author helped with sample processing for genomic, metabolomic and proteomic
analyses and preparation of the figures presenting proteomic and metabolomic
results.
The author has made the most contributions to the review in Chapter 1 and
the studies described in Chapter 3, 4, and 5 and some contributions to the studies
in Chapter 2, 6, 7, and 8, as described above and further indicated throughout the
thesis. Genome annotation and phylogenetic analyses described in Chapter 2 were
conducted by Dr. J. M. Palmer. Growth characterization, in vitro stress assays,
and virulence testing in zebrafish model described in Chapter 2 were conduced by
Dr. B. P. Knox. All proteomic analyses described in Chapter 3, 4, 5, 6, and 8 were
conducted at City of Hope by A. Chiang and Dr. M. Kalkum. The Simulated
Mars conditions experiments described in Chapter 4 were conducted by Dr. A.
Elsaesser and Dr. P. Ehrenfreund at the Leiden Institute of Chemistry in the
Netherlands. Genome annotation and analyses described in Chapter 4, 5, 6, and 8
- 6 -
1.3. Evaluation of fungal adaptation mechanisms A. Błachowicz
were conducted or supervised by Dr. J. E. Stajich. Dr. J. Romsdahl was the lead
researcher responsible for conducting the experiments and drafting the manuscripts
presented in Chapter 6, 7, and 8.
- 7 -
Chapter2
Characterization of
Aspergillus fumigatus isolated from
air and surfaces of the International
Space Station
1
2.1 Abstract
One mission of the Microbial Observatory Experiments on the International
Space Station (ISS) is to examine the traits and diversity of fungal isolates to gain
a better understanding of how fungi may adapt to microgravity environments and
how this may affect interactions with humans in closed habitat. Here, we report an
initial characterization of two isolates, ISSFT-021 and IF1SW-F4, of Aspergillus fu-
migatus collected from the ISS in comparison to the experimentally established
clinical isolates Af293 and CEA10. Whole genome sequencing of ISSFT-021 and
IF1SW-F4 shows 54,960 and 52,129 single nucleotide polymorphisms, respectively,
compared to Af293 which is consistent with observed genetic heterogeneity amongst
sequenced A. fumigatus isolates from diverse clinical and environmental sources.
Assessment of in vitro growth characteristics, secondary metabolite production,
1
Knox

, Blachowicz

, Palmer

et al., mSphere, 2016;

equal contributing; the author
conducted secondary metabolite (SM) analysis and drafted corresponding results, materials and
methods, and discussion sections.
2.2. Introduction A. Błachowicz
and susceptibility to chemical stresses reveal no outstanding differences between
ISS and clinical strains that would suggest special adaptation to life aboard the ISS.
Virulence assessment in a neutrophil-deficient larval zebrafish model of invasive
aspergillosis revealed that both ISSFT-021 and IF1SW-F4 were significantly more
lethal compared to Af293 and CEA10.
Importance
As durations of manned space missions increase, it is imperative to understand
the long-term consequence of microbial exposure on human health in closed human
habitat. To date, studies aimed at bacterial and fungal contamination of space
vessels has highlighted species compositions biased towards hardy, persistent
organisms capable of withstanding harsh conditions. In the current study, we
assess traits of two independent Aspergillus fumigatus strains isolated from the
International Space Station. Ubiquitously found in terrestrial soil and atmospheric
environments, A. fumigatus is a significant opportunistic fungal threat to human
health particularly among the immunocompromised. Using two well-known clinical
isolates of A. fumigatus as comparators, we find that both ISS isolates exhibit
normalin vitro growth and chemical stress tolerance yet cause more lethality in
a vertebrate model of invasive disease. These findings substantiate the need for
additional studies of physical traits and biological activities of microbes adapted
to microgravity and other extreme extraterrestrial conditions.
2.2 Introduction
Microorganisms are unavoidable inhabitants of human-made structures in
space due to anthropogenic sources including human and cargo movement [71].
Our understanding of how the stressors of such environments, which include
microgravity and increased exposure to irradiation, influence microbial biology
over time remains in its infancy [72]. Changes in microbial community composition
and microbial species characteristics has the potential to affect human health and
safety, particularly in light of the fact that extended periods of time in space have
been shown to alter vertebrate and human immunity [73, 74]. Furthermore, as the
- 9 -
2.2. Introduction A. Błachowicz
duration of manned space missions increase, such as going to Mars, it becomes
of heightened importance to understand the breadth and potential consequences
of host-microbe interactions in crew habitation. There exists an unmet need for
studies characterizing individual microbial species isolated directly from space
environments as sampling experiments to date aim at understanding changes
in microbial community composition at the species level [74, 75, 76], and it
has been documented that simulated space environments are providing a poor
comparison to what is actually observed in orbit [8], highlighting a need for
additional experimentation of samples derived from space environments.
While, unsurprisingly, most of the sampled bacterial diversity from space
environments align with commensal organisms [77], many fungi represent a unique
component of microbial communities in space environments as their populations
are not replenished by virtue of human presence suggesting they have exploited
or adapted to a proliferative niche aboard these human-made structures. Fungal
colonization of space vessels is nothing new as various species have been isolated
from the Skylab, Mir, and various modules of the International Space Station
(ISS) (US, Japan’s KIBO, and the Russian segments) [75, 76, 78, 79, 39, 80, 81].
Fungi have been reported to cause damage to electrical and structural components
through the decomposition of wire insulation and window gaskets [81]. The most
commonly sampled fungal genera from space environments include the terrestrially
ubiquitous sporulating molds Cladosporium, Penicillium, and Aspergillus. Airborne
spores, also known as conidia, are ubiquitous in human environments and can
exacerbate pulmonary allergic reactions [82] and cause life-threatening invasive
infections after germinating in immunocompromised individuals [83, 84]. Among
airborne fungi, Aspergillus fumigatus is the most frequently encountered agent of
pulmonary complications and invasive infections resulting in invasive aspergillosis
(IA) in immunocompromised populations with average mortality rates of 50% even
with proper diagnosis and treatment [46].
Globally encountered in soil and air, A. fumigatus is well adapted to colonizing
diverse environments through its metabolic diversity, broad stress and thermal
tolerances, and easily dispersed conidia [85, 86, 87]. Likely underlying the ubiquity
- 10 -
2.3. Results A. Błachowicz
and pathogenic capacity of A. fumigatus is a great degree of genetic diversity
observed amongst strains from diverse environmental and clinical sources [88,
89, 90]. While many factors may contribute to the ubiquity of A. fumigatus,
the production of small bioactive molecules, or secondary metabolites (SMs),
has become of particular interest as these compounds have been shown to play
central roles in niche exploitation, stress tolerance, and virulence [46, 91, 92].
Considering A. fumigatus as a ubiquitously encountered opportunistic pathogen
with great metabolic and genetic diversity, it is an organism of particular interest
to monitor and examine as a contaminant of human space vessels. Here, we
report an initial characterization of two A. fumigatus strains isolated from different
sources of the ISS. Our experimental approach aimed to investigate each strain’s
genetic origins and characteristics,in vitro growth and stress tolerance, secondary
metabolite production, and virulence. Given the importance of A. fumigatus as
an opportunistic pathogen, both ISS strains were studied in comparison to two
well-known clinical isolates to assess pathogenic traits that may be of consequence
to human health.
2.3 Results
2.3.1 Identification of A. fumigatus samples from the ISS
Air and surface sampling during the Microbial Observatory Experiments on
the ISS identified numerous bacterial and fungal isolates [8]. For this study,
we chose to focus on two independently sampled strains of A. fumigatus which
were initially identified by morphological characteristics and later verified by
internal transcribed spacer (ITS) region sequencing (ITS sequences for ISSFT-021
and IF1SW-F4 are available under GenBank accession numbers KT832787 and
KX675260, respectively). Strain ISSFT-021 was sampled from a high-efficiency
particulate arrestance (HEPA) filter, and strain IF1SW-F4 was obtained from a
hard surface adjacent to the cupola window (Fig. 2.1A) via wiping surface materials.
By nature of this sampling method, it is impossible to know exact residence times
aboard the ISS for each strain.
Whole-genome sequencing (WGS) of ISSFT-021 and IF1SW-F4 [93] reveal
- 11 -
2.3. Results A. Błachowicz
Figure2.1: IsolationandphylogeneticcharacterizationofISSstrains. (A)IF1SW-F4was
isolated from the wall area outlined in blue adjacent to the cupola window aboard the ISS.
ISSFT-021 was independently isolated from a HEPA filter (not shown). (B) Frequency
distribution of total SNPs found in 94 sequenced clinical and environmental isolates of
A. fumigatus in comparison to a reference genome (Af293). Colored arrows designate the
bin groups to which each strain included in this study belongs. (C) Phylogenetic tree
of 95 sequenced isolates of A. fumigatus, showing mating type (MAT1-1 or MAT1-2),
clinical or environmental origin, and geographical sampling location. Strains of interest
used in this study are highlighted in yellow. Figure courtesy of Dr. J. M. Palmer.
- 12 -
2.3. Results A. Błachowicz
54,960 and 52,129 single nucleotide polymorphisms (SNPs) compared to the clinical
isolate and model laboratory strain Af293, respectively, which is not outside the
genetic diversity observed among 95 sequenced A. fumigatus isolates (Fig. 2.1B).
Figure 2.1C shows a phylogeny of these isolates of A. fumigatus inferred using
maximum likelihood from SNP sequences covered in every genome (147,792 total
SNP positions). The tree also includes information on individual isolate mating
type, clinical or environmental origin, and also geographical location. This analysis
illustrates the genomic variation that exists with A. fumigatus and, moreover,
suggests that we are unable to predict phenotypic characteristics (i.e., virulence)
based on clinical/environmental origin, mating type, or geographical isolation
source. Withregardtomatingtype, strainsISSFT-021andCEA10(anotherclinical
isolate and model laboratory strain) are MAT1-1, while IF1SW-F4 and Af293 are
MAT1-2. Interestingly, ISSFT-021 and IF1SW-F4 show a close relationship to the
patient isolate Af300 [94], suggesting these three strains may have arisen from a
common origin. As it has been suggested previously that genetic consequences
resulting from irradiation exposure during time in space may manifest as insertions
and deletions (INDELs) over point mutations [95, 96, 97], we analyzed ISSFT-021,
IF1SW-F4, and CEA10 and averages from all sequenced isolates included in this
study against the reference genome (Af293) and found no obvious enrichment for
INDELs in the ISS isolates.
2.3.2 Visual characterization and growth rates of ISS strains in vitro
To assess basic physiological phenotypes of strains ISSFT-021 and IF1SW-F4,
growth characteristics of the ISS strains were investigated on defined glucose
minimal medium (GMM) [98]. Gross visual assessment of point-inoculated GMM
plates revealed slight differences in colony diameter and pigment production after a
5 day incubation (Fig. 2.2A), indicating that each strain possesses unique physical
and chemical properties under these conditions. An examination of radial growth
rates revealed that both ISS strains significantly outgrew both Af293 and CEA10 at
all time points investigated (Fig. 2.2B), which is consistent with previous reports
of strain-dependent variations in growth rates of A. fumigatus isolates [88] as
well as increased biomass production for some microbes during exposure to space
- 13 -
2.3. Results A. Błachowicz
Figure 2.2: In vitro growth of ISS isolates compared to growth of the clinical isolates
Af293 and CEA10. (A) Growth on GMM at 37

C, showing colony morphology and
color. (B) Radial growth at 37

C on GMM. Statistical analyses were performed by
one-way ANOVA. (C) Germination rates in liquid GMM at 37

C, 250 rpm. Spores were
considered germinated after germ tube lengths were observed to be greater than or equal
to the swollen spore base. Figure courtesy of Dr. B. P. Knox.
environments [99]. As early spore germination rates may favor niche exploitation
and establishment of robust growth [88], we next sought to determine whether
ISS isolates exhibited different germination dynamics in vitro. Figure 2.2C shows
there were no differences in germination rates between ISSFT-021, IF1SW-F4, and
CEA10 in liquid GMM, as nearly all spores germinated after an 8 hr incubation,
while Af293 showed a marked delay in germination.
- 14 -
2.3. Results A. Błachowicz
2.3.3 ISS strains show no enhanced resistance to chemical stresses
in vitro
While unstressed growth (Fig. 2.2) revealed several differences between ISS and
patient isolate strains, it is reasonable to posit that resistance to stressful conditions
may play a greater role in colonization and propagation aboard harsh space vessel
environments. Therefore, we challenged all strains to a variety of classical chemical
stresses on GMM to assay susceptibility to osmotic stress (sodium chloride [NaCl]),
DNA damage stress (methyl methanesulfonate [MMS]), cell wall stress (Congo
red), and oxidative stress (hydrogen peroxide [H
2
O
2
]) (Fig. 2.3). Figure 2.3A
shows colony appearance and growth reduction on supplemented media compared
to that under unstressed growth conditions. No significant differences in growth
reduction were observed for either ISS strain in comparison to Af293 or CEA10
(Fig. 2.3B). For oxidative stress tests, we chose a diffusion assay [100] to reduce
experimental variation in working with this chemical (unpublished observations).
Zones of inhibition (Fig. 2.3C) were measured to infer sensitivity to hydrogen
peroxide. No significant differences were observed between ISS strains regarding
hydrogen peroxide sensitivity (Fig. 2.3D); however, both were significantly more
resistant than Af293 and less resistant than CEA10, demonstrating an intermediate
phenotype of ISS isolates between the two clinical strains.
2.3.4 Secondary metabolite analysis among ISS and clinical isolates
SM profiles of ISSFT-021, IF1SW-F4, CEA10, and Af293 were examined after
cultureonsolidGMMbyusinghigh-performanceliquidchromatography–photodiode
array detection–mass spectroscopy (HPLC-DAD-MS) analysis. Examination of the
SM profiles revealed a distinct chemical signature for each strain under the condi-
tion tested (Fig 2.4A). Detailed yield analysis of each SM produced was carried
out (Fig. 2.4B). Compared to Af293, an increase in fumigaclavine A production
was observed with IF1SW-F4 (P = 0:0001) but not with ISSFT-021, whereas a
significant decrease in fumigaclavine C production was noticed in both strains
(P = 0:04 and 0.0001 in ISSFT-021 and IF1SW-F4, respectively). Fumiquinazoline
production increased in ISSFT-021 (P =0:0004) but not in IF1SW-F4 compared
- 15 -
2.3. Results A. Błachowicz
Figure 2.3: ISS isolates showed no enhanced resistance to chemical stresses in vitro.
(A) Colony appearance after point inoculations of 10 μL containing 110
4
conidia on
solid GMM supplemented with the following stressors: 1.0 M NaCl, 0.02% MMS, and
25 mg/mL Congo red (CR). (B) Quantification of growth inhibition was measured by
colony diameters after a 72 hr incubation at 37

C. Data shown are the radial growth
versus that in controls. (C) Hydrogen peroxide sensitivity was assayed by diffusion
assay with 1-cm holes filled with 100 μL of 4% H
2
O
2
in plates containing 510
7
spores
suspended in top agar. (D) Zones of inhibition were measured as diameters after 48 hr
of growth, and significance determined via one-way analysis of variance. Figure courtesy
of Dr. B. P. Knox.
- 16 -
2.3. Results A. Błachowicz
to the two controls. Pyripyropene A production increased in both ISSFT-021 and
IF1SW-F4 (P = 0:0063 and 0.0018, respectively). Observed SM yields for CEA10
were lower than for any other strain, with an observed decrease in production of
all but two compounds (pyripyropene A and fumagillin) (Fig. 2.4A). Nevertheless,
as production of SMs in media does not necessarily replicate the pattern of SM
production during infections, the potential in vivo SM profiles of the ISS strains
remain unclear.
While a detailed analysis of SNPs with potential consequence on SM regulation
and production was beyond the scope of the present study, polymorphisms within
26 SM gene clusters that produce either known SMs or have strong bioinformatic
support to produce a likely SM were observed [101]. The combined analysis
of CEA10, ISSFT-021, and IF1SW-F4 identified 1,578 variants in comparison
with Af293 within the boundaries of the 26 SM gene clusters, and 504 of these
variants were predicted to result in nonsynonymous substitution in an SM cluster
gene (data not shown). Importantly, our analysis corroborated the previously
published point mutation R202L in FtmD (Afu8g00200), which results in loss
of fumitremorgin production in Af293 [102], as well as the previously described
frameshift mutation in TpcC in the CEA10 trypacidin cluster that leads to loss
of function of the trypacidin polyketide synthase [103]. A metabolite between
pyripyropene A (peak no. 5) and fumagillin (peak no. 6) (Fig. 2.4A, starred) was
observed via liquid chromatography-mass spectrometry (LC/MS) in the CEA10,
ISSFT-021, and IF1SW-F4 strains (see Fig. 2.5). We used high-resolution mass
spectrometry (494.2276 in positive mode) to obtain a proposed molecular formula
of C
27
H
31
O
6
N
3
for the compound (see Fig. 2.6). A chemical database (Reaxys)
search using the proposed formula revealed no known A. fumigatus metabolite that
matched this formula. However, the metabolites versicamide F from Aspergillus
versicolor [104] and taichunamides C and F from Aspergillus taichungensis [105]
matched the proposed formula. These findings might indicate that the compound is
a previously uncharacterized prenylated indole alkaloid produced by A. fumigatus.
- 17 -
2.3. Results A. Błachowicz
Figure 2.4: Secondary metabolite production of ISS strains. (A) Secondary metabolite profiles of ISSFT-021, IF1SW-F4,
CEA10, and Af293 when grown on GMM. Individual metabolite production is reported as either increased, decreased, or
no difference. compared to that of Af293. (B) Metabolite quantification, showing the percent change for each metabolite in
relation to Af293; significance was determined using a one-way ANOVA. Figure prepared by the author.
- 18 -
2.3. Results A. Błachowicz
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Time (min)
0
200000
400000
uAU
0
200000
400000
uAU
0
200000
400000
uAU
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Time (min)
0
50
100
0
50
100
0
50
100
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Time (min)
0
50
100
0
50
100
0
50
100
CEA10
ISSFT-021
IF1SW-F4
CEA10
c + ESI
ISSFT-021
c + ESI
IF1SW-F4
c + ESI
CEA10
c + ESI
493.5-494.0
ISSFT-021
c + ESI
493.5-494.0
IF1SW-F4
c + ESI
493.5-494.0
500 1000 1500
m/z
0
100000000
200000000
300000000
400000000
500000000
600000000
Intensity
1044.91
493.96
533.92
352.15
580.21
227.08
615.98
1126.79 915.73
500 1000 1500
m/z
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
Intensity
1044.82
493.89
533.85
352.09
227.06 754.02
1297.49
500 1000 1500
m/z
0
200000000
400000000
600000000
800000000
1000000000
1200000000
Intensity
493.92
1044.82
533.85
352.10
227.13 573.74 786.56
1297.82
IF1SW-F4 ISSFT-021 CEA10
PDA
Total ion counts
Extracted ion counts
Figure 2.5: Secondary metabolite production of ISS strains, with a focus on an unknown
A. fumigatus compound. Secondary metabolite profiles shown are for ISSFT-021, IF1SW-
F4, and CEA10 when grown on glucose minimal medium. Traces present PDA, total,
and extracted ion counts and ESIMS (positive mode) data for the unknown compound
analyzed by LC/MS. Figure prepared by the author.
- 19 -
2.3. Results A. Błachowicz
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
494.2276
79.0211
156.1201
C27H31O6N3
Figure 2.6: High-resolution mass spectrometry of unknown compound. ESIMS (positive
mode) spectrum of a new, unknown compound, with its proposed molecular formula.
Figure prepared by the author.
Figure 2.7: Virulence assessment in a larval zebrafish model of invasive aspergillo-
sis. (A) Survival outcome through 7 days postinfection (dpi) of neutrophil-deficient
mpx:mCherry-2A-Rac2D57N larvae, in which neutrophils specifically are unable to reach
the site of infection. (B) Survival outcome of the second ISS isolate, IF1SW-F4, compared
to that of CEA10 and ISSFT-021 in mpx:mCherry-2A-Rac2D57N larvae. Shown are
data pooled from three (A) or four (B) independent experimental replicates. Statistical
analyses were performed using the Cox proportional hazard regression analysis. Figure
courtesy of Dr. B. P. Knox.
- 20 -
2.4. Discussion A. Błachowicz
2.3.5 ISS strains exhibit increased virulence in a vertebrate model of
invasive aspergillosis
Given the ISS’s intimate environment and potential for frequent exposure
between astronauts and A. fumigatus, it is of high importance to investigate the
virulence potential of these isolates. Therefore, we tested the virulence of both
ISS strains against CEA10 and Af293 in a larval zebrafish model of IA, which has
been shown to recapitulate key aspects of disease observed in murine models and
human disease [106, 107]. As immunocompetent zebrafish larvae, like humans and
mice, do not succumb to lethal infection following A. fumigatus challenge [106],
we utilized neutrophil-deficient larvae [Tg(mpx:mCherry-2A-Rac2D57N)] [108] to
investigate virulence differences between strains. Recapitulating previous reports in
a murine infection model [94], we found that CEA10 was more virulent than Af293
(P = 0:0001), while ISSFT-021 caused significantly more lethality than CEA10
(P = 0:0075) (Fig. 2.7A). Furthermore, no significant difference was observed in
virulence between ISSFT-021 and IF1SW-F4 (P = 0:1), while IF1SW-F4, like
ISSFT-021 (Fig. 2.7A), was significantly more virulent than CEA10 (P = 0:0025)
(Fig. 2.7B).
2.4 Discussion
In the present study, we investigated two independently isolated strains of
A. fumigatus from the ISS for differences in genetic makeup, growth characteri-
zations, stress tolerance, secondary metabolite production, and virulence. While
microbial colonization of space vessels is unavoidable and is well-established in
the literature [71, 77], less understood is how the unique stresses found aboard
space vessels, such as increased irradiation and microgravity, affect microorganisms
and their biology. Therefore, as A. fumigatus is the most significant airborne
opportunistic mold pathogen of humans [109], it became prudent to investigate
the ISS A. fumigatus strains for factors that may affect health-related interactions
with astronauts aboard the ISS and initiate lines of investigation into how space
flight may influence this particular pathogen.
Samples from HEPA filters, 16 surface locations, 12 air samples, and debris
- 21 -
2.4. Discussion A. Błachowicz
collected using vacuum cleaners across multiple spaceflights during the Microbial
Observatory Experiments [8] revealed the presence of many microbial species
aboard the ISS. Among 200 bacterial and fungal isolates identified using molec-
ular methods [8], fungal strains were particularly enriched with members of the
genera Penicillium, Aspergillus, and Rodoturulla. However, only two isolates were
identified as A. fumigatus from these samples; one each from a 40-month-old
HEPA filter (ISSFT-021) and a surface location adjacent to the cupola window
(IF1SW-F4) (Fig. 2.1A). Interestingly, since the inception of the ISS,20 years
of environmental monitoring did not reveal the presence of A. fumigatus [110].
Considering the environmental ubiquity of this species, this anomaly might be
partly due to collections from small surface areas (25 cm
2
), compared to the larger
1 m
2
sampling areas adapted during this study. In addition, regular ISS operation
and environmental monitoring protocols utilized conventional methods, in which
the isolation of Aspergillus was reported but not identified to the species level [110].
Unfortunately, ISS operations did not archive any fungal strains isolated during
these 20 years of operation; therefore, we could not address whether or not any
of the Aspergillus isolates were A. fumigatus. Hence, even though A. fumigatus
is ubiquitous in the terrestrial atmospheres and would thus be an unsurprising
contaminant of the ISS, this is the first report about the isolation of A. fumigatus
from the ISS.
Following initial culture and ITS sequence-based identification of the isolates
as A. fumigatus, we undertook WGS to facilitate the current and future studies
of ISSFT-021 and IF1SW-F4 [93]. Analysis of SNPs among all publicly available
sequenced isolates (> 100 total; 95 unique isolates included in the present study)
across a global range of clinical and environmental sources shows considerable
genetic diversity, consistent with previous reports of genetic variance in A. fumi-
gatus [89]. Given that we are unable to know how, when, and where ISSFT-021
and IF1SW-F4 initially colonized the ISS, it is curious that both strains bare
the closest relationship to the strains Af300 and JN10 [94] (Fig. 2.1C). Af300
was isolated in 1995 in Manchester, United Kingdom, from a leukemia patient
(David Denning, personal communication), and JN10 is an environmental isolate
of unknown sampling origin. Further clouding the origins of these strains is that
- 22 -
2.4. Discussion A. Błachowicz
cargo shipments to the ISS are manufactured and launched from all over the world.
Notably, both ISS isolates show no enhanced accumulation of SNPs (Fig. 2.1B),
suggesting that life aboard the space station was not accompanied by an accu-
mulation of mutations presumably from enhanced exposure to irradiation and
microgravity; however, since the proper terrestrial control strains do not exist for
ISSFT-021 and IF1SW-F4, we cannot determine/quantify mutations that may
have accumulated during time aboard the ISS. Interestingly, previous data suggest
that DNA damage resulting from time aboard the ISS may favor chromosomal
aberrations and large deletions over point mutations [95, 96, 97], a conclusion that
may have been fueled by studies that reported finding no detectable mutations
from time in space, although these experiments utilized experimental setups that
would favor detection of point mutations over large genetic lesions, or the studies
were possibly too short in duration for mutations to accumulate [111]. Sequence
analysis with ISS A. fumigatus strains suggested that there is no enrichment for
any type of mutation we could identify through our resequencing-based mapping
approach, namely, INDELs, in comparison with Af293, yet it remains to be deter-
mined whether A. fumigatus possesses inherent characteristics that would facilitate
maintenance of genomic integrity over other biological systems in space (including
humans and other eukaryotes) from which mutation data have been inferred.
Our reported in vitro analysis showing increased radial growth rates for space
isolates versus clinical isolates (Fig. 2.2) is not without precedent, as enhanced
fungal growth from Aspergillus and Penicillium species recovered from Mir, com-
pared to terrestrial reference strains, has previously been reported [77]. However,
there is a considerable difference among growth rates for terrestrial strains of
A. fumigatus, and so whether or not the increased growth rate observed in this
report is a consequence of life in space (i.e., mutation) or simply due to natural
variation is not possible to determine without comparison to original terrestrial
isolates. While a detailed analysis of specific SNPs in the ISS isolates was beyond
the scope of our present study, the number total SNP differences of ISSFT-021
and IF1SW-F4 compared to Af293 were within the range of documented variance
among existing sequences of environmental A. fumigatus strains (Fig. 2.1), sug-
- 23 -
2.4. Discussion A. Błachowicz
gesting that, without a more detailed analysis, the ISS strains likely reflect genetic
diversity already found on Earth [89].
While it is conceivable that differential growth rates among A. fumigatus
isolates [88] may favor more rapidly growing strains in gaining a niche foothold, the
stresses aboard the ISS likely influence microbial colonization to a greater extent
than de novo growth rates. As such, stress tolerance may play a more dominant
role in the ability of A. fumigatus to colonize harsh niches. Given that the stress
response pathways of A. fumigatus are unarguably involved in this organism’s
environmental ubiquity and its leading position as an opportunistic pathogen [85,
112, 113], it is still unclear how existing genetic heterogeneity plays a role in
stress tolerance. While limited in scope, we found no enhanced ability of the ISS
strains over Af293 and CEA10 to tolerate a diversity of chemical stresses (Fig. 2.3),
suggesting that stress tolerance may not play a dominant role in A. fumigatus
persistence aboard the ISS. Future studies aimed at guiding enhanced disinfectant
protocols aboard space vessels should consider these data and pursue effective
methods for clearing surfaces of A. fumigatus. However, it was observed that
both ISSFT-021 and IF1SW-F4 were significantly more resistant to UV irradiation
than clinical isolates [93], and current studies are under way that will leverage
sequence data and molecular approaches to elucidate these mechanisms. It is
critical to note that care must be taken in interpreting these results, as terrestrial
equivalents or reference strains, even if identified as the same species, do not
represent experimental controls. Future studies aimed at determining the effects
of microgravity and space irradiation on A. fumigatus biology will require direct
comparison to terrestrial clones not subjected to time in space.
ExaminationofsecondarymetaboliteprofilesofclinicalandISSstrainsrevealed
differences in SM production (Fig. 2.4). Whether these changes are due to adapta-
tions to unfavorable environmental conditions, such as low nutrient availability,
enhanced irradiation, and microgravity, remains to be determined and will be the
focus of future studies. One clear example linking genomic data to SM production
was found with strain IF1SW-F4, with which increased levels of fumigaclavine A
(47) were detected, whereas fumigaclavine C [114] production was significantly
- 24 -
2.4. Discussion A. Błachowicz
decreased (Fig. 2.4). The prenyl transferase FgaPT1 in A. fumigatus is known to
be responsible for the prenylation of fumigaclavine A to form fumigaclavine C [115],
and in the IF1SW-F4 strain there exists a frameshift mutation in fgaPT1 that
might be responsible for the observed accumulation of fumigaclavine A. Additional
differences in metabolite production between the two ISS strains compared to
Af293 could be due to SNPs found within regions of the secondary metabolite
gene clusters (see Data Set S1). Fumagillin, a toxic SM [116], is produced by
both Af293 and CEA10, while production was significantly decreased in both
of the ISS strains (Fig. 2.4). While fumagillin can alter neutrophil responses to
pathogenic stimuli [117, 118], among other elements of host defense [46], it has
been proposed as a potential virulence factor. Interestingly, the more virulent
ISS strains showed lower fumagillin production, suggesting that in vitro fumag-
illin production profiles may not be accurate predictors of virulence potential,
based on our in vivo findings with the neutrophil-deficient larval zebrafish model.
Monomethylsulochrin, a proposed precursor in the trypacidin pathway, is produced
by both ISS strains and Af293 but not by CEA10, despite the ability of this strain
to produce trypacidin [103]. However, our study was insufficient to pinpoint which,
if any, of the metabolites has influence on the increased virulence observed in
the two ISS strains, and additional experiments will be necessary to identify the
connection between increased virulence and specific metabolite production.
As A. fumigatus is a ubiquitous environmental organism and multifaceted
opportunistic pathogen [86], many studies have aimed at understanding traits
underlying pathogenesis and whether a strain’s virulence potential can be inferred
preemptively and indirectly from isolation source, physical traits, or genetic data.
Interestingly, studies have been unable to consistently support the hypothesis that
clinicalstrainshaveundergoneaselectivebottleneckandarethereforemorevirulent
than environmental isolates [88, 89, 119]. As both ISSFT-021 and IF1SW-F4 are
environmental isolates, albeit from a highly unusual environment, our finding
that both ISS strains are more virulent than the two clinical strains reinforces
the idea that isolation source is not predictive of pathogenic potential and also
reinforces the conclusion that all strains of A. fumigatus, regardless of origin,
possess an infective potential [120]. Alternatively, previous reports have suggested
- 25 -
2.4. Discussion A. Błachowicz
a correlation between in vitro growth rates and virulence [88, 121]. While we did
observe higher growth and germination rates of ISS isolates in vitro (Fig. 2.2), the
greatest difference was the slower growth and germination of Af293 compared to
all others, which may contribute to the lower virulence of this strain; however,
the identical germination rates and slightly reduced radial growth of CEA10 are
less suggestive of an underlying influence on virulence potential. Additionally, the
growingbodyofliteraturecharacterizing A. fumigatus strain-dependentphenotypes
has revealed broad phenotypes, including toxin production [120, 122] and host
immune responses [94], that suggest a complex milieu of factors involved in
predicting the outcome of host-A. fumigatus interactions. Furthermore, there has
been speculation that mating type (MAT1-1, MAT1-2) may offer insight into the
pathogenic potential of A. fumigatus isolates [119, 123], yet this supposition has
been challenged by more recent studies using isogenic mating-type pairs [124].
Both ISS strains are closely related (Fig. 2.1C) but possess opposite mating types
(Fig. 2.1C) and were found to be more virulent than either Af293 (MAT1-2) or
CEA10 (MAT1-1) and equally virulent as each other (Fig. 2.7). Regardless of
these strains coming from uncontrolled genetic backgrounds, our finding supports
current data that suggest there is no link between mating type and virulence.
Despite the ISS isolates coming from an extreme environment and possessing
opposite mating types, different growth rates in vitro, and distinct SM profiles
between themselves and Af293 and CEA10, our cumulative data are unable to
offer a predictive potential of enhanced virulence for these strains.
This study has shown the existence of distinct A. fumigatus isolates on the
ISS. The origin of these strains remains unknown, yet analysis of genome sequence
data showed their relationship to 93 other sequenced genomes of this species and
revealed a close relationship to a known patient isolate, Af300. Considering the
genetic diversity and environmental ubiquity of A. fumigatus, it is not surprising
to observe significant phenotypic variation among isolates, including growth rates,
stress tolerance, SM production, and virulence, with virulence being a multifaceted
phenotype in this opportunistic pathogen and likely a culmination of traits that
render the pathogenic potential of any given isolate difficult to predict. Altogether,
the present study reinforces the idea that all A. fumigatus strains, regardless of
- 26 -
2.5. Materials and Methods A. Błachowicz
isolation source or genetic origin, represent potential pathogens and should serve
to guide current and future sampling and maintenance regimens for space vessels.
2.5 Materials and Methods
2.5.1 Isolation and verification of A. fumigatus isolates from the ISS
Particulates associated with HEPA filters were scraped, and approximately 1
mg of material was resuspended in sterile phosphate-buffered saline (PBS; pH 7.4)
before being spread onto potato dextrose agar (PDA) plates [8]. To collect samples
from cupola surfaces, sterile polyester wipes were used. The sampling wipes
were assembled and manifested in the Jet Propulsion Laboratory (JPL, Pasadena,
CA) prior to space flight. Briefly, each polyester wipe (9 by 9 in; ITW Texwipe,
Mahwah, NJ) was folded two times and soaked in 15 mL of sterile molecular-
grade water (Sigma-Aldrich, St. Louis, MO) for 30 min, followed by transfer
to a sterile zip lock bag [125]. The wipes were packed along with the other kit
elements at Ames Research Center (ARC; Moffett Field, CA) and included TC
(total count, tryptic soy agar) and SDA (Sabouraud dextrose agar) contact slides
(Hycon, EMD Millipore, Billerica, MA), Opsite adhesive tape (Smith & Nephew,
Inc., London, United Kingdom), and an air sampling device with gelatin filters
(Sartorius, Göttingen, Germany). Each sampling kit was sent to the ISS as a
part of the Space-X cargo and was returned to Earth on Soyuz TM-14 or the
Dragon capsule. The kits were delivered to JPL immediately after returning to
Earth. During each sampling session on the ISS, only one astronaut collected
samples from eight different locations, using wipes and contact slides. Each wipe
was used to collect a sample 1 m
2
. The control wipe (environmental control) was
only taken out from the zip lock bag, unfolded, and packed back into the zip lock.
The samples were stored at 4

C until the return trip to Earth and subsequent
processing.
The sample processing took place in a class 10K cleanroom at JPL immediately
upon delivery of the return kits. Each wipe was aseptically taken out from the zip
lock bag and transferred to a 500 mL sterile bottle containing 200 mL of sterile
PBS. The bottle with the wipe was shaken for 2 min followed by concentration
- 27 -
2.5. Materials and Methods A. Błachowicz
with a concentrating pipette (InnovaPrep, Drexel, MO) using 0.45 μm hollow
fiber polysulfone tips and PBS elution fluid. The environmental control and each
sample were concentrated to 4 mL. A 200 μL aliquot was serially diluted in PBS
to estimate the cultivable population.
Concentrated samples were diluted in PBS (up to 10
6
of each original sam-
ple), plated on the media (100 μL; in duplicates) Reasoner’s 2A agar (R2A) for
environmental bacteria and PDA for fungi, and incubated at 25

C for 7 days;
CFU were then counted. A minimum of five isolates of distinct morphologies
was picked up for each location from each type of medium. The isolates were
archived in the semisolid R2A or PDA slants (agar media diluted 1:10) and stored
at room temperature. For identification purposes, each fungal isolate was revived
on PDA medium. Once a culture was confirmed to be pure, DNA extraction
was performed by colony PCR (UltraClean DNA kit [Mo Bio, Carlsbad, CA] or
Maxwell automated system [Promega, Madison, WI]). Concurrently, two cryobead
stocks (Copán Diagnostics, Murrieta, CA) were prepared for each isolate. Fungal
DNA was used for PCR to amplify ITS regions with primers ITS1F (5
0
TTGGT-
CATTTAGAGGAAGTAA 3
0
) [126] and Tw13 (5
0
GGTCCGTGTTTCAAGACG
3
0
) following an established protocol [127]. The fungal sequences were searched
against the UNITE database and identified based on the closest similarity to ITS
sequences of fungal type strains [128].
Publicly available raw sequencing reads of all A. fumigatus strains used in this
study were downloaded from either the NCBI Sequence Read Archive (SRA) or
the EBI European Nucleotide Archive (ENA) for comparison to the ISS isolates.
SNPs were called against the Af293 genome reference (NCBI accession number
GCA_000002655.1) using the Snippy pipeline and default settings. Briefly, the
Snippy program aligns reads to the genome reference sequence by using BWA
v0.7.12-r1044 [129], and variants are called using the FreeBayes program v0.9.21-7-
g7dd41db [130]. The variants are then quality filtered, and a “core” set of SNP
variants (defined as those SNPs with sufficient sequencing coverage for the genomic
location for all isolates) are extracted from the data using Snippy. A custom
Python script was used to convert the SNP data to binary format, and a maximum
- 28 -
2.5. Materials and Methods A. Błachowicz
likelihood phylogeny was inferred using RAxML v8.2.8 [131] with 1,000 bootstrap
replicates. Snippy variant call files (VCF) from CEA10, ISSFT-021, and IF1SW-
F4 were imported into CLC Genomics Workbench v9.01, and the variants were
filtered and annotated based on overlap with secondary metabolite gene cluster
predictions [101]. To determine which variants would result in nonsynonymous
changes to coding genes, the variants were annotated using the Amino Acid
Changes module of CLC Genomics Workbench.
2.5.2 Fungal culture
After initial isolation on PDA plates, all A. fumigatus strains were grown
on solid glucose minimal medium at 37

C, unless otherwise noted, for conidial
preparation, physiological analysis, and stress tests. Conidial preparations for
in vitro analyses were harvested after approximately 72 hr with sterile 0.01%
Tween–water and gentle agitation with an L-shaped spreader before being passed
through a double layer of sterile mica cloth into a 50 mL screw-cap tube. Conidia
wereenumeratedwithahemocytometerbeforeadjustmenttovariousconcentrations
as needed. Original spore suspensions maintained as glycerol stocks were stored at
80

C.
2.5.3 Physiological analysis
All GMM plates for physiological analysis and stress testing were measured
to contain 25 mL. For radial growth assessment, 110
4
conidia in a volume of
10 μL were centrally inoculated onto GMM plates. Radial growth was measured
daily at the time points indicated. For spore germination rate assays, 100 mL
of liquid GMM was inoculated with 510
6
spores/mL and grown at 37

C and
250 rpm. At 2, 4, 6, and 8 hr, 1 mL samples were drawn, pelleted in a tabletop
centrifuge for 1 min at maximum rpm and resuspended in a final volume of 100 μL
to concentrate spores and germlings for facile enumeration. As germlings can
clump, clouding clear counts, each sample was briefly sonicated in a water bath
for 10 s. One hundred cells per condition were counted and scored. A spore was
considered germinated when the germ tube diameter was greater than or equal to
- 29 -
2.5. Materials and Methods A. Błachowicz
the diameter of the swollen base.
2.5.4 Stress tests
Radial growth was assayed as described above but after supplementation with
various stressors. Concentrations for stress chemicals were as follows: 1 M NaCl,
4 μM menadione, 25 μg/mL Congo red, and 0.01% methyl methanesulfonate. For
assessment of H
2
O
2
sensitivity, it is easier to obtain consistent results by using a
modified diffusion assay [100] over direct medium supplementation, as done for
the stressors listed above. Briefly, spores were evenly suspended in 55

C GMM
while still liquid at a final count 1:510
4
per 25 mL plate. Following solidification
of the agar, a 1 cm circular core was removed from the center of each plate, and
the resulting hole was inoculated with 100 μL of 4% H
2
O
2
. Zones of inhibition
were measured after 48 hr.
2.5.5 Secondary metabolite extraction and analysis
Fungal isolates were cultivated at 30

C on GMM agar plates (6 g/liter NaNO
3
,
0.52 g/L KCl, 0.52 g/L MgSO
4
7H
2
O, 1.52 g/L KH
2
PO
4
, 10 g/L D-glucose,
15 g/L agar supplemented with 1 mL/L of trace elements) at 10
7
spores/μL per
plate (10 cm). After 5 days, agar was chopped into small pieces and extracted with
25 mL methanol (MeOH), followed by 1 hr sonication and filtration. Extraction
and sonication steps were repeated with 25 mL of 1:1 MeOH-dichloromethane.
After a second filtration, combined crude extracts of each isolate were evaporated in
vacuo to yield a residue that was then suspended in 25 mL of water and partitioned
with ethyl acetate (EtOAc; 25 mL). The EtOAc layer was evaporated in vacuo,
redissolved in 2 mL of 20% dimethyl sulfoxide–MeOH, and 10 μL was examined
by HPLC-DAD-MS analysis. HPLC-MS was carried out using a ThermoFinnigan
LCQ Advantage ion trap mass spectrometer with a reverse-phase C
18
column (3μm;
2.1 by 100 μm; Alltech Prevail) at a flow rate of 125 μL/min. The solvent gradient
for LC/MS was 95% MeCN–H
2
O (solvent B) in 5% MeCN–H2O (solvent A), both
of which contained 0.05% formic acid, as follows: 0% solvent B from 0 to 5 min, 0
to 100% solvent B from 5 min to 35 min, 100% solvent B from 35 to 40 min, 100
- 30 -
2.5. Materials and Methods A. Błachowicz
to 0% solvent B from 40 to 45 min, and reequilibration with 0% solvent B from 45
to 50 min.
2.5.6 Zebrafish care and maintenance
Adult zebrafish were reared as described previously [106]. Briefly, adults were
maintained on a dedicated aquatic system and exposed to a light/dark cycle of
14 hr and 10 hr, respectively, and fed twice daily. After spawning, embryos were
collected in E3 buffer and stored at 28.5

C. Methelyne blue, an ingredient of
E3 buffer that inhibits fungal growth, was omitted from E3 buffer (E3-MB) at
24 hr postfertilization. All larval zebrafish procedures and adult husbandry were
performed in compliance with NIH guidelines and approved by the University of
Wisconsin-Madison Institutional Animal Care and Use Committee.
2.5.7 Larval zebrafish virulence assay
Virulence assays were performed using the larval zebrafish model of invasive
aspergillosis as described previously [106] with slight modifications. Larval immune
suppression was obtained genetically through the use of transgenic mpx:mCherry-
2A-Rac2D57N larvae [108]. Prior to infection, larvae were screened and selected for
mCherry expression in neutrophils to identify individuals harboring the dominant
negative allele. Briefly, hindbrain ventricle infections were performed at 48 hr
postfertilization, versus 36 hr postfertilization, as originally reported. Briefly,
conidial stocks in PBS at a concentration of 1:510
8
conidia/mL were mixed 2:1
with 1% phenol red to 10
8
conidia/mL to visualize injection success. Larvae were
anesthetized in E3-MB supplemented with 0.2 nM tricaine (ethyl 3-aminobenzoate;
Sigma-Aldrich) prior to microinjection of 3 nL spore suspension or PBS vehicle
control into the hindbrain ventricle through the otic vesicle. Following microinjec-
tion, larvae were rinsed several times to remove anesthetic and housed individually
in wells of a 96-well plate in100 μL E3-MB. Survival of individual larvae was
scored daily using loss of heartbeat as the readout for mortality.
- 31 -
2.5. Materials and Methods A. Błachowicz
2.5.8 Statistical analysis
In order to compare differences in SM production between isolates, the area
under the electrospray ionization curve (ESI) was integrated for each compound.
SM data collected from three independent biological replicates were used for
statisticalanalysis. Ordinaryone-wayanalysisofvariance(ANOVA)wasconducted
to compare the level of production of seven identified SMs between Af293 (treated
as a control) and CEA10, ISSFT-021, and IF1SW-F4. The data are presented
as column charts with corresponding error bars. Data analysis was conducted
using GraphPad Prism version 7. Survival analysis for larval zebrafish infection
experiments was performed as previously described [106] by pooling experimental
replicatesandgeneratingPvaluesviaCoxproportionalhazardsregressionanalysis.
- 32 -
Chapter3
Proteomic characterization of
Aspergillus fumigatus isolated from
air and surfaces of the International
Space Station
1
3.1 Abstract
The on-going Microbial Observatory Experiments on the International Space
Station (ISS) revealed the presence of various microorganisms that may be affected
by the distinct environment of the ISS. The low-nutrient environment combined
with enhanced irradiation and microgravity may trigger changes in the molecular
suite of microorganisms leading to increased virulence and resistance of microbes.
Proteomic characterization of two Aspergillus fumigatus strains, ISSFT-021 and
IF1SW-F4, isolated from HEPA filter debris and cupola surface of the ISS, re-
spectively, is presented, along with a comparison to well-studied clinical isolates
Af293 and CEA10. In-depth analysis highlights variations in the proteome of both
ISS-isolated strains when compared to the clinical strains. Proteins that showed
increased abundance in ISS isolates were overall involved in stress responses, and
carbohydrate and secondary metabolism. Among the most abundant proteins were
1
Blachowicz et al., Fungal Genetics and Biology, 2019
3.2. Introduction A. Błachowicz
Pst2 and ArtA involved in oxidative stress response, PdcA and AcuE responsible
for ethanol fermentation and glyoxylate cycle, respectively, TpcA, TpcF, and TpcK
that are part of trypacidin biosynthetic pathway, and a toxin Asp-hemolysin. This
report provides insight into possible molecular adaptation of filamentous fungi to
the unique ISS environment.
3.2 Introduction
The International Space Station (ISS) is a man-made closed habitat that
functions as a platform to study the impact of the distinct space environment,
which includes enhanced irradiation and microgravity on humans [132, 133, 134,
135, 37], animals [34, 35], plants [32, 33, 136, 137, 138] and microorganisms [30, 139,
31, 50, 140]. Most experiments conducted on board the ISS are precisely planned.
Studied organisms are intentionally sent to the ISS to investigate the possible
alterations in their physiology, using ground controls for comparison. However,
one on-going ISS Microbial Observatory (ISS-MO) experiment focuses on studying
hitchhikers that have followed humans and cargo aboard the ISS [141]. Thorough
investigation of microbiological characteristics of closed habitats, like the ISS,
are indispensable to National Aeronautics and Space Administration (NASA), as
manned long-term space flight missions are within reach. A deeper understanding
of microbes that coexist in closed habitats with humans remains imperative to
astronauts’ health and the overall maintenance of closed systems.
Strict scrutiny of the microbiome and mycobiome of the ISS [141, 79, 8, 81,
39, 80], Mir [142, 76], and Skylab [75], in the past, has revealed prevalence of
fungal genera: Cladosporium, Penicillium, and Aspergillus in space environments.
These fungi can be both beneficial and detrimental to mankind, as they produce
a myriad of commercially useful bioactive compounds [143, 144, 145, 146, 147,
148], while also causing allergies [45], infections [149, 150] and biodeterioration of
habitats [151, 152, 27]. Aspergillus fumigatus, one of many fungal isolates identified
in a recent ISS-MO study [8], is a ubiquitous saprophytic fungus [153]. Its enormous
adaptation capacity enables it to not only be omnipresent in the environment,
but also to be a successful opportunistic pathogen [86]. A. fumigatus causes
- 34 -
3.3. Results A. Błachowicz
variety of health conditions spanning from allergies to potentially life-threatening
invasive aspergillosis (IA) in immunocompromised individuals [46, 154]. Initial
characterization of two A. fumigatus ISS isolates, ISSFT-021 and IF1SW-F4,
showed no outstanding differences in their genomes and secondary metabolites
profiles when compared to clinical isolates CEA10 and Af293, however both isolates
were significantly more lethal in a larval zebrafish model of IA [155]. Considering
that A. fumigatus becomes more virulent in space and therefore potentially more
dangerous to astronauts’ health, it was pertinent to further investigate molecular
changes of ISS-isolated strains.
Presented in this study are the unique differences observed in the proteome of
two ISS-isolated A. fumigatus strains, ISSFT-021 and IF1SW-F4, when compared
to Af293 and CEA10. The goal of this study was to understand if the unique
environmentoftheISS(low-nutrients, enhancedirradiationandmicrogravity)alters
the proteome of A. fumigatus. Due to an existing gap in our understanding of how
filamentous fungi molecularly adapt to space conditions, proteome investigation of
these two ISS-isolated A. fumigatus strains was prudent.
3.3 Results
3.3.1 Proteome analysis overview
Proteins with altered abundance in ISS-isolated strains ISSFT-021 and IF1SW-
F4, and clinical isolates Af293 and CEA10, were investigated upon extraction of
total protein from each strain. Extracted proteins were digested into peptides
and labeled using tandem mass tags (TMT), fractionated, and analyzed via LC-
MS/MS followed by spectrum/sequence matching using A. fumigatus Af293 protein
database (NCBI). The abundance ratios of all identified proteins were normalized
to Af293, that enabled identification of 553, 464 and 626 increased and 314, 289 and
317 decreased in abundance proteins in CEA10, ISSFT-021 and IF1SW-F4 strains,
respectively (Fig. 3.1). When compared to both, Af293 and CEA10, 60 proteins
showed increased and 32 decreased abundance in space strains only (fold change
(FC) >j2jº. AspGD GO Slim terms [156] were used to study the distribution of
differentially abundant proteins in space strains. Analysis of proteins with increased
- 35 -
3.3. Results A. Błachowicz
100 75 50 25 0 25 50 75 100
Protein catabolic process
DNA metabolic process
Protein folding
Transcription,
DNA-templated
Signal transduction
Pathogenesis
Cytoskeleton organization
Cellular homeostasis
Vesicle-mediated transport
Ribosome biogenesis
Sexual sporulation
Asexual sporulation
Cellular respiration
Secondary
metabolic process
Lipid metabolic process
Cell cycle
Filamentous growth
Cellular protein
modification process
Translation
RNA metabolic process
Organelle organization
Transport
Developmental process
Response to stress
Cellular amino acid
metabolic process
Response to chemical
Carbohydrate
metabolic process
No. of differentially expressed proteins
GO SLIM category
CEA10 Up-regulated
CEA10 Down-regulated
ISSFT-021 Up-regulated
ISSFT-021 Down-regulated
IF1SW-F4 Up-regulated
IF1SW-F4 Down-regulated
Figure 3.1: AspGD GO Slim terms of proteins normalized to Af293. Differentially
expressed proteins in CEA10, ISSFT-021 and IF1SW-F4 are categorized into GO Slim
categories using AspGD. Categories containing at least 5 up- or down-regulated proteins
are presented.
- 36 -
3.3. Results A. Błachowicz
abundance revealed involvement of 14 proteins in carbohydrate metabolic processes,
eight in stress responses, five in secondary metabolism and toxins biosynthesis, and
two in pathogenesis whereas five, three, one and zero proteins showed, respectively,
decreased abundance in these categories. Proteins associated with cellular amino
acid metabolic process (6), lipid cellular homeostasis (3), metabolic processes (2),
pathogenesis (2), and translation (2) exhibited increased abundance in space strains
only (Fig. 3.2). FungiDB [157] was used to carry out GO term enrichment analysis
to gain a general understanding of biological processes possibly affected by unique
environment of the ISS. The results revealed that significantly over-represented up-
regulated biological processes included secondary metabolic processes (40% of all
up-regulated proteins), carbohydrate metabolic processes (23%), and response to
chemical (15%), whereas significantly over-represented down-regulated processes
included carbohydrate metabolic processes (15%), response to heat (6%), and
mRNA metabolic processes (6%).
3.3.2 Secondary metabolism and toxins
The proteomic analysis revealed altered abundance of proteins involved in sec-
ondary metabolism (Table 3.1). Proteins involved in trypacidin biosynthetic path-
way, includingemodinO-methyltransferaseTpcA(AFUA_4G14580), glutathioneS-
transferaseTpcF(AFUA_4G14530), anddehydrataseTpcK(AFUA_4G14470)[158,
159, 103], were at least threefold more abundant in ISS-isolated strains than in
clinical isolates. Arp1 (AFUA_2G17580), a scytalone dehydratase involved in
conidial pigment biosynthesis [160, 161, 162] was threefold more abundant, whereas
Asp-hemolysin (Asp-HS; AFUA_3G00590) [163] was eight times more abundant
in ISS strains.
To confirm observed up-regulation of proteins involved in the trypacidin
biosynthesis secondary metabolite profiles of the clinical and ISS isolates were
acquiredusinghighperformanceliquidchromatography-photodiodearraydetection-
mass spectroscopy (HPLC-DAD-MS) analysis. Examination of the production
yields of monomethylsulochrin, the final intermediate in trypacidin biosynthesis,
(trypacidin is not detectable until 7–8 days of growth [103]) showed600% and
- 37 -
3.3. Results A. Błachowicz
15 10 5 0 5 10 15
Cellular respiration
Filamentous growth
Lipid metabolic process
Pathogenesis
Translation
Transport
Cellular homeostasis
Organelle organization
RNA metabolic process
Secondary
metabolic process
Cellular protein
modification process
Developmental process
Response to stress
Cellular amino acid
metabolic process
Response to chemical
Carbohydrate
metabolic process
No. of differentially
expressed proteins
GO SLIM category
Up-regulated
space strains
Down-regulated
space strains
Figure 3.2: AspGD GO Slim terms of proteins differentially expressed in space strains
when compared to Af293 and CEA10. Differentially expressed proteins in ISSFT-021 and
IF1SW-F4 are categorized into GO Slim categories using AspGD. Categories containing
at least 2 up- or down-regulated proteins are presented.
200% increased production in ISSFT-021 and IF1SW-F4, respectively (Fig. 3.3).
Additionally, proteome of clinical and ISS strains was examined in liquid CD and
PD media and confirmed significant up-regulation of Asp-HS (data not shown).
Lastly, observed differences in sporulation capacity between the strains do not
seem to be directly correlated with conidia-associated protein expression levels
(Fig. 3.4), suggesting that the observed variations are due to exposure to space
environment rather than different growth rates.
- 38 -
3.3. Results A. Błachowicz
Table 3.1: Proteins involved in secondary metabolism and toxin biosynthesis that
revealed increased or decreased abundance
ORF Protein Putative function / ac-
tivity
Relative protein abundance

P-value
CEA10 ISSFT-021 IF1SW-F4
AFUA_4G141530 TpcF Glutathione S-transferase
involved in trypacidin
biosynthesis
0:83 3:02 2:46 2:7610
6
AFUA_3G00590 Asp-HS Asp-hemolysin; hemolytic
toxin
0:00 3:01 3:24 7:4810
9
AFUA_4G14580 TpcA Emodin O-
methyltransferase
0:14 2:95 3:06 6:7710
7
involved in trypacidin
biosynthesis
AFUA_4G14470 TpcK Dehydratase involved in 0:27 2:58 2:17 3:0410
3
trypacidin biosynthesis
AFUA_2G17580 Arp1 Scytalone dehydratase in-
volved in conidial pigment
biosynthesis
0:58 2:49 2:69 2:2410
7
AFUA_4G00860 DprA Dehydrin-like protein / ox-
idative, osmotic and pH
stress responses
3:54 5:64 4:62 1:3710
7

Log2 fold change of CEA10, ISSFT-021, and IF1SW-F4 compared to Af293 (P < 0:05).
1 6 1 8 20 22 24 26 28 30 32 34 36 38 40
T ime (min)
0
50000000
Intensity
0
50000000
Intensity
0
50000000
Intensity
0
50000000
Intensity

+ EIC 347.3
IF1SW-F4

+ EIC 347.3
ISSFT-021

+ EIC 347.3
CEA10

+ EIC 347.3
Af293
A B
Figure 3.3: Monomethylsulochrin yields in clinical and ISS-isolated strains. (A) Ex-
tracted ion count (EIC) traces of monomethylsulochrin in a positive mode. (B) Quantifi-
cation of of monomethylsulochrin production using positive mode EIC when compared
to Af293. Significance was determined using Welch’s t-test.
- 39 -
3.3. Results A. Błachowicz
Day 3 Day 4 Day 5
0
2×10
5
4×10
5
6×10
5
8×10
5
1×10
6
Total spore count
Af293
CEA10
ISSFT-021
IF1SW-F4
*
*
*
*
*
*
Figure 3.4: Sporulation capacity of clinical and ISS-isolated strains. Average values of
conidial counts from 3 biological replicates are plotted after 3, 4 and 5 days of growth.
Statistical significance was determined using multiple t-test with Holm-Sidak correction.
3.3.3 Stress response
Among proteins with altered abundance, 11 were involved in the stress re-
sponse of A. fumigatus (Table 3.2). AFUA_5G11430, a quinone oxidoreduc-
tase, and Pst2 (AFUA_1G02820), an NADH-quinone oxidoreductase, involved
in oxidative stress response [164] were three and four times more abundant in
space strains. Erythromycin esterase, AFUA_1G05850, and 3
0
exoribonuclease
AFUA_2G15980 [165] were at least twofold more abundant in ISS strains. Among
proteins with decreased abundance several were heat shock proteins including
Scf1 (AFUA_1G17370) [166, 167], and Awh11 (AFUA_6G12450) [165]. Other
down-regulated protein was dehydrin-like protein DprA (AFUA_4G00860) that is
known to play a role in oxidative stress response [168].
3.3.4 Carbohydrate metabolic processes
Comparative analysis of proteomes of ISS-isolated strains, IF1SW-F4 and
ISSFT-021, with clinical isolates CEA10 and Af293 revealed changes in abundance
of proteins involved in carbohydrate metabolic processes (Table 3.3). Pyruvate
decarboxylase PdcA (AFUA_3G11070), which catalyzes the first step in anaerobic
conversion of pyruvate to ethanol [169], was about three times more abundant
in space strains when compared to clinical isolates. Proteins involved in glyc-
- 40 -
3.3. Results A. Błachowicz
Table 3.2: Proteins involved in stress response that revealed increased or decreased
abundance
ORF Protein Putativefunction/ac-
tivity
Relative protein abundance

P-value
CEA10 ISSFT-021 IF1SW-F4
AFUA_3G08470 Glucose-6-phosphate
1-dehydrogenase
1:41 3:06 3:33 8:6610
7
AFUA_4G141530 TpcF Glutathione S-transferase
involved in trypacidin
biosynthesis
0:83 3:02 2:46 2:7610
6
AFUA_4G11730 GldB Glycerol dehydrogenase 1:29 2:83 3:70 5:3410
7
AFUA_5G11430 Quinone oxidoreductase 0:95 2:55 2:51 1:4510
9
AFUA_2G03290 ArtA 14–3-3 family protein 0:67 2:21 2:82 1:9010
7
AFUA_1G02820 Pst2 NADH-quinone oxidore-
ductase
0:37 2:10 2:55 9:3310
6
AFUA_2G15980 3
0
exoribonuclease family
protein; pre-miRNA pro-
cessing
0:02 1:24 2:20 4:7310
3
AFUA_1G05850 Erythromycin esterase 0:16 1:22 2:48 6:6110
3
AFUA_1G17370 Scf1 Heat shock protein 2:95 5:23 4:26 4:2510
7
AFUA_6G12450 Awh11 Heat shock protein 4:05 5:81 5:12 5:4610
5
AFUA_4G00860 DprA Dehydrin-like protein /
oxidative, osmotic and pH
stress responses
3:54 5:64 4:62 1:3710
7

Log2 fold change of CEA10, ISSFT-021, and IF1SW-F4 compared to Af293 (P < 0:05).
erol metabolism, including glycerol dehydrogenase GldB (AFUA_4G11730), and
glycerol kinase AFUA_4G11540 [164] were at least 2.5-fold more abundant in
ISS-isolated strains. AFUA_5G10540 and AFUA_1G02140, which are homo-
logues of A. niger An14g04190 and GdbA (An01g06120) [170], respectively, and
involved in glycogen biosynthesis and metabolism were at least twofold more abun-
dant. Protein abundance of AFUA_3G08470, AFUA_4G08880, AFUA_7G01830,
and AFUA_1G14710 involved in glucose metabolism, increased twofold at mini-
mum. Both, the 14-3-3 family protein ArtA (AFUA_2G03290) [171] and malate
synthase AcuE (AFUA_6G03540) [172] were threefold more abundant when
compared to clinical isolates. At minimum twofold increased abundance of phos-
phoketolase (AFUA_3G00370) and hexokinase HxkA (AFUA_2G05910) [173]
was observed. Among the decreased in abundance proteins were hydrolase Exg17
(AFUA_6G14490)[174]and-1,3-glucanmodifyingenzymeSun1(AFUA_7G05450)[175].
- 41 -
3.3. Results A. Błachowicz
Table 3.3: Proteins involved in carbohydrate metabolism that revealed increased or
decreased abundance
ORF Protein Putative function / ac-
tivity
Relative protein abundance

P-value
CEA10 ISSFT-021 IF1SW-F4
AFUA_3G11070 PdcA Pyruvate decarboxylase in-
volved in ethanol fermenta-
tion pathway
1:95 3:42 3:43 9:5010
8
AFUA_4G11540 Glycerol kinase 1:52 3:18 2:94 5:9910
5
AFUA_3G08470 Glucose-6-phosphate
1-dehydrogenase
1:41 3:06 3:33 8:6610
7
AFUA_4G11730 GldB Glycerol dehydrogenase 1:29 2:83 3:70 5:3410
7
AFUA_1G14710 Beta-glucosidase 1:63 2:70 3:52 4:3410
5
AFUA_5G10540 1,4-alpha-glucan branch-
ing enzyme activity, glyco-
gen biosynthesis
0:91 2:23 3:06 1:3910
7
AFUA_2G03290 ArtA 14–3-3 family protein 0:67 2:21 2:82 1:9010
7
AFUA_6G03540 AcuE Malate synthase 0:38 2:10 2:16 1:2210
7
AFUA_1G02140 Glycogen debranching en-
zyme
0:87 1:98 3:18 1:0810
3
AFUA_3G00370 Phosphoketolase 0:51 1:88 2:84 3:5010
5
AFUA_4G04680 FGGY-family carbohy-
drate kinase
0:81 1:82 2:61 1:7310
5
AFUA_4G08880 Glucose-6-phosphate
1-epimerase
0:32 1:46 2:61 7:0010
5
AFUA_7G01830 Ugp1 UTP-glucose-1-phosphate
uridylyltransferase
0:00 1:35 2:11 4:4310
5
AFUA_2G05910 HxkA Hexokinase 0:01 1:20 2:12 8:6810
7
AFUA_7G05450 Sun1 Beta-1,3-glucan modifying
enzyme
0:61 1:87 2:15 4:1310
2
AFUA_1G06910 Arabinogalactan endo-1,4-
beta-galactosidase activity
0:43 1:53 2:04 9:0510
6
AFUA_6G05030 Polysaccharide deacetylase 0:09 1:21 1:23 1:5310
3
AFUA_6G14490 Exg17 O-glycosyl hydrolase 0:21 1:41 1:33 4:6810
5
AFUA_1G03140 Glycosyl hydrolase 0:31 1:31 1:44 1:3910
6

Log2 fold change of CEA10, ISSFT-021, and IF1SW-F4 compared to Af293 (P < 0:05).
- 42 -
3.4. Discussion A. Błachowicz
3.4 Discussion
The proteome of A. fumigatus has been studied under various conditions,
including short- [176], and long-term hypoxia [177], following exposure to antifungal
agents like amphotericin B [178], and voriconazole [179], and during different
developmental stages [167]. However, in this report we present unique proteome
differences observed in the two ISS-isolated strains when compared to clinical
isolates. To date, there is no report which elucidates the molecular response of
filamentous fungi to the distinct environment of the ISS, despite previous reports
of their presence on board of the ISS [8] and Mir [76, 44]. In-depth understanding
of alterations triggered in the proteome of omnipresent A. fumigatus remains
imperative for astronauts’ health, as it is an opportunistic pathogen that affects
individuals with impaired immune system functions [153].
Both ISS-isolated A. fumigatus strains displayed higher abundance of several
proteins involved in trypacidin biosynthesis. Trypacidin is a potent mycotoxin
produced by A. fumigatus conidia. It has been shown to have cytotoxic activity
against A549 alveolar lung cells suggesting its importance during the infection [180].
Further, it has been reported that A. fumigatus conidia with disrupted produc-
tion of trypacidin exhibit higher susceptibility to macrophage clearance [159].
Increased abundance of proteins involved in biosynthesis of trypacidin in both
ISS-isolated strains may be possible cause of the reported increased virulence in
the larval zebrafish model of both ISS-isolated strains when compared to clinical
isolates [155]. Moreover, emodin and questin, precursors of trypacidin, are pig-
mented anthraquinones that have been reported to have a protective effect against
UV in other organisms (Xanthoria elegans and Cetraria islandica) that produce
these types of compounds [103, 181]. It is therefore reasonable to assume that
up-regulation of trypacidin production may be possible adaptation to the enhanced
irradiation environment of the ISS, however this hypothesis has to be validated by
further experiments.
Further, the level of Asp-HS, which is a known toxin produced by A. fumiga-
tus [163, 182] was highly increased in both ISS-isolates. While several studies have
reported its hemolytic [183] and cytotoxic [184, 185] activities in the past, a recent
- 43 -
3.4. Discussion A. Błachowicz
report showed lack of attenuated virulence when Asp-HS gene was deleted [186].
This discrepancy may be strain specific as the studies used different A. fumigatus
strains to carry out the experiments or related to experimental models used in
both studies, as at times results observed for in vitro analyses do not correlated
with the outcomes observed in in vivo studies. The exact role of Asp-HS in
the pathogenicity remains to be determined during future studies. Nonetheless,
increased abundance of Asp-HS observed in both ISS-isolated strains appears to
be a part of the A. fumigatus adaptation response to the unique ISS environment.
One of the increased in abundance proteins in ISS-isolated A. fumigatus
strains was Arp1, which is one of the six enzymes involved in the DHN-melanin
production [160, 161, 162]. Arp1 disruption resulted in production of reddish pink
conidia with induced C3 binding that led to phagocytosis and killing of conidial
spores during infection [160, 161]. Increased abundance of Arp1 protein may,
therefore, be another cause of the previously reported increased virulence in the
larval zebra fish model [155]. Additionally, higher AlbA abundance, involved in
DHN-melanin production [187], was observed in JSC-093350089 A. niger isolated
from the ISS [188]. Seemingly, both ISS-isolated species, A. fumigatus and A. niger,
responded with increased melanin production to enhanced irradiation on board
of the ISS. This observation is in agreement with previous reports of increased
melanin production in fungi isolated from high-radiation environments such as
the Chernobyl Power Plant accident sites or “Evolution Canyon” [7, 53]. However,
because none of the other enzymes involved in the melanin biosynthesis showed
increased abundance when compared to both clinical isolates simultaneously,
further assessment of melanin production is necessary to definitively conclude its
increased production under ISS conditions.
Proteome analysis of both ISS-isolated A. fumigatus strains revealed altered
levels of proteins involved in oxidative stress response. ArtA, a regulatory protein,
was reported to be increased in abundance in response to incubation with H
2
O
2
,
suggesting its importance in overcoming oxidative stress [171]. Pst2, which is
also present in Candida albicans and Saccharomyces cerevisiae, was shown to be
induced in response to oxidative stress [189, 190]. Additionally, Pst2 was reported
- 44 -
3.4. Discussion A. Błachowicz
to be a post-translational modification target protein that is ubiquitinated under
peroxide stress conditions in C. albicans [191]. Interestingly, such post-translational
modifications play a role in several cellular processes in eukaryotes, including cell
growth regulation and environmental adaptation, suggesting their importance
in gaining survival advantage [192]. These data are in agreement with previous
reports of induced oxidative stress response in humans, mice and yeast while in
space [193, 42, 194, 195, 196]. Overall, increased abundance of proteins involved in
oxidative stress response was also observed in Acinetobacter sp. Ver3 when exposed
to UV irradiation [197] and JSC-093350089 A. niger [188]. Although increased
abundance of several oxidative stress-correlated proteins was observed in this study,
it was previously reported that ISS-isolated strains were more resistant than Af293
to H
2
O
2
exposure, but less resistant than CEA10, suggesting no adaptation [155].
Among the more abundant proteins in both ISS-isolated strains several were
involved in carbohydrate metabolism. Malate synthase AcuE (AFUA_6G03540)
is one of the three key enzymes involved in glyoxylate cycle [172], which has
been shown to be crucial for fungal growth on C
2
compounds and fatty acids as
a sole carbon source [198]. In our earlier study it was documented that these
two ISS-isolated strains significantly outgrew both clinical isolates [155]. Further,
more abundant PdcA (AFUA_3G11070), a pyruvate decarboxylase, has been
shown to be involved in ethanol fermentation [169]. These data therefore suggest
possible adaptation of A. fumigatus to low-nutrient environment of the ISS [141, 8],
which is in agreement with changes observed in proteome of A. fumigatus during
starvation [60]. Similarly, up-regulation of proteins involved in starvation response
was observed in ISS-isolated JSC-093350089 A. niger [188].
This study presents comparative proteomic analysis of two ISS-isolated A. fu-
migatus strains, ISSFT-021 and IF1SW-F4 when compared to well-studied isolates
Af293 and CEA10. Such comparison has enabled the identification of possible
adaptation responses to the unique microgravity environment of the ISS, which
includes increased abundance of stress response related proteins, and modulation
of proteins involved in carbohydrate and secondary metabolism. To our knowl-
edge this is the first report that focused on studying the proteomic changes in
- 45 -
3.5. Materials and Methods A. Błachowicz
filamentous fungus isolated from on board of the ISS. Complex analyses of possible
molecular alterations triggered by microgravity and enhanced irradiation will be
pertinent to the future long-term manned space flights, as such an understanding
is crucial for astronauts’ health and biodeterioration of the closed habitat.
3.5 Materials and Methods
3.5.1 Isolation and identification of A. fumigatus
Procedures to isolate and identify A. fumigatus collected from the ISS were
described previously [155]. In brief, HEPA filter associated particulates were
scraped, resuspended in sterile phosphate-buffered saline (PBS; pH 7.4) and spread
onto potato dextrose agar (PDA) plates [8]. Cupola surfaces were sampled with
sterile polyester wipes assembled at Jet Propulsion Laboratory (JPL) prior to
space flight. After each sampling event on board the ISS, wipes were returned to
JPL for subsequent processing. During that process, two of multiple isolates were
identified via ITS region and subsequently whole genome sequencing (WGS) as
A. fumigatus.
3.5.2 Growth conditions
Af293, CEA10, ISSFT-021 and IF1SW-F4 were cultivated for 5 days at 30

C
on glucose minimal medium (GMM) agar plates (6 g/L NaNO
3
, 0.52 g/L KCl,
0.52 g/L MgSO
4
7H
2
O, 1.52 g/L KH
2
PO
4
, 10 g/L D-glucose, 15 g/L agar
supplemented with 1 mL/L of Hutner’s trace elements) covered with cellophane
membrane. Each Petri plate (D =10 cm) was inoculated with 10
7
spores/plate.
For supplementary comparison, each strain was also cultured in potato-dextrose
(PD, BD Difco, Franklin Lakes, NJ) and Czapek-dox (CD, BD Difco) liquid media
for 2 days and 10 days, respectively.
3.5.3 Protein extraction
Mycelia and spores from GMM agar plates were collected. The hyphae from
the liquid media were collected on Whatman paper filter and washed with cell
- 46 -
3.5. Materials and Methods A. Błachowicz
culture grade water. All samples were stored at80

C prior to protein extraction.
The lysis buffer consisted of 100 mM triethylammonium bicarbonate (TEAB)
with 1:100 Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL) and
200 μg phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO). The hyphae
from the liquid media were first homogenized on ice using a Polytron (Kinematica
AG, Bohemia, NY) with a speed setting of 5 for 3–5 times (1 min/time, 30 s pause).
Subsequently, the crude homogenates were subjected to a Precellys 24 homogenizer
(Bertin, Rockville, MD) in which each sample was processed inside a 2 mL cryotube
with 0.5 mm glass beads three times (at 4

C , 6500 rpm, 1 min, repeated 3 times
with 15 s pauses in between). Mycelia from GMM were homogenized directly
by bead beating due to their small volume. The lysed fungi were centrifuged at
17,000 g for 15 min. Protein concentrations in the supernatants were measured by
Bradford assay (Bio-Rad Laboratories, Inc. Hercules, CA).
3.5.4 Tandem mass tag (TMT) labeling
200μg proteins from each group were TCA-precipitated. Obtained protein pel-
lets were washed with ice-cold acetone, and resolubilized in 25 μL TEAB (100 mM)
and 25 μL 2,2,2-trifluoroethanol (TFE). Subsequently, proteins were reduced with
tris(2-carboxyethyl)phosphine (TCEP, 500 mM), alkylated with iodoacetamide
(IAA) and digested overnight using mass spec grade trypsin/lysC (Promega, Madi-
son, WI) at 37

C. The digested peptides were quantified using the Pierce Quan-
titative Colorimetric Peptide Assay (Thermo Scientific). 40 μg of peptides from
each specific sample was labeled with the Thermo Scientific TMTsixplex Isobaric
Mass Tagging Kit (Af293 with TMT
6
-126, CEA10 with TMT
6
-127, ISSFT-021
with TMT
6
-128, and IF1SW-F4 with TMT
6
-130) according to the manufacturer’s
protocol. The TMT
6
-131 label was used as a reference that contained an equal
amount of peptides from each of the samples. All labeled-peptide mixtures were
combined into a single tube, mixed, and fractionated into eight fractions using the
Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit. The
fractionated samples were dried using a SpeedVac concentrator and re-suspended
in 1% (v/v) formic acid prior to LC-MS/MS analysis.
- 47 -
3.5. Materials and Methods A. Błachowicz
3.5.5 LC-MS/MS analysis
AnOrbitrapFusionTribridmassspectrometerwiththeThermoEASY-nLCion
source, 75μm2 cm Acclaim PepMap100 C
18
trapping column, and 75μm25 cm
PepMap RSLC C
18
analytical column was used to analyze the samples. Peptides
were eluted at 45

C with a flow rate of 300 nL/min over a 110 min gradient,
from 3 to 30% solvent B (100 min), 30–50% solvent B (3 min), 50–90% solvent B
(2 min), and 90% solvent B (2 min). The solvent A was 0.1% formic acid in water
and the solvent B was 0.1% formic acid in acetonitrile.
The full MS survey scan (m/z 400–1500) was acquired at a resolution of
120,000 and an automatic gain control (AGC) target of 210
5
in the Orbitrap
with the 50 ms maximum injection time for MS scans. Monoisotopic precursor
ions were selected for fragmentation with charge states 2-7, within a ±10 ppm
mass window, using a 70 s dynamic exclusion function. MS
2
scans (m/z 400–2000)
were performed using the linear ion trap with the 35% CID collision energy. The
ion trap scan rate was set to “rapid”, with an AGC target of 4 10
3
, and a
maximum injection time of 150 ms. Subsequently, ten fragment ions from each
MS
2
experiment were subjected to an MS
3
experiment. The MS
3
scan (m/z
100–500) generated the TMT reporter ions in the linear ion trap using HCD at
a 55% collision energy, a rapid scan rate and an AGC target of 510
3
, and a
maximum injection time of 250 ms.
3.5.6 Proteome data processing
The Proteome Discoverer (version 2.1.0.81, Thermo Scientific) with searching
engines Sequest-HT against an A. fumigatus Af293 protein database from NCBI
containing 9845 non-redundant sequences was used to search all MS/MS spectra.
The following parameters: 5 ppm tolerance for precursor ion masses and 0.6 Da
tolerance for fragment ion masses were selected. The static modification settings
included carbamidomethyl of cysteine residues, whereas dynamic modifications
included oxidation of methionine, TMT6plex modification of lysine -amino groups
and peptide N-termini, and acetyl modification of peptide N-terminus. A false
discovery rate (FDR) of 1% for peptides and proteins was obtained using a target-
- 48 -
3.5. Materials and Methods A. Błachowicz
decoy database search. The reporter ions integration tolerance was 0.5 Da while
the co-isolation threshold was 75%. The average signal-to-noise threshold of all
reporter peaks was greater than 10. The sum of all detected reporter ions of
associated peptides from a protein was used to determine the total intensity of
a reporter ion for that protein. The ratios between reporter and the reference
reporter ions (TMT
6
-131) were used to estimate the abundance ratio of each
protein. For the statistical analysis, the sum of reporter ion intensities for each
protein was Log2 transformed and technical triplicate measurements for each
protein were averaged. Only the proteins that were identified with at least one
peptide detected in each technical replicate, and quantified in all three technical
replicates, were considered for the analysis. One-way ANOVA was performed to
identify proteins that are differentially expressed. Proteins with p-value 0.05
were further evaluated for up- and down-regulation using a cut-off value of1
fold (Log2) change.
3.5.7 Secondary metabolie analysis
After 5 days of growth in the conditions described above, spores and mycelia
were scrapped from the cellophane and the agar was chopped into small pieces.
Samples were extracted with 25 mL MeOH, and 25 mL of 1:1 MeOH/DCM
each followed by 1 hour sonication and filtration. Combined crude extracts were
evaporated in vacuo and ectracted with EtOAc (25 mL). The EtOAc extracts were
evaporated in vacuo, re-dissolved in 2 mL of 20% DMSO/MeOH and 10 μL was
examined by high performance liquid chromatography-photodiode array detection-
mass spectrometry (HPLC-DAD-MS) analysis. HPLC-MS was carried out using
ThermoFinnigan LCQ Advantage ion trap mass spectrometer with an RP C
18
column (Alltech Prevail C
18
3 mm 2:1100 mm) at a flow rate 125 μL/min. The
solvent gradient for LC/MS was 95% MeCN/H2O (solvent B) in 5% MeCN/H
2
O
(solvent A) both containing 0.05% formic acid, as follows: 0% solvent B from 0
to 5 min, 0 to 100% solvent B from 5 min to 35 min, 100% solvent B from 35
to 40 min, 100 to 0% solvent B from 40 to 45 min, and re-equilibration with 0%
solvent B from 45 to 50 min.
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3.5. Materials and Methods A. Błachowicz
3.5.8 Sporulation capacity assessment
To examine sporulation capacity, 1 10
4
conidia in 10 μL were centrally
inoculated on GMM agar plates (20 mL) and grown at 30

C. Conidia were
collected with 4 mL of sterile 0.1% (v/v) Tween solution in water using L-shaped
spreader and enumerated on day 3, 4 and 5 at indicated time with hemocytometer.
Total number of conidia was assessed based on the counts from three biological
replicates.
- 50 -
Chapter4
Proteomic and metabolomic
characteristics of extremophilic
fungi under simulated Mars
conditions
1
4.1 Abstract
Filamentous fungi have been associated with extreme habitats, including
nuclear power plant accident sites and the International Space Station (ISS). Due to
immense adaptation and phenotypic plasticity capacities, fungi may thrive in what
seems like uninhabitable niches. This study is the first report of fungal survival
after exposure of monolayers of conidia to simulated Mars conditions (SMC).
Conidia of several Chernobyl nuclear accident-associated and ISS-isolated strains
were tested for UV-C and SMC sensitivity, which resulted in strain-dependent
survival. Strains surviving exposure to SMC for 30 min, ISSFT-021-30 and
IMV 00236-30, were further characterized for proteomic and metabolomic changes.
Differential expression of proteins involved in ribosome biogenesis, translation,
and carbohydrate metabolic processes was observed. No outstanding metabolome
alterations were revealed. Lastly, ISSFT-021-30 conidia re-exposed to UV-C
1
Blachowicz et al., Frontiers in Microbiology, in revision
4.2. Introduction A. Błachowicz
exhibited enhanced UV-C resistance when compared to the conidia of unexposed
ISSFT-021.
4.2 Introduction
Extremophiles are of interest to the National Aeronautics and Space Admin-
istration (NASA) due to their potential to survive hostile and extraterrestrial
conditions [199]. Bacteria, fungi, and archaea have been shown to thrive and pro-
liferate in habitats characterized by low nutrient availability [200], desiccation [6],
high and low temperatures [201, 2], acidic and alkaline pH [202, 203], radiation [7,
204, 205, 206], and other extreme conditions [207, 208, 140]. There is a need
for studies that focus on elucidating microbial survival mechanisms in extreme
habitats, as a primary goal of planetary protection policy is to prevent forward
and backward contamination of any celestial bodies and the Earth [199]. So far,
the majority of such studies under various simulated extraterrestrial conditions
have focused on investigating extremophilic bacteria, mainly spore-formers [209,
210], and rocks containing fungi [211, 212] however, monolayers of fungal conidia
were not studied.
One investigation revealed that survival of spacecraft-associated bacteria under
simulated Mars UV irradiation is strain-specific and depends on the isolation
site [213]. Spacecraft-associated bacteria were more resistant than, used as dosi-
metric control, Bacillus subtillis 168 [213]. Additionally, exposure of B. subtilis 168
spores to dark space conditions (no UV) outside the International Space Sta-
tion (ISS) and simulated Mars conditions (SMC) for 559 days induced expression
of genes involved in DNA and protein damage response, and oxidative and en-
velope stress [209]. Further, the first-generation of spores of another bacterium,
Bacillus pumillus SAFR-032, isolated from the Jet Propulsion Laboratory (JPL)
spacecraft assembly facility (SAF) was more UV-C resistant than the ground
control counterparts following exposure to space UV conditions for 18 months. Ex-
tensive studies into the whole genome of SAFR-032 revealed the presence of several
DNA repair-associated genes that may have facilitated its survival and adaptation
to harsh environmental conditions. Further, proteome analysis showed that stress
- 52 -
4.2. Introduction A. Błachowicz
response proteins, like superoxide dismutase, were increased in abundance when
compared to control [140].
Fungi produce conidia or spores as part of their life cycle. Some of these
structures can be more resistant to environmental impact than the typical fungal
coenocytic cell. Therefore, molecular characterization and further understanding
of fungal resistance to UV-C and SMC are of high importance. Additionally, fungi
possess a wide variety of mechanisms to protect against solar radiation, which
include enzymes removing reactive oxygen species (ROS), DNA repair mechanisms,
including nucleotide excision repair (NER) and photoreactivation (PR), and the
production of pigments such as melanins and melanin-like compounds, and UV-
absorbing metabolites that act as sunscreens [214]. The hardy nature, adaptability
and plasticity of fungi enable their persistence in extreme conditions, making them
potential contaminants of cleanrooms and, therefore, a forward contamination
source.
On April 25, 1986 one of the most significant nuclear accidents in history took
place. Reactor 4 of the Chernobyl nuclear power plant (ChNPP) exploded and high
levels of released radioactivity turned the surrounding area into a hostile environ-
mentanduninhabitableforhumans. Withinthefollowing18yearsover2,000fungal
isolates were collected from the nuclear power plant, its 30-km Exclusion Zone and
beyond, representing some 200 species in over 90 genera [206]. The observed fungal
communities were of low complexity and dominated by melanin-containing strains.
Approximately 20% of the isolates displayed a previously unknown phenomenon
of growing towards the radiation source referred as positive radiotropism [215,
206]. Therefore, this unique characteristic of Chernobyl fungi makes them an ideal
fungal model for studying adaptation to SMC. Further, the fungal species isolated
from Chernobyl have also been detected in built environments, including JPL clean
rooms [216, 217], simulated closed habitat [49], and on board of the ISS [8], which
further emphasizes the need to study their potential for forward contamination.
The ISS is another type of environment that may be considered hostile for
microorganisms, featuring constant temperature and humidity, controlled airflow,
enhanced irradiation, and microgravity [29]. Several reports examining microbial
- 53 -
4.3. Results A. Błachowicz
burden aboard the ISS have showed that fungal populations thrive in this environ-
ment [218, 8]. Other studies have investigated molecular adaptations of selected
species to space conditions, revealing changes in metabolome and proteome of
ISS-isolated strains [219, 155, 188]. In other investigations, several multilayered
or embedded cryptoendolithic fungal communities were exposed to space condi-
tions [220, 211, 221, 222, 223]. Tested isolates that adapted to environmental
extremes of their habitats survived SMC for extended periods of time and revealed
a high stability of the DNA in the surviving cells [220, 221, 222]. Despite these
studies there are significant gaps in our understanding of the molecular mechanisms
that facilitate survival of filamentous fungi under SMC and their potential to adapt
and survive in outer space.
This is the first report that evaluated the survival of Chernobyl-associated
and ISS-isolated fungal conidia exposed in monolayers to SMC. Dried monolayers
of conidia of four filamentous fungi were exposed to SMC and two strains, As-
pergillus fumigatus and Cladosporium cladosporoides, which survived exposure to
SMC for 30 min, were further analyzed for phenotypic, proteomic and metabolomic
changes.
4.3 Results
4.3.1 Identification of fungal strains
Twelve fungal strains isolated from Chernobyl nuclear accident sites, belonging
to nine genera, and one ISS-isolated strain were included in this study. Eight
of the Chernobyl isolates were collected over time from the wall surface of the
exploded Unit-4 of ChNPP. The other four fungi isolated from soil within the
exclusion zone were included for comparison. The strains used in the study were
selected based on their ecological and biological significance from the collection
of over 2,000 isolates. Speciation was determined by classical cell and colony
morphology-based identification techniques. A smaller set of fungi underwent whole
genome sequencing. Names, GenBank accession numbers, and their significance
are presented in Table 4.1.
- 54 -
4.3. Results A. Błachowicz
Table 4.1: Fungal isolates used in the study and their significance
Strain no. Species identifica-
tion
Accession no. Isolation site Significance
IMV 00034

Cladosporium
herbarum
Wall surface,
Unit-4, ChNPP
Exhibits radiotropism
IMV 00045

Cladosporium
sphaerospermum
MSJI00000000
#
Wall surface,
Unit-4, ChNPP
Exhibits radiotropism
IMV 00236

Cladosporium MSJH00000000
#
Wall surface,
Unit-
Exhibits radiotropism
cladosporioides 4, ChNPP
IMV 00253

Acremonium muro-
rum
Soil, 10-km ChEZ Exhibits radiotropism
IMV 00265 Beauveria bassiana MSJG00000000
#
Wall surface,
Unit-4, ChNPP
Produces tenellin
IMV 00293 Fusarium oxysporum MSJJ00000000
#
Wall surface,
Unit-4, ChNPP
Produces dihydronaph-
thoquinone
IMV 00454 Trichoderma virens MSJK00000000
#
Soil, 10-km ChEZ Exhibits auxin-dependent
lateral root growth/ my-
coparasitism
IMV 00738 Penicillium Soil in the Ter-
napol
European Patent 2 333
088
citreonigrum region
IMV 00882 Aureobasidium MSJF00000000
#
Wall surface,
Unit-
Produces diketopiper-
azine
pullulans 4, ChNPP
IMV 01167 Aspergillus terreus MSJE00000000
#
Soil, Kirovograd
region
Produces citreoviridin
IMV 01221 Aspergillus sydowii Wall surface,
Unit-4, ChNPP
Produces sesquiterpe-
onids
IMV 01851 Apiospora montagnei Wall surface,
Unit-4, ChNPP
Produces TMC-95A
ISSFT-021 Aspergillus fumigatus KT832787
#
ISS HEPA filter Opportunistic pathogen
IMV - Institute for Microbiology and Virology (Academy of Sciences), Kiyv, Ukraine
ISSFT - International Space Station Filter
ChNPP - Chernobyl nuclear power plant
ChEZ - Chernobyl exclusion zone
radiotropism
# whole genome sequencing
- 55 -
4.3. Results A. Błachowicz
0 500 1000 1500 2000
0.0001
0.001
0.01
0.1
1
UVC (254) Dose [J/m
2
]
N/N
0

IMV 00265
IMV 00293
IMV 00236*
IMV 00034*
IMV 00045*
IMV 01167
IMV 01851
IMV 00253
ISSFT-021
IMV 00454
IMV 00738
IMV 00882
IMV 01221
Figure 4.1: UV-C resistance of Chernobyl and ISS-isolated fungal strains. Purified
spores of 13 strains were exposed to various UV-C doses. The UV-C survival rates were
calculated using formula: NN
0
= # of conidia survived at any given dose / # of conidia
exposed at time 0.
4.3.2 Survival of desiccated conidial spores under UV-C irradiation
The impact of UV-C irradiation on the survival of dried fungal conidia is
presented in (Figure 4.1). Out of 13 irradiated strains all but three (Beauve-
ria bassiana IMV 00265, Fusarium oxysporum IMV 00293 and Aureobasidium pul-
lulans IMV 00882) survived exposure to the dose of 2,000 J/m
2
. Two radiotropic
strains, Cladosporium sphaerospermum IMV 00045 and Cladosporium cladospori-
oides IMV 00236, and the non-radiotropic Penicillium citreonigrum IMV 00738
showed survival at the level of 3.48%, 3.60% and 2.18%, respectively, which was
higher than the0.1% survival rate observed for the other strains (Figure 4.1).
Rapid decrease in conidia survivability was observed at dose of 500 J/m
2
( 2
to 3 log reduction). From that point until the doses of 1000 or 2000 J/mm
2
, the
decrease in survival was less pronounced.
- 56 -
4.3. Results A. Błachowicz
Table 4.2: Quantitative analysis of the simulated Mars Conditions (SMC) tolerance of
selected extremotolerant Chernobyl- and ISS-isolated fungi
Strain
a
SMC % of survival
N/A 5 min 30 min 5 min 30 min
IMV 00034

+ + 21:11 0:00
IMV 00236

+ + + 4:14 2:83
IMV 01851 + + 4:17 0:00
ISSFT-021 + + + 15:00 2:50
Growth after exposure of cultures to simulated Mars
conditions (SMC)
b
a
IMV - Institute for Microbiology and Virology (Academy
of Sciences), Kiyv, Ukraine
a
ISSFT - International Space Station Filter
b
“+” growth after exposure
b
“” no growth after exposure
4.3.3 Survival of desiccated conidia under simulated Mars conditions
(SMC)
Based on the initial analysis of survival under SMC (not shown) four fun-
gal strains were exposed to SMC for 5 and 30 min at the Leiden Institute of
Chemistry, the Netherlands. All exposed strains, C. cladosporioides IMV 00236,
Apiospora montagnei IMV 01851, C. herbarum IMV 00034 and Aspergillus fumi-
gatus ISSFT-021, survived exposure to SMC for 5 min, whereas only two strains:
C. cladosporioides IMV 00236 and A. fumigatus ISSFT-021 survived exposure
for 30 min (Table 4.2). Probably the most striking observation was the highest
survival rate of IMV 00034 after 5 min exposure to SMC followed by complete
eradication after 30 min exposure to SMC. However because the main focus of the
manuscript was to discuss the potential of fungi to survive long-term exposure to
SMC we did not further investigate why IMV 00034 may be highly tolerant to
SMC-exposure for the short time but it fails to survive during longer exposure.
Nevertheless, it remains interesting question and a topic for further investigation.
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4.3. Results A. Błachowicz
4.3.4 Secondary metabolite (SM) profiling of Aspergillus fumigatus
and Cladosporium cladosporioides exposed to SMC
Organic extracts of unexposed ISSFT-021 and IMV 00236, and 30 min SMC-
exposed ISSFT-021-30 and IMV 00236-30 strains were examined to test if exposure
to SMC alters SM production. No significant differences were observed in SM
production when comparing samples before and after SMCs exposure, including
yield or type of compound produced, in either of the strains (Figure 4.2). However,
there appeared to be a tendency of increased SM production yield in both strains
followingexposuretoSMCswhencomparedtounexposedcounterparts(Figure4.2B
and 4.2D).
4.3.5 Proteome profiling of Aspergillus fumigatus exposed to SMC
The proteomic characterization of SMC-exposed ISSFT-021-30 revealed 51-up
and 24 down-regulated proteins when compared to unexposed ISSFT-021 (fold-
change (FC)>j2j, P < 0:05). Analysis of the distribution of differentially expressed
proteins among biological processes revealed that 27 proteins were involved in
translation and ribosome biogenesis, 11 in carbohydrate metabolism, and 10 in
stress response (Figure 4.3). Further, significantly over-represented up-regulated
biological processes included translation (50% of all up-regulated proteins), and
carbohydrate metabolic processes (15%), whereas significantly over-represented
down-regulated processes included carbohydrate derivative metabolic processes
(12%).
Approximately 50% of all up-regulated proteins in ISSFT-021-30 were in-
volved in translation and ribosome biogenesis, including proteins that comprise
the small and large ribosomal subunit (Table 4.3). The majority of these pro-
teins were reported to be differentially expressed during the early development
of A. fumigatus [224]. Both Rpl3 (AFUA_2G11850) and ribosomal protein P0
(AFUA_1G05080), whose expression during conidiation are regulated by BrlAp,
were more than twofold up-regulated [225]. A number of differentially expressed
proteins were involved in carbohydrate metabolism (Table 4.4), including isocitrate
lyase AcuD (AFUA_4G13510), one of the key enzymes in the glyoxylate cy-
- 58 -
4.3. Results A. Błachowicz
Figure 4.2: Secondary metabolite production of SMC-exposed ISSFT-021-30 and IMV 00236-30 when compared to
unexposed ISSFT-021 and IMV 00236. (A) Secondary metabolite profiles of ISSFT-021-30 and ISSFT-021 when grown
on GMM. (B) Secondary metabolite profiles of IMV 00236-30 and IMV 00236 when grown on MEA. (C) Metabolite
quantification, showing the percent change for each metabolite in relation to unexposed ISSFT-021; significance was
determined using Welch’s t-test. (D) Metabolite quantification, showing the percent change for each metabolite in relation
to unexposed IMV 00236; significance was determined using Welch’s t-test.
- 59 -
4.3. Results A. Błachowicz
30 25 20 15 10 5 0 5 10 15 20 25 30
Response to chemical
Filamentous growth
Cellular homeostasis
Cellular amino acid
metabolic process
Transport
Response to stress
RNA metabolic process
Organelle organization
Carbohydrate
metabolic process
Ribosome biogenesis
Translation
No. of differentially
expressed proteins
GO SLIM category
Up-regulated
Down-regulated
Figure 4.3: AspGD GO Slim terms of differentially expressed proteins in ISSFT-021-
30. Differentially expressed proteins in (FC >j2j, P < 0:05) were mapped to terms
representing various biological processes using AspGD Gene Ontology (GO) Slim Mapper.
cle [172]. Cellobiohydrolases CbhB (AFUA_6G11610) and AFUA_8G01490,
both involved in cellulose degradation, and hydrolase AFUA_5G07080 were
more than twofold up-regulated in SMC-exposed ISSFT-021-30 [226]. A twofold
increase in protein abundance was observed for glycerol dehydrogenase GldB
(AFUA_4G11730) [164], phosphoglycerate kinase (AFUA_1G10350), and hexoki-
nase HxkA (AFUA_2G05910) [173]. Among the proteins involved in carbohydrate
metabolism that exhibited decreased abundance were malate and alcohol de-
hydrogenases (AFUA_6G05210 [164], AFUA_5G06240 [224], respectively) and
mannose-6-phosphate isomerase (AFUA_4G08410) [178]. Several proteins with
increased abundance were involved in response to stress (Table 4.5). Dehydrin-like
protein DprC (AFUA_7G04520), which is known to play a role in protecting
cells against freezing [168], and AFUA_1G14090, which is predicted to be in-
volved in histidine biosynthesis [165], were twofold up-regulated. Down-regulated
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4.3. Results A. Błachowicz
Table 4.3: Differentially expressed proteins involved in translation and ribosome biogen-
esis in ISSFT-021-30 subjected to SMC
ORF Protein Relative protein P-value Putative function / ac-
tivity
abundance

AFUA_5G05630 2:15 1:1910
3
60S ribosomal protein L23
AFUA_6G05200 2:01 4:6710
2
60S ribosomal protein L28
AFUA_4G03880 1:66 7:8610
3
60S ribosomal protein L7
AFUA_4G07435 1:60 2:6810
4
60S ribosomal protein L36
AFUA_5G06360 1:59 1:4610
3
60S ribosomal protein L8
AFUA_2G03380 1:58 2:3710
3
large ribosomal subunit
AFUA_4G07730 1:56 3:3210
2
60S ribosomal protein L11
AFUA_1G03390 1:54 4:0610
3
60S ribosomal protein L12
AFUA_1G09100 1:47 1:3810
3
60S ribosomal protein L9
AFUA_6G11260 1:46 6:2110
3
Ribosomal protein L26
AFUA_5G03020 1:43 3:8410
2
60S ribosomal protein L4
AFUA_2G11850 Rpl3 1:39 2:3610
4
Allergenic ribosomal L3
protein
AFUA_2G16370 1:39 4:9310
3
60S ribosomal protein L32
AFUA_1G14410 Rpl17 1:36 5:8210
3
60S ribosomal protein L17
AFUA_2G09210 1:34 1:1710
2
60S ribosomal protein L10
AFUA_2G03040 1:34 4:8410
2
Ribosomal protein L34
AFUA_3G06760 1:32 1:4810
2
Ribosomal protein L37
AFUA_1G05080 1:27 5:6610
3
60S ribosomal protein P0
AFUA_4G04460 1:25 5:1510
3
60S ribosomal protein L13
AFUA_3G13480 1:22 3:3010
2
Translation initiation factor
2 alpha subunit
AFUA_6G03830 1:22 3:2210
3
Ribosomal protein L14
AFUA_6G12660 1:18 2:4610
2
40S ribosomal protein S10b
AFUA_1G11130 1:18 2:2010
2
60S ribosomal protein L6
AFUA_1G12890 1:17 1:8910
3
60S Ribosomal protein L5
AFUA_2G09200 1:10 1:8310
3
60S ribosomal protein L30
AFUA_2G16010 1:07 2:9710
2
Prolyl-tRNA synthetase
AFUA_2G03590 Rps21 1:05 8:3010
4
Ribosomal protein S21e

Log2 fold change of ISSFT-021-30 when compared to ISSFT-021 (P < 0:05).
stress response proteins included the proliferating cell nuclear antigen (PCNA)
(AFUA_1G04900), and the formaldehyde dehydrogenase (AFUA_2G01040), and
AFUA_8G04890 with predicted role in response to salt stress [165].
- 61 -
4.3. Results A. Błachowicz
Table 4.4: Differentially expressed proteins involved in carbohydrate metabolism in
ISSFT-021-30 subjected to SMC
ORF Protein Relative protein P-value Putative function / ac-
tivity
abundance

AFUA_4G13510 AcuD/Icl1 1:78 4:6010
3
Isocitrate lyase involved in
the glyoxylate cycle
AFUA_6G11610 CbhB 1:42 3:8510
3
Cellobiohydrolase
AFUA_5G07080 1:39 4:3710
2
Hydrolase
AFUA_8G01490 1:32 1:6410
2
Cellobiohydrolase
AFUA_4G11730 GldB 1:08 3:7010
2
Glycerol dehydrogenase
AFUA_1G10350 1:03 4:7410
3
Phosphoglycerate kinase
AFUA_2G05910 HxkA 1:01 1:0710
2
Hexokinase
AFUA_1G06960 1:00 3:7610
2
Pyruvate dehydrogenase
complex subunit alpha
AFUA_6G05210 1:18 1:3610
4
Malate dehydrogenase
AFUA_5G06240 AlcC 1:22 1:8310
4
Alcohol dehydrogenase
AFUA_4G08410 2:23 1:5310
2
Mannose-6-phosphate
isomerase

Log2 fold change of ISSFT-021-30 when compared to ISSFT-021 (P < 0:05).
4.3.6 Proteome profiling of Cladosporium cladosporioides exposed to
SMC
The proteomic characterization of C. cladosporioides upon exposure to SMC
for 30 min revealed that 51 proteins were up-regulated and 218 proteins were
down-regulatedwhencomparedtounexposedIMV00236(FC >j2j, P < 0:05)inre-
sponsetoSMC.ThedistributionofdifferentiallyexpressedproteinsinSMC-exposed
IMV 00236-30 among biological processes is shown in Figure 4.4. Among differen-
tially expressed proteins 22 were involved in post-translation modification, protein
turnover, and chaperones, 21 in carbohydrate transport and metabolism, 20 in en-
ergy production and conversion, and 17 in translation and ribosomal structure and
biogenesis. Interestingly, the majority of the proteins involved in the translation
and ribosomal structure and biogenesis in SMC-exposed IMV 00236-30 exhibited
down-regulation (Table 4.6), which is the opposite expression pattern to ISSFT-021-
30. Additionally, a number of proteins involved in post-translational modification
and chaperones (Table 4.7) showed decreased abundance, including an aspartic en-
- 62 -
4.3. Results A. Błachowicz
Table4.5: Differentially expressed proteins involved in response to stress in ISSFT-021-30
subjected to SMC
ORF Protein Relative protein P-value Putative function / ac-
tivity
abundance

AFUA_1G14410 Rpl17 1:36 5:8210
3
60S ribosomal protein L17
AFUA_3G13480 1:22 3:3010
2
Translation initiation factor
2 alpha subunit
AFUA_7G04520 DprC 1:21 5:7410
3
Dehydrin-like protein, acts
downstream of SakA to con-
fer cold tolerance
AFUA_1G11130 1:18 2:2010
2
60S ribosomal protein L6
AFUA_1G14090 1:15 5:5610
3
Histidine biosynthesis
AFUA_4G11730 GldB 1:08 3:7010
2
Glycerol dehydrogenase
AFUA_8G04890 1:21 4:0610
2
Role in response to salt
stress
AFUA_1G15450 1:31 4:6210
3
Adenylosuccinate synthase
AFUA_1G04900 1:57 4:2610
3
Proliferating cell nuclear
antigen (PCNA)
AFUA_2G01040 2:67 8:0610
4
Formaldehyde dehydroge-
nase

Log2 fold change of ISSFT-021-30 when compared to ISSFT-021 (P < 0:05).
dopeptidase (BS090_008183/ENOG410PH8I), which is an ortholog of A. fumigatus
AFUA_5G13300 [165]. BS090_010805/ENOG410PMR5, an ortholog of mitochon-
drial matrix cochaperone Mge1p (YOR232W) in Saccharomyces cerevisiae [227]
was fourfold down-regulated when compared to SMC-unexposed IMV 00236.
Proteins involved in carbohydrate metabolism displayed differential abundance
(Table 4.8), including the fourfold up-regulated exo-polygalacturonase involved
in pectin degradation BS090_001871/ENOG410PG7M, which is an ortholog of
An12g07500 in Aspergillus niger [228]. Chitin deacetylases BS090_000013 and
BS090_000044/ENOG410PMJX,andthechitinrecognitionproteinBS090_010953
/ENOG410PMF7 were at least threefold up-regulated. Down-regulated pro-
teins involved in carbohydrate metabolism included phosphoglycerate mutase
BS090_004087 /ENOG410QEDC, which is an ortholog of Aspergillus nidulans
AN8720 with a predicted role in gluconeogenesis and glycolysis [229], glucanase
BS090_003291/ENOG410PM6Handalpha-amylaseBS090_011829/ENOG410PMDW.
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4.3. Results A. Błachowicz
20 15 10 5 0 5 10 15 20
Cell cycle control, cell division,
chromosome partitioning
Cytoskeleton
Defense mechanisms
Chromatin structure and dynamics
RNA processing and modification
Intracellular trafficking, secretion,
and vesicular transport
Signal transduction mechanisms
Transcription
Lipid transport and metabolism
Inorganic ion
transport and metabolism
Translation,
ribosomal structure and biogenesis
Secondary metabolites biosynthesis,
transport, and catabolism
Post-translational modification,
protein turnover, and chaperones
Amino acid transport
and metabolism
Energy production
and conversion
Carbohydrate transport
and metabolism
No. of differentially
expressed proteins
COG category
Up-regulated
Down-regulated
Figure 4.4: Biological process COG categories of differentially expressed proteins in
IMV 00236-30. Differentially abundant proteins (FC >j2j, P < 0:05) were mapped to
terms representing various biological processes using Cluster of Orthologous Genes (COG)
database in CloVR.
Differentially expressed proteins involved in energy production and conversion
(Table 4.9) included up-regulated BS090_001715 / ENOG410PGTG, an or-
tholog of A. niger NADPH dehydrogenase (An11g08510), and isocytrate lyase
and dehydrogenase BS090_001881 / ENOG410PGND, and BS090_000939 /
ENOG410PFHR, respectively. Proteins with decreased abundance included nitrate
reductase BS090_004112 / ENOG410PUCE and ATP synthase BS090_005644 /
ENOG41KOG1758.
- 64 -
4.3. Results A. Błachowicz
Table 4.6: Differentially expressed proteins involved in translation, ribosomal structure
and biogenesis in IMV 00236-30 subjected to SMC
Accession Relative protein P-value EggNog EggNog annotation
abundance

BS090_000406 1:44 2:6710
3
ENOG410PN3M 40S ribosomal protein S12
BS090_001853 1:13 1:8910
3
ENOG410PG8Y Ribosomal protein L15
BS090_003273 1:03 1:1910
2
ENOG410PN86 La domain
BS090_000015 1:12 3:6010
3
ENOG410PMV6 40s ribosomal protein S17
BS090_011340 1:16 5:2710
3
ENOG410PNPS Translation initiation fac-
tor
BS090_009099 1:18 3:8810
2
ENOG410PGC2 Eukaryotic translation ini-
tiation factor 5
BS090_002173 1:23 7:8410
3
ENOG410PP8V 60S ribosomal protein L31
BS090_005915 1:24 6:1510
3
ENOG410PHV2 Prolyl-tRNA synthetase
BS090_006863 1:35 2:1910
4
ENOG410PPAS 60s ribosomal protein
BS090_002466 1:35 3:6510
3
ENOG410PQS0 Processing of the 20S
rRNA-precursor to mature
18S rRNA
BS090_003537 1:35 7:2410
3
ENOG410PI34 Component of the eukary-
otic translation initiation
factor 3 (eIF-3) complex
BS090_000480 1:48 1:3610
2
ENOG410PFEB Seryl-tRNA synthetase
BS090_001464 1:53 1:7810
3
ENOG410PRUB 60S acidic ribosomal pro-
tein P2
BS090_006330 1:65 3:3010
3
ENOG410PQ50 Ribosome biogenesis pro-
tein Nhp2
BS090_009967 1:81 1:1910
2
ENOG410PRWG 60S acidic ribosomal pro-
tein P1
BS090_006228 2:13 5:6910
3
ENOG410PP4P L-PSP endoribonuclease
family protein (Hmf1)
BS090_007862 2:52 1:5610
3
ENOG410PQSK 60S ribosomal protein L22

Log2 fold change of IMV 00236-30 when compared to IMV 00236 (P < 0:05).
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4.3. Results A. Błachowicz
Table 4.7: Differentially expressed proteins involved in post-translational modification,
protein turnover, and chaperones in IMV 00236-30 subjected to SMC
Accession Relative protein P-value EggNog EggNog annotation
abundance

BS090_010341 1:55 5:8010
3
ENOG410PJAB Thioredoxin reductase
BS090_002416 1:42 3:6810
3
ENOG41KOG1339 Aspartic
BS090_010922 1:12 3:3410
3
ENOG410PX4S OsmC-like protein
BS090_011023 1:01 1:5210
3
ENOG410PNQ9 Peptidyl prolyl cis-
trans isomerase
Cyclophilin
BS090_008028 1:12 4:8810
2
ENOG410PPYQ Ubiquitin conjugating
enzyme
BS090_005834 1:20 6:6410
4
ENOG410PJ50 26S proteasome non-
ATPase regulatory sub-
unit 11
BS090_010452 1:24 1:8310
2
ENOG410PQY3 Peptidyl-prolyl cis-
trans isomerase
BS090_010972 1:28 3:6710
3
ENOG410PP80 Cupin domain protein
BS090_011316 1:32 7:3110
3
ENOG410PKHZ Protein-L-isoaspartate
O-methyltransferase
BS090_005149 1:33 2:6310
3
ENOG410PP3T Subunit 3
BS090_009030 1:34 1:9110
3
ENOG410PP19 Peptidyl-prolyl cis-
trans isomerase
BS090_004399 1:59 1:8710
3
ENOG410PPJH Heat shock protein
BS090_007304 1:63 2:9810
3
ENOG410PHFF Protease S8 tripeptidyl
peptidase I
BS090_009384 1:63 1:0110
3
ENOG410PI5I Tripeptidyl-peptidase
BS090_008147 1:81 3:0610
3
ENOG41KOG0541 Peroxiredoxin
BS090_005718 1:90 1:7210
4
ENOG410PGPE Disulfide-isomerase
BS090_010805 2:03 1:0310
2
ENOG410PMR5 Component of the
PAM complex
BS090_010009 2:07 3:5210
2
ENOG410PQV2 Prefoldin subunit 6
BS090_008183 2:19 :6910
3
ENOG410PH8I Aspartic endopepti-
dase
BS090_009852 2:47 6:1710
3
ENOG410PSDM Glutaredoxin
BS090_008141 3:13 6:9610
3
ENOG410PRTR Heat shock protein

Log2 fold change of IMV 00236-30 when compared to IMV 00236 (P < 0:05).
- 66 -
4.3. Results A. Błachowicz
Table 4.8: Differentially expressed proteins involved in carbohydrate transport and
metabolism in IMV 00236-30 subjected to SMC
Accession Relative protein P-value EggNog EggNog annotation
abundance

BS090_010953 2:31 8:9410
4
ENOG410PMF7 Chitin recognition pro-
tein
BS090_001871 2:14 3:3810
3
ENOG410PG7M Exo-polygalacturonase
BS090_000013 1:95 1:8910
2
ENOG410PMJX Chitin deacetylase-like
mannoprotein MP98
BS090_003824 1:93 9:3410
3
ENOG410PMRY LysM domain
BS090_001404 1:67 6:9210
3
ENOG41KOG1458 Fructose-1,6-
bisphosphatase
BS090_000044 1:58 8:2210
3
ENOG410PMJX Chitin deacetylase-like
mannoprotein MP98
BS090_000502 1:28 7:0710
3
ENOG410PJKF Catalyzes the epimeriza-
tion of the S- and R-
forms of NAD(P)HX
BS090_011896 1:21 9:0410
3
ENOG410PK51 Glyco_18
BS090_010859 1:09 2:8010
2
ENOG410PGWP Mannose-6-phosphate
isomerase
BS090_011008 1:10 5:2710
4
ENOG410PG84 Beta-glucosidase
BS090_007695 1:13 2:3610
3
ENOG410PJ6P Glucan 1,4-alpha glucosi-
dase
BS090_008700 1:16 5:4010
3
ENOG410PK8I WSC domain
BS090_006133 1:17 5:1810
3
ENOG410PF9K Glyceraldehyde-3-
phosphate dehydroge-
nase
BS090_002903 1:37 3:4310
2
ENOG410PJIN Glycolipid transfer pro-
tein HET-C2
BS090_003039 1:47 9:1310
4
ENOG410PH6W Cell Wall
BS090_008425 1:49 2:1210
3
ENOG410PIQS Snf1 kinase complex
beta-subunit Gal83
BS090_003291 1:51 4:2210
3
ENOG410PM6H Glucanase
BS090_011829 1:66 1:4310
3
ENOG410PMDW Alpha-amylase
BS090_011280 1:91 8:4010
3
ENOG410PKN9 Oxalate decarboxylase
BS090_004931 2:02 3:6110
2
ENOG410PM9J Major intrinsic protein
BS090_004087 2:34 9:9310
5
ENOG410QEDC Phosphoglycerate mu-
tase

Log2 fold change of IMV 00236-30 when compared to IMV 00236 (P < 0:05).
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4.3. Results A. Błachowicz
Table 4.9: Differentially expressed proteins involved in energy production and conversion
in IMV 00236-30 subjected to SMC
Accession Relative protein P-value EggNog EggNog annotation
abundance

BS090_001053 1:45 2:2410
3
ENOG410PGVS Oxidoreductase
BS090_001715 1:25 3:0010
3
ENOG410PGTG NADH flavin oxidoreductase
NADH oxidase family pro-
tein
BS090_001881 1:23 8:7110
3
ENOG410PGND Isocitrate lyase
BS090_000071 1:16 1:9510
3
ENOG410PFIA Component of the ubiquinol-
cytochrome c reductase com-
plex
BS090_007434 1:04 1:2510
3
ENOG410PI78 Phosphoenolpyruvate
carboxykinase
BS090_000939 1:03 1:8710
2
ENOG410PFHR Isocitrate dehydrogenase
NADP
BS090_000792 1:02 1:2710
2
ENOG410PN6K Mitochondrial membrane
ATP synthase (F(1)F(0)
ATP synthase or Complex
V)
BS090_004086 1:11 4:0610
2
ENOG410PFM5 Inorganic pyrophosphatase
BS090_011745 1:14 4:0410
2
ENOG410PNH4 Conserved hypothetical pro-
tein
BS090_003155 1:19 5:6510
3
ENOG410PNBT Regulatory protein
SUAPRGA1
BS090_010935 1:23 1:2510
2
ENOG410PFFW Electron transfer flavopro-
tein
BS090_011347 1:26 1:2910
3
ENOG410PNPT Cytochrome c oxidase
polypeptide VIa
BS090_003274 1:27 8:9810
5
ENOG410PFBB Stomatin family
BS090_005666 1:28 4:5910
3
ENOG410PH2F Mitochondrial membrane
ATP synthase (F(1)F(0)
ATP synthase or Complex
V)
BS090_006999 1:33 2:0010
3
ENOG410PFI6 Electron transfer flavopro-
tein
BS090_004112 1:51 1:1610
2
ENOG410PUCE Nitrate reductase
BS090_011473 1:53 5:6310
3
ENOG410PJA9 Vacuolar ATP synthase sub-
unit e
BS090_008223 1:59 8:2610
3
ENOG410PNQY Iron sulfur cluster assembly
protein
BS090_008509 2:04 2:6210
3
ENOG410PS16 Mitochondrial ATP syn-
thase epsilon chain domain-
containing protein
BS090_005644 2:40 4:7010
3
ENOG41KOG1758 ATP synthase

Log2 fold change of IMV 00236-30 when compared to IMV 00236 (P < 0:05).
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4.3. Results A. Błachowicz
0 500 1000 1500 2000 2500 3000 3500 4000
0.001
0.01
0.1
1
UVC (254) Dose [J/m
2
]
N/N
0

IF1SW-F4
CEA10
ISSFT-021-30 min
Af293
ISSFT-021
Figure 4.5: UV-C resistance of A. fumigatus ISS-isolated and clinical strains. Purified
conidia of ISS-isolated, ISSFT-021 and IF1SW-F4, SMC-exposed, ISSFT-021-30, and
clinical isolates, Af293 and CEA10, strains were exposed to varying doses of UV-C. The
UV-C survival rates were calculated using formula: NN
0
= # of conidia survived at any
given dose / # of conidia exposed at time 0. The average fungal conidia survival rates
from three different experiments are plotted.
4.3.7 Increased resistance to UV-C of SMC exposed Aspergillus fumi-
gatus conidia
Survival rates of several ISS-isolated and clinical isolates of A. fumigatus
following exposure to UV-C are presented in Figure 4.5. SMC exposed ISSFT-
021-30 exhibited increased UV-C resistance ( 20% of conidia survived the UV-C
dose of 4,000 J/m
2
) when compared to unexposed ISSFT-021 and another A.
fumigatus ISS isolate IF1SW-F4. Additionally, IF1SW-F4 was more resistant
to UV-C exposure, than ISSFT-021 and clinical CEA10 strain, which displayed
similar resistance patterns. The clinical isolate Af293 was the most sensitive,
showing 2-log reduction when exposed to the highest tested UV-C dose. Exposure
experiments were repeated 3 times and showed the same trends.
- 69 -
4.4. Discussion A. Błachowicz
4.4 Discussion
Although it has been well documented that bacteria are associated with the
spacecraft environment [230, 231, 213], few studies address the persistence of fungi
in this environment [216, 217]. Prepared in monolayers spacecraft-associated spore-
forming bacteria have been shown to survive exposure to UV-C and SMC [213, 232],
but similar studies have not been performed for fungal conidia. Microorganisms
exposed as multilayers are shielded from UV penetration (submicron level) and
subsequent UV-damage is prevented, therefore generation of monolayers of tested
cells/spores/conidia is crucial for characterizing irradiation-induced microbial
lethality. The observed strain-dependent UV-C sensitivity of fungal conidia was not
surprising, as similar conclusions were drawn for UV-exposed bacterial species [232].
Interestingly, ten out of thirteen selected fungal species survived the UV-C dose
of 2,000 J/m
2
, while bacterial species in a similar study survived exposure to
1,000 J/m
2
[232], suggesting that some extremophilic fungi are as hardy, if not
more, than tested bacteria. It should be noted, that conducting experiments
that will investigate protective nature of fungi for bacteria could help understand
the microbial survival in extreme niches, as bacteria and fungi coexist in the
environment and form communities to survive harsh conditions.
The space environment varies significantly from Earth. It is characterized
by enhanced irradiation and distinct atmospheric conditions. Therefore, it was
imperative to assess fungal survival under SMC, as fungi are known to be present
during manned space missions. Among the four strains that survived 5 min
exposure to SMC two, ISSFT-021 and IMV 00236, survived exposure to SMC for
30 min. Most studies testing survival of microorganisms under SMC have been
conducted using bacterial spores or fungal communities. In one such study several
Bacillus spp. were tested for simulated Mars UV irradiation tolerance, which
resulted in no survival beyond 30 min exposure for all strains except B. pumilus
SAFR-032, which was inactivated after 180 min of exposure [233]. However, when
B. subtilis spores were exposed to SMC, including irradiation and atmospheric
conditions, 99.9% of spores were eradicated within 30 s and a 15 min exposure
resulted in no recovery of viable spores from aluminum coupons [234]. Interestingly,
- 70 -
4.4. Discussion A. Błachowicz
when the more extremotolerant B. pumilus SAFR-032 was tested under SMC,
no spores were recovered from the aluminum coupon after 30 min exposure, but
bacterial growth was observed once coupons were placed in tryptic soy broth
(TSB) [232]. In this study, recovery of fungal conidia exposed in monolayers from
aluminum coupons was possible for both ISSFT-021 and IMV 00236 even after
30 min exposure, suggesting an enhanced ability of fungal conidia to withstand
such environments. The results from this study, combined with those revealing that
cryptoendolithic fungal communities embedded in rocks can withstand SMC for
an extended period of time [211, 221, 222], imply that fungi should be considered
as possible forward contamination source. This is further supported by the fact
that the omnipresence of filamentous fungi has been documented in spacecraft
assembly facilities [216, 217].
It has been reported that upon exposure to space conditions bacteria become
more UV resistant [140]. Similarly, this study showed that SMC-exposed ISSFT-
021-30 had a higher tolerance to UV irradiation than its unexposed counterpart
ISSFT-021. Additionally, it appeared to be more tolerant to UV exposure than any
of the additionally tested A. fumigaus strain, including another ISS-isolated strain
IF1SW-F4 and two clinical isolates Af293 and CEA10. These results suggest that
exposure to an enhanced irradiation environment may lead to adaptive alterations
that give fungi increased environmental advantage when exposed to unique space
conditions.
One major way that filamentous fungi respond to external stimuli is through
alterations in SM production. Although these bioactive molecules are not directly
necessary for survival, they often confer environmental advantage [57]. Both fungal
species subjected to SMC, ISSFT-021-30 and IMV 00236-30, displayed slightly
increased yield of produced SMs. Such tendency is in agreement with previously
observed elevated yields of SM in ISS-isolated JSC-093350089 A. niger when
compared to culture collection strain ATCC 1015 (Romsdahl et al, submitted).
Additionally, when the metabolome of ISSFT-021 was characterized and compared
tothewell-studiedAf293, productionyieldsofpyripyropeneAandfumiquinazolines
increased [155]. Therefore, the observed tendency of increased SM production in
- 71 -
4.4. Discussion A. Błachowicz
strains following exposure to SMC supports the hypothesis that space conditions
might have altered secondary metabolite production yields.
This study revealed that exposure to SMC altered the proteome of both
ISSFT-021-30 and IMV 00236-30 when compared to unexposed counterparts. In-
terestingly, in both species, the highest number of differentially expressed proteins
were translation-related ribosomal components. Interestingly, exposed to ionizing
radiation C. sphaerospermum, Wangiella dermatitidis, and Cryptococcus neo-
formans showed increased growth when compared to unexposed controls due to
electronic changes in melanin [56], however in our study only ISSFT-021-30 seemed
to follow that pattern, revealing up-regulation of translation-related proteins. Ob-
served opposite expression patterns of translation-related proteins, which underlay
species-related unique defense system, may have been shaped by the varying envi-
ronmental origins of each isolate [235, 236]. This discrepancy suggests that different
species of filamentous fungi alter their growth and development in response to
adverse environmental conditions in a species/strain-specific manner. Furthermore,
the difference in the expression levels of translation-related ribosomal protein may
lead to the overall up- and down-regulation of other proteins in ISSFT-021-30 and
IMV 00236-30, respectively. Interestingly, ribosomal protein Rpl17, has been indi-
cated as crucial for survival in A. fumigatus [237], Cryptococcus neoformans [238],
and S. cerevisiae [239] especially once grown on glucose. Induced abundance of
Rpl17 upon exposure to SMC may suggest that it modulates A. fumigatus response
to harsh conditions depending on a carbon source. Several differentially expressed
proteins were involved in carbohydrate metabolism and energy conversion, includ-
ing isocitrate lyase AcuD (AFUA_4G13510) in proteome of ISSFT-021-30. AcuD
is one of the key enzymes in glyoxylate cycle, which facilitates fungal growth on
alternative C
2
carbon sources [198]. In addition, AcuE, another enzyme in the
glyoxylate cycle, exhibited increased abundance in ISS-isolated ISSFT-021 and
IF1SW-F4 when compared to clinical isolates Af293 and CEA10 [219]. Further,
increased abundance of proteins involved in starvation response were observed
in the ISS-isolated JSC-093350089 A. niger when compared to the well-studied
culture collection strain ATCC 1015 [188]. These findings suggest that increased
production of starvation-response enzymes plays a role in adaptation to space
- 72 -
4.5. Materials and Methods A. Błachowicz
conditions. Several enzymes involved in chitin recognition and degradation were
up-regulated in IMV 00236-30. These enzymes enable using chitinous debris as
an alternative carbon source and allow morphogenetic changes during growth
and differentiation [61], which further suggests that alterations in carbohydrate
metabolism is an adaptive response to SMC. Interestingly, when protein patterns
of Cryomyces antarcticus, Knufia perforans, and Exophiala jeanselmei exposed
in multilayers to SMC were analyzed by 2D gel electrophoresis no additional
stress-induced proteins were observed [212].
This study affirms the enormous capability of filamentous fungi to adapt to
extreme environmental conditions and thrive in a wide variety of ecological niches.
To our knowledge this is the first report of shotgun proteomic and metabolomic
analyses of filamentous fungi in response to SMC. Such complex state of the art
analyses of fungal adaptive responses to space conditions are essential for ensuring
safety in the era of future outer space explorations, as fungi will undoubtedly
accompany people during space voyages. Thorough understanding of how filamen-
tous fungi adapt to space conditions is important for both maintaining crew health
and preventing biocorrosion of the spacecraft, as both opportunistic pathogenic
fungi [155] and technophiles [44] have been reported on board of the ISS and Mir
space stations.
4.5 Materials and Methods
4.5.1 Sample collection sites
Subcultures of the isolated strains were obtained from the Institute of Mi-
crobiology and Virology, Ukrainian Academy of Sciences, within the framework
of a multiyear collaborative research program, to the Center for Environmental
Biotechnology at Lawrence Berkeley National Laboratory (LBNL). For this study
12 Chernobyl nuclear accident-associated isolates were selected (Table 4.1).
- 73 -
4.5. Materials and Methods A. Błachowicz
4.5.2 Preparation of aluminum coupons with monolayers of dried fun-
gal conidia
High-grade aluminum coupons (Al 6061-T6) were precision cleaned for sterility
as previously described [232]. Each coupon was seeded with 100 μL of conidia
suspension to contain approximately 10
5
conidia per coupon. Conidia were counted
using a hemocytometer (Double Neubauer Counting Chamber, Hausser Scientific,
Horsham, PA) after harvesting 5 days grown cultures at 26

C on Potato Dextrose
Agar (PDA). Conidial suspensions were diluted in molecular biology grade water
(Fisher Scientific, Waltham, MA) and approximately 10
5
conidia were added to
each coupon followed by drying overnight at the room temperature in a bio-hood.
The monolayers of conidia were confirmed by scanning electron microscopy (data
not shown).
4.5.3 UV-C exposure and recovery
Aluminum coupons with dried fungal conidia were placed in a plastic Petri
dish, without a lid, and exposed to UV-C using a low-pressure handheld mercury
arc UV lamp (model UVG-11; UVP Inc., Upland, CA). The lamp was placed
above the sample, and the UV flux at the surface of exposed sample was measured
using UVX digital radiometer (UVP Inc.). The exposure time required to produce
doses: 0, 50, 100, 500, 1000, and 2,000 J/m
2
was calculated at 100 μW/cm
2
. After
exposure to UV-C 100 μL of 10% polyvinyl alcohol (PVA) was applied on each
coupon and dried at 37

C for 50 min. Dried PVA along with fungal conidia
was peeled using sterile forceps and added to 1 mL of molecular biology grade
water (Fisher Scientific). The PVA extraction step was repeated. When PVA was
dissolved, serial dilutions were prepared and plated on PDA in duplicates. Colony
forming units (CFUs) were counted after 7 days of incubation at 26

C.
4.5.4 Simulated Martian conditions (SMC)
Survival and response of fungal strains under SMC were tested in a Mars
simulation chamber equipped with a UV transparent fused silica window according
to a previously described set up [240, 232, 241]. Coupons prepared following the
- 74 -
4.5. Materials and Methods A. Błachowicz
protocol described above were placed in the sterile Falcon tubes and sent to the
Netherlands. After arrival (1.5 weeks) samples deposited on aluminum coupons
were placed inside the simulation chamber and subsequently the chamber was
evacuated via an oil-free scroll pump (XDS5, Edwards Vacuum, Crawley, United
Kingdom) to reach a base pressure of 20 Pa. While continuously being pumped,
the chamber was purged five times with high purity CO
2
(99.995%, H
2
0 < 5 ppm,
O
2
< 5 ppm, Praxair, Danbury, CT, USA), before establishing a continuous CO
2
gas flow to maintain a stable chamber pressure of approximately 600 Pa. Samples
were exposed at room temperature to simulated solar light (SF150 with a xenon
arc lamp, 150W, Sciencetech Inc., London, Canada) via the fused silica window
of the simulation chamber. The 200-400 nm wavelength integrated irradiance at
the sample distance was 58.7 W/m
2
(Figure 4.6). Samples were exposed for 5 and
30 min to cumulative doses of 2,670 J/m
2
and 16,110 J/m
2
, respectively, before
being removed from the chamber and placed in sterile Falcon tubes for shipment
back to JPL (Pasadena, CA, USA) for further analysis. Upon return to JPL
SMC-exposed samples were processed following the aforementioned PVA protocol
to assess the survival rates.
4.5.5 Secondary metabolite extraction and analysis
A. fumigatus strains were cultivated at 30

C on GMM agar plates while
C. cladosporioides strains were grown at 26

C on MEA agar plates starting
with 10
7
conidia/per Petri plate (D = 10 cm). After 5 days, agar was chopped,
extracted with 25 mL methanol and 25 mL of 1:1 methanol/dichloromethane each
followed by 1 hr sonication and filtration. After the second filtration, combined
crude extracts from each isolate were evaporated in vacuo, suspended in 20 mL
of water and partitioned with ethylacetate (20 mL). The ethylacetate layer was
evaporated in vacuo, re-dissolved in 1 mL of 20% dimethyl sulfoxide/methanol
and 10 μL was examined by high performance liquid chromatography-photodiode
array detection-mass spectrometry (HPLC-DAD-MS) analysis. HPLC-MS was
carried out using ThermoFinnigan LCQ Advantage ion trap mass spectrometer
with an RP C
18
column (Alltech Prevail C
18
3 mm 2:1 100 mm) at a flow
rate 125 μL/min. The solvent gradient for LC/MS was 95% acetonitrile/H
2
O
- 75 -
4.5. Materials and Methods A. Błachowicz
Figure 4.6: UV spectra (200 to 400 nm) of the solar simulator employed in this study.
Other lighting spectra of Mars models are presented along with the integrated irradiance
over the wavelength range from 200 to 400 nm. The figure courtesy of Dr. A. Elsaesser.
(solvent B) in 5% acetonitrile/H
2
O (solvent A) both containing 0.05% formic acid,
as follows: 0% solvent B from 0 to 5 min, 0 to 100% solvent B from 5 min to 35
min, 100% solvent B from 35 to 40 min, 100 to 0% solvent B from 40 to 45 min,
and re-equilibration with 0% solvent B from 45 to 50 min.
4.5.6 Proteome samples extraction and processing
Exposure of fungal conidia to SMC required using about 10
7
conidia/coupon
to avoid shadowing effect [232], however such amount of biomass was not enough to
perform detailed proteome analyses. Therefore, to observe permanent alterations in
proteomes of SMC-exposed strains when compared to unexposed ones A. fumigatus
strains were regrown at 30

C on GMM and C. cladosporioides strains at 26

C on
MEA starting with 10
7
conidia/per Petri plate (D = 10 cm). After 5 days, mycelia
- 76 -
4.5. Materials and Methods A. Błachowicz
and spores from agar plates were collected and stored at80

C prior to protein
extraction at City of Hope (Duarte, CA). The protein was extracted as previously
described [188]. In brief, mycelia and spores were lysed and homogenized using
a bead beater. Protein concentrations were measured by Bradford assay with a
bovine serum albumin standard curve (Bio-Rad Laboratories, Inc., Hercules, CA).
The samples were processed for a tandem mass tag (TMT) labeling as described
by [188] with modification. The proteomic profiling of A. fumigatus and C.
cladosporioides strains was carried out in two separate TMT LC/MS experiments.
A. fumigatus strains ISSFT-021 and ISSFT-021-30 were labeled with TMT
6
-128
and TMT
6
-129, respectively. Two biological samples of C. cladosporioides strains
IMV00236 and IMV00236-30 were labeled with TMT
6
-128/130 and TMT
6
-129/131,
respectively.
The samples were analyzed on an Orbitrap Fusion Tribrid mass spectrome-
ter with an EASY-nLC 1000 Liquid Chromatograph, a 75 μm2 cm Acclaim
PepMap100 C
18
trapping column, and a 75 μm25 cm PepMap RSLC C
18
analyt-
ical column, and an Easy-Spray ion source (Thermo Fisher Scientific) as previously
described (Romsdahl et al., 2018).
4.5.7 Quantitative proteomics analysis
All MS spectra were analyzed using Proteome Discoverer (version 2.2.0.388,
Thermo Fisher Scientific) with Sequest-HT search engines. Protein databases were
either A. fumigatus Af293 database from NCBI containing 9845 non-redundant
sequences or an in-house annotated draft genome sequence of C. cladosporioides
(MSJH00000000). The search parameters were described by Romsdahl, J. et al.
Technicaltriplicatemeasurementsforeachproteinwereaveraged. Onlyproteins
that were identified with at least one peptide detected in each technical replicate,
and quantified in all technical and biological replicates, were considered for the
analysis. TheidentifiedproteinswerethenaveragedandLog2transformed. Student
t-test was performed to identify proteins that are differentially expressed between
each SMC-exposed and unexposed group. Proteins with p 0:05 were further
evaluated for up- and down-regulation using a cut-off value of 2 fold change.
- 77 -
4.5. Materials and Methods A. Błachowicz
AspGD Gene Ontology (GO) Slim terms [156] were used to study the distribution
of differentially expressed proteins among biological processes in SMC-exposed
ISSFT-021-30 while the Cluster of Orthologous Genes (COG) database [242]
used in CloVR [243] was used to study the distribution of differentially expressed
proteins in SMC-exposed IMV 00236-30.
4.5.8 Genome annotation
Genome annotation of C. cladosporioides IMV 00236 was performed on the
deposited assembly (MSJH00000000) with Funannotate (v1.5.1) [244]. Proteins
from Capnodiales fungi (Dothideomycetes), Swissprot database (SIB Swiss In-
stitute of Bioinformatics Members, 2016), and transcripts from Cladosporium
sphaerospermum UM 843 [245] were used as informant sequences. Conserved genes
were identified from BUSCO core set “ascomycota_odb9” and were used to create
training set for ab initio gene prediction by Augustus [246, 247]. The ab initio
predictor GenemarkHMM-ES was trained using its self-training procedures [248].
These predictions along with splice-aware aligned proteins using DIAMOND [249]
followed by refinement with exonerate to improve spliced alignment accuracy [250].
Consensus gene models were generated from the combined evidence with Evidence
Modeler [251]. Predicted gene function from conserved protein domains [252],
Swissprot (SIB Swiss Institute of Bioinformatics Members, 2016) and inferred ho-
mology to conserved protein clusters in eggNOGdb [253] and secondary metabolite
cluster prediction [254].
- 78 -
Chapter5
The International Space Station
environment triggers molecular
responses in Aspergillus niger
1
5.1 Abstract
Due to immense phenotypic plasticity and adaptability, Aspergillus niger
is a cosmopolitan fungus that thrives in versatile environments, including the
International Space Station (ISS). This is the first report of genomic, proteomic
and metabolomic alterations observed in JSC-093350089 A. niger following a
controlled experiment aboard the ISS. Whole genome sequencing (WGS) revealed
that ISS conditions triggered mutations in specific regions of the JSC-093350089
genome when compared to the ground-grown control. Proteome analysis showed
altered abundance of proteins involved in carbohydrate metabolism, stress response,
and cellular amino acid and protein catabolic processes following growth aboard
the ISS. Metabolome analysis further confirmed that space conditions altered
molecular suite of ISS-grown JSC-093350089 A. niger.
1
Blachowicz et al., in review
5.2. Introduction A. Błachowicz
5.2 Introduction
The International Space Station (ISS) is a research facility orbiting at an
approximate altitude of 250 miles that is utilized to study physiological, psycho-
logical, and immunological responses of humans living in isolation [132, 134, 73,
36]. However, the distinct ISS environment, which includes microgravity and
enhanced irradiation, affects all other living organisms aboard the ISS in addition
to humans. There is a growing body of research that focuses on molecular charac-
terization of animal [34, 35], plant [32, 33, 136], and microbial [30, 31] responses to
the conditions encountered in the ISS. Among the most studied microorganisms
are various species of bacteria [255, 256, 140, 257] and yeast [258, 42, 259, 260,
261]. However, there is an unsubstantial number of studies that characterize the
molecular responses of fungi [188, 262, 219].
Filamentous fungi are producers of a myriad of bioactive compounds, or sec-
ondary metabolites (SMs), which often environmental advantage despite not being
directly essential for survival, and therefore facilitate survival in hostile niches [59,
58, 263, 57, 264]. SMs span from potent bioactive molecules used in the drug
discovery processes [265, 266, 267] or other branches of the industry [268, 269,
270, 271] to health hazardous toxins [272, 273, 274, 275]. Altered production
of various SMs is one potential mechanism of fungal adaptation to extreme en-
vironments. For example, increased production of melanin, a pigment with UV
protective properties, was observed in fungi isolated from Chernobyl nuclear power
plant [54] and “Evolution Canyon” [53]. One such highly melanized fungal species
is Aspergillus niger.
Industrially important A. niger [276] is a saprophytic organism that has been
isolated from various ecological niches, including decaying leaves [277], common
households [278, 279], and the ISS [8]. The A. niger strain JSC-093350089 isolated
from the surface of the US compartment of the ISS was previously characterized
using multi-omic techniques. Performed analyses revealed genetic variance typical
forthatoftheA. niger clade, increasedabundanceofproteinsinvolvedinstarvation
response, oxidative stress, and cell wall modulation [188], and alteration in SM
production levels when compared to well-studied ATCC 1015 strain (Romsdahl, et
- 80 -
5.3. Results A. Błachowicz
al., submitted). However, definite ascribing of observed molecular alterations to
the ISS environment was impossible, due to the lack of an appropriate control in
these initial characterization studies. Nevertheless, in-depth characterization of
ISS–isolated JSC-093350089 A. niger provided insight into potential space-induced
molecular phenotypes.
This study is the first report of the multi-omic characterization of A. niger
JSC-093350089 following the growth aboard the ISS when compared to ground
controls. To study the impact of the enhanced irradiation and microgravity on
JSC-093350089, the strain was transported to and grown aboard the ISS. Upon
return to Earth, ISS-grown samples, along with ground controls, were immediately
processed for metabolome, proteome and genome analyses with theaim of obtaining
important insights into the adaptive responses of A. niger to space conditions.
ISS-grown samples were then regrown on Earth to identify any conserved molecular
alterations.
5.3 Results
5.3.1 Genome variation in the ISS-grown JSC-093350089 A. niger.
To identify genetic variations occurring in the ISS-grown JSC-093350089 when
compared to ground-grown samples, paired-end whole genome sequencing (WGS)
was performed on both ISS- and ground-grown samples. Obtained reads were
aligned to the CBS 513.88 reference genome and single nucleotide polymorphisms
(SNPs) present in the ground control were filtered. This revealed presence of 375
SNPs and 620 INDELs that occurred because of growth on the ISS (Table 5.1).
Distribution of non-synonymous point mutations among genes is presented in Ta-
ble 5.2. Interestingly, about 80% of these mutations occurred in chromosome VIII
and 13% occurred in chromosome XII, while the remaining 7% were distributed
evenly amongst other chromosomes. The majority of missense point mutations
were observed within genes of unknown function. However, several character-
ized genes containing missense SNPs have DNA binding activity (An06g01180,
An08g11890, An12g00840), DNA polymerase activity (An08g11520), protein kinase
and transferase activity (An08g12110), phospholipase activity (An08g12250), and
- 81 -
5.3. Results A. Błachowicz
Table 5.1: Summary of genetic variations observed in ISS-grown JSC-093350089 when
compared to ground control
Type of mutation Occurence number
SNPs Intergenic 205
Missense 79
Splice region 4
Start gained 1
Stop lost 1
5 prime UTR 1
Synonymous 84
Total no. of SNPs 375
INDELs Intergenic 444
5 prime UTR 10
Conservative inframe deletion 8
Disruptive inframe deletion 14
Frameshift 109
3 prime UTR 15
Splice region 11
Start lost 1
Stop gained 8
Total no. of INDELs 620
RacA protein binding activity (An12g06420) (Table 5.2). Most of the observed
SNPs (55%) and INDELs (71%) were located in intergenic regions (Table 5.1).
Interestingly, unlike SNPs, INDELs were distributed evenly throughout all chro-
mosomes.
Table 5.2: Single nucleotide polymorphisms (SNPs) in ISS-grown JSC-093350089 when
compared to ground control
Function Gene Base mutation
compared to gnd
control
Mutation type
- 82 -
5.3. Results A. Błachowicz
RNA polymerase II transcrip-
tion factor activity, sequence-
specific DNA binding
An06g01180 An06_G279214A 5 prime UTR
RNA-directed DNA poly-
merase
An08g11520 An08_G2725926T Missense
activity and role in RNA- An08_T2726169G
dependent DNA replication An08_C2726328A
An08_T2726340C
An08_A2726541G
An08_A2726556G
An08_C2726566A
An08_G2726574T
An08_G2726997A
An08_C2727012T
An08_G2728798A
An08_C2729323T
An08_G2726128T Stop lost
DNA binding activity An08g11890 An08_A2824556C Missense
An08_C2824566G
Protein kinase and trans-
ferase
An08g12110 An08_G2869126A Missense
activity An08_T2869564C
Phospholipase An08g12250 An08_C2920251G Missense
An08_A2920254C
An08_C2920298T
An08_A2920340G
DNA binding, RNA poly-
merase
An12g00840 An12_T222942C Missense
II transcription factor activ-
ity
An12_G222974T
An12_G222982A
An12_G223213A
An12_G223422C
An12_G223434A
An12_A223471G
An12_G222400A Splice region
RacA binding protein, polar-
ized
An12g06420 An12_A1548559G Missense
cell growth An12_G1548931A
An12_A1548987G
An12_A1549053C
Unknown function An08g08380 An08_C1997243T Missense
- 83 -
5.3. Results A. Błachowicz
Unknown function An08g11220 An08_A2664300G Missense
An08_C2664502A
Unknown function An08g11230 An08_G2666099A Splice region
An08_T2666106C
Unknown function An08g11540 An08_A2731686T Missense
An08_T2731695C
An08_T2733283C
An08_C2733499T
Unknown function An08g11550 An08_G2734928C Missense
Unknown function An08g11570 An08_C2737667T Missense
Unknown function An08g11650 An08_G2765994T Missense
An08_G2766002T
Unknown function An08g11670 An08_G2768423A Missense
Unknown function An08g11830 An08_T2814800G Missense
Unknown function An08g11840 An08_T2817189C Missense
An08_A2817202G
Unknown function An08g11860 An08_G2818780C Missense
An08_G2819091A
An08_T2819644G
Unknown function An08g11870 An08_G2820964A Missense
An08_C2821036T
An08_C2821061T
An08_T2821094C
An08_A2821104C
An08_C2821119A
An08_T2821648G
An08_C2821664T
An08_T2821693A
An08_G2821697A
An08_A2821958T
An08_G2821978A
An08_T2822640C
An08_G2821995A
Unknown function An08g11880 An08_G2824016C Missense
Unknown function An08g11910 An08_C2827842A Missense
An08_C2828992G
Unknown function An08g11940 An08_A2835378G Missense
Unknown function An08g11950 An08_T2836585A Missense
An08_T2836586C
An08_A2837163G
Unknown function An08g11960 An08_A2839341G Missense
An08_G2839738A
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5.3. Results A. Błachowicz
Unknown function An08g12230 An08_C2911588T Missense
An08_T2911676C
An08_G2912446C
An08_T2912451G
An08_A2913614T
An08_T2911894A Splice region
Unknown function An08g12230 An08_G2913874C Start gained
Unknown function An08g12240 An08_A2916240C Missense
Unknown function An12g05800 An12_T1429831C Missense
5.3.2 ProteomiccharacterizationofISS-grownJSC-093350089A.niger
Differentially expressed proteins in ISS-grown JSC-093350089 strain were in-
vestigated following the extraction of total protein from 3 biological replicates
of ISS- grown and ground control counterpart strains. Due to the low yields of
extracted proteins, biological replicates were combined and divided into two parts
that were then TMT labeled and subtracted to analysis via LC-MS/MS followed by
spectrum/sequence matching using A. niger CBS 513.88 protein database (NCBI).
Protein abundance ratios in ISS-grown JSC-093350089 were normalized to Earth-
grown counterparts, which enabled identification of 70 up- and 142 down-regulated
proteins (fold-change (FC)>j2j, P < 0:05) in response to space conditions. AspGD
Gene Ontology (GO) Slim terms [156] were used to study the distribution of differ-
entially expressed proteins in ISS-grown JSC-093350089 when compared to ground
controls (Figure 5.1). Among differentially expressed proteins 29 were involved in
carbohydrate metabolism, 18 in stress responses, 21 in amino acid metabolism,
and 15 in protein catabolic processes (Figure 5.1). Protein GO term enrichment
analysis was conducted using FungiDB [157], which revealed that significantly
over-represented up-regulated biological processes included carbohydrate metabolic
processes (28% of all up-regulated proteins) and stress response (10%), whereas
significantly over-represented down-regulated processes included cellular amino
acid metabolic processes (13%), proteasomal ubiquitin-independent (10%) and
dependent processes (10%), and proteasomal protein catabolic processes (10%).
- 85 -
5.3. Results A. Błachowicz
20 15 10 5 0 5 10 15 20
Cellular protein
modification process
Cellular respiration
Cytoskeleton organization
DNA metabolic process
Organelle organization
Protein catabolic process
Protein folding
Ribosome biogenesis
RNA metabolic process
Vesicle-mediated transport
Vitamin metabolic process
Cell adhesion
Cellular homeostasis
Conjugation
Signal transduction
Toxin metabolic process
Asexual sporulation
Cellular amino acid
metabolic process
Pathogenesis
Secondary
metabolic process
Translation
Transport
Cell cycle
Filamentous growth
Lipid metabolic process
Response to chemical
Sexual sporulation
Developmental process
Response to stress
Carbohydrate
metabolic process
No. of differentially
expressed proteins
GO SLIM category
Up-regulated
Down-regulated
Figure 5.1: AspGD GO Slim terms of differentially expressed proteins in ISS-grown
JSC-093350089. Differentially expressed proteins in (FC >j2j, P < 0:05) were mapped to
terms representing various biological processes using AspGD Gene Ontology (GO) Slim
Mapper.
- 86 -
5.3. Results A. Błachowicz
The majority of differentially expressed proteins in ISS-grown JSC-093350089
A. niger were involved in carbohydrate metabolism (Table 5.3). Interestingly, eight
of these genes, including cellobiohydrolases A and B (An07g09330, An01g11660),
XlnA 1,4--xylanase (An03g00940) and D-xylose reductases XyrA and XdhA
(An01g03740, An12g00030) were regulated by XlnR. XlnR is a transcriptional
regulator involved in degradation of polysaccharides, xylan, cellulose and D-
xylose [280]. -glucanases An11g01540 and An02g00850, which are involved in
carbonstarvationresponseinA. niger [62], wereatminimumthreefoldup-regulated
in ISS-grown strain. -1,2-mannosidases An08g08370, An13g01260 were at least
3.5 fold up-regulated, whereas pyruvate decarboxylase PdcA (An02g06820) was
nearly threefold up-regulated. Pyruvate kinase PkiA (An07g08990), pyruvate
dehydrogenase Pda1 (An07g09530), and isocitrate lyase AcuD (An01g09270) were
at least twofold less abundant in ISS-grown samples. Several proteins involved
in the stress response were differentially expressed in ISS-grown JSC-093350089
(Table 5.4). Proteins exhibiting at least twofold up-regulation included cell wall
organization protein EcmA (An04g01230) and An16g07920, whose orthologs play
a role in salt stress response. Down-regulated stress- response proteins included
heat shock protein An06g01610, DNA-binding protein HttA (An11g11300), and
quinone reductase An12g06300. Lastly, a variety of proteins involved in cellular
amino acid processes (Table 5.5), and protein catabolic processes (Table 5.6) were
down-regulated.
5.3.3 MetabolomiccharacterizationofISS-grownJSC-093350089A.niger
Alterations in SM production in response to ISS conditions were assessed by
extracting organic compounds from three biological replicates of ISS- and ground-
grown JSC-093350089. The extracts were analyzed using high-performance liquid
chromatography coupled with diode-array detection and electrospray ionization
tandem mass spectrometry (HPLC-DAD-MS). Compounds were identified based
on mass, UV absorption, and retention time, which were agreement with litera-
ture [187] (Figure 5.2). Assessment of SM production yields revealed slight decrease
in production of bicoumanigrin A, fonsecinones B and C, aurasperones C and B,
and kotanin in ISS-grown isolate. Production levels of pestalamide B, nigerazine B,
- 87 -
5.3. Results A. Błachowicz
Table 5.3: Differentially expressed proteins involved in carbohydrate metabolism
ORF Protein CAZy family Function / activity Relative protein P-value
abundance

An03g00940 XlnA/XynA GH10 1,4--xylanase 2:20 2:6610
3
An01g11660 CbhB GH7, CBM1 Cellobiohydrolase B 2:00 2:4910
3
An03g00500 GH30 1,6--glucosidase 1:97 6:9110
3
An11g01540 GH16 -glucanase 1:93 4:2010
3
An08g08370 GH92 -1,2-mannosidase 1:93 5:2210
3
An13g01260 GH92 -1,2-mannosidase 1:83 4:1610
3
An15g04900 AA9, CBM1 -1,4-glucanase D 1:77 2:1810
2
An11g03340 AamA GH13 acid -amylase 1:71 8:9510
4
An02g00850 GH16 -glucanase 1:70 4:2810
2
An11g01120 Alr – Erythrose reductase 1:65 7:9410
4
An15g07800 AgtC GH13 4--glucanotransferase 1:61 2:6410
3
An03g00960 AxhA GH62 -L-
arabinofuranosidase
1:60 9:6610
3
An02g06820 PdcA – Pyruvate decarboxylase 1:57 5:7610
3
An02g11150 AglB GH27 -galactosidase II 1:37 2:8910
3
An08g01710 AbfC GH51 Arabinofuranosidase 1:36 4:9410
4
An14g02760 EglA GH12 -1,4-glucanase 1:27 6:7010
5
An14g02070 CEnc Acetylxylan esterase 1:27 8:6710
3
An05g02410 GH2 Glycoside hydrolase 1:10 1:7710
2
An07g09330 CbhA GH7 Cellobiohydrolase A 1:07 1:4410
3
An01g03740 XyrA – D-xylose reductase 1:08 2:5910
2
An12g00030 XdhA – D-xylulose reductase 1:10 1:7210
2
An12g00030 XdhA – D-xylulose reductase 1:10 1:7210
2
An07g08990 PkiA – Pyruvate kinase 1:12 2:9210
3
An18g06500 – Phosphomannomutase 1:14 6:8410
3
An12g03070 GlaB GH15 Glucoamylase 1:38 7:8710
3
An11g02550 – Phosphoenolpyruvate
carboxykinase
1:51 3:4410
3
An15g01920 McsA – 2-methylcitrate syn-
thase
1:55 2:1810
2
An01g09270 AcuD – Isocitrate lyase 1:59 9:8310
3
An15g03550 GH43 Hydrolase 1:60 9:1810
4
An07g09530 Pda1 – Pyruvate dehydroge-
nase
2:19 2:0010
3

Log2 fold change of ISS-grown JSC-093350089 compared to Earth-grown counterpart (P < 0:05).
- 88 -
5.3. Results A. Błachowicz
Table 5.4: Differentially expressed proteins involved in stress response
ORF Protein Function / activity Relative protein P-value
abundance

An01g14960 Asparaginase 2:21 6:4610
4
An14g02460 FhbA Flavohemoglobin 1:56 1:4510
3
An01g02320 RasA Ras GTPase 1:21 8:7610
3
An07g04620 Hypothetical protein 1:11 5:8110
3
An16g07920 Hypothetical protein 1:08 2:8710
2
An04g01230 EcmA Cell wall organization
protein
1:02 8:3810
4
An04g04130 Ornithine transaminase 1:03 1:3110
2
An16g07110 Ach1 Acetyl-CoA hydrolase 1:06 2:2510
2
An04g04870 Superoxide dismutase 1:08 4:7110
3
An01g03740 XyrA D-xylose reductase 1:08 2:5910
2
An07g08990 PkiA Pyruvate kinase 1:12 2:9210
3
An08g00970 Rps28 Ribosomal protein of the
small subunit
1:16 4:0910
3
An02g07210 PepE Acid aspartic protease 1:21 1:2710
4
An12g06300 Quinone reductase 1:25 3:2810
2
An11g11300 HttA DNA binding activity
and role in DNA repair
1:30 2:8110
3
An09g05870 Ndk1 Nucleoside-diphosphate
kinase
1:53 5:7910
4
An02g06560 Glutathione peroxidase /
transferase activity
1:69 9:5610
3
An06g01610 Heat shock protein 2:08 5:3510
3

Log2 fold change of ISS-grown JSC-093350089 compared to Earth-grown counterpart
(P < 0:05).
and nigragillin exhibited a reduction of approximately 50% when compared to
control strains.
Both ISS- and ground-grown JSC-093350089 were regrown at 29

C and
SMs production was evaluated. Due to the difference in growth temperature,
obtained SM profiles of regrown isolates differed from those grown on the ISS
(Figure 5.3). Interestingly, the trend in the production of SMs was the opposite of
the one observed immediately after growth on the ISS. The majority of identified
compounds showed slight increase in the production levels. Further, statistically
- 89 -
5.3. Results A. Błachowicz
Table 5.5: Differentially expressed proteins involved in cellular amino acid metabolic
processes
ORF Protein Function / activity Relative protein P-value
abundance

An01g14960 Asparaginase 2:21 6:4610
4
An12g00160 Malate dehydrogenase 1:27 2:7210
3
An14g06010 Chorismate mutase 1:01 1:5010
2
An04g04130 Ornithine transaminase 1:03 1:3110
2
An16g02970 Glycine/Serine hydrox-
ymethyltransferase
1:05 2:7410
4
An11g09510 Aspartate semialdehyde de-
hydrogenase
1:06 2:3510
3
An01g06530 Branched-chain-amino-acid
transaminase activity
1:18 2:4910
2
An15g05770 Hydrogen sulfide / sulfur
amino acid biosynthetic pro-
cess
1:19 1:4310
3
An15g00610 Imidazoleglycerol-
phosphate dehydratase
1:19 1:4010
3
An05g00410 Glycine/Serine hydrox-
ymethyltransferase
1:22 8:6310
3
An13g00550 3-deoxy-7-
phosphoheptulonate syn-
thase
1:36 6:7710
3
An09g03940 Ilv2 Ketol-acid reductoisomerase 1:40 1:0810
3
An02g10750 Cysteine synthase 1:45 3:8410
3
An11g02170 Fumarylacetoacetate hydro-
lase
1:47 1:6410
3
An15g02490 Homoisocitrate dehydroge-
nase activity, role in lysine
biosynthesis
1:48 8:4710
3
An02g07250 AgaA Arginase 1:54 2:8610
3
An02g12430 IcdA Isocitrate dehydrogenase 1:59 1:3810
3
An08g01960 Adenosylhomocysteinase 1:62 4:4310
3
An17g00910 Gamma-aminobutyrate
transaminase
1:72 5:6910
3
An11g02160 Maleylacetoacetate iso-
merase
1:83 4:4910
3
An03g04280 Similarity to pyridoxine syn-
thesis component pyroA of
A. nidulans
1:88 9:7210
3

Log2 fold change of ISS-grown JSC-093350089 compared to Earth-grown counterpart
(P < 0:05).
- 90 -
5.3. Results A. Błachowicz
Table 5.6: Differentially expressed proteins involved in protein catabolic processes
ORF Protein Function / activity Relative protein P-value
abundance

An02g07210 PepE Acid aspartic protease 1:21 1:2710
4
An18g06800 Pre10 20S CP alpha subunit of
the proteasome
1:27 1:0810
3
An04g01800 Hypothetical protein 1:53 2:5010
3
An02g07040 Scl1 20S CP alpha subunit of
the proteasome
1:84 4:3710
3
An02g03400 Pup2 20S CP alpha subunit of
the proteasome
1:84 5:6210
3
An11g04620 Endopeptidase 1:96 1:2010
2
An07g02010 Pre8 20S CP alpha subunit of
the proteasome
1:99 2:8310
3
An11g06720 Pre9 20S CP alpha subunit of
the proteasome
1:99 9:4810
3
An18g06680 Role in proteasomal
ubiquitin-independent
protein catabolic process
2:03 4:6510
3
An04g01870 Endopeptidase activator
activity
2:05 5:9910
3
An18g06700 Pre7 20S CP beta subunit of
the proteasome
2:08 2:5910
3
An11g01760 Protein similar to Pre2p
proteasome 20S subunit
2:10 9:9610
4
An13g01210 Endopeptidase 2:12 4:5510
3
An15g00510 Pre5 20S CP alpha subunit of
the proteasome
2:13 3:0110
3
An02g10790 Pre6 20S CP alpha subunit of
the proteasome
2:18 6:0410
3

Log2 fold change of ISS-grown JSC-093350089 compared to Earth-grown counterpart
(P < 0:05).
- 91 -
5.3. Results A. Błachowicz
10 12 14 16 1 8 20 22 2 4 26 28 30 32 3 4 36 38 4 0 42 44
Tim e (m in)
0
1000 00
2000 00
3000 00
4000 00
uA U
0
1000 00
2000 00
3000 00
4000 00
uA U
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8,9,10
8,9,10
11
11
A. niger
ground
JSC-093350089 A. niger
ISS-grown
1 Nigragillin
2 Nigerazine B
3 Pestalamide B
4 Bicoumanigrin A
5 Funalenone
6 [M-H]- = 351.5
7 Aurasperone C
8 Aurasperone B
9 Kotanin
10 Fonsecinone B
11 Fonsecinone C

JSC-093350089 A. niger
control
A
B
Unknown
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Nigragillin
% of production of control
*
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Aurasperone C
% of production of control
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Nigerazine B
% of production of control
**
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Aurasperone B
% of production of control
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Pestalamide B
% of production of control
*
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Kotanin
% of production of control
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Bicoumanigrin A
% of production of control
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Fonsecinone B
% of production of control
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Funalenone
% of production of control
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Fonsecinone C
% of production of control
JSC-093350089
control
JSC-093350089
ISS-grown
0
50
100
150
Unknown
% of production of control
Figure 5.2: Secondary metabolite production of ISS-grown JSC-093350089 when compared to ground controls. (A)
Secondary metabolite profiles of ISS- and ground-grown JSC-093350089 when grown on GMM.(B) Metabolite quantification,
showing the percent change for each metabolite in relation to ground-grown JSC-093350089; significance was determined
using Welch’s t-test.
- 92 -
5.3. Results A. Błachowicz
10 15 2 0 25 30 35 40 4 5
Tim e (m in)
0
1000 00
2000 00
uA U
0
1000 00
2000 00
uA U
1 Nigragillin
2 Pyranonigrin A
3 Nigerazine B
4 Pestalamide B
5 Bicoumanigrin A
6 Unknown
7 Aurasperone C
8 Aurasperone B
9 Kotanin
10 Fonsecinone B
11 Fonsecinone C
12 Fonsecinone C derivative
13 Fonsecinone A
14 Asperpyrone B
15 Aurasperone A
16 Asperpyrone C
JSC-093350089
control
JSC-093350089
ISS-grown
1
1
2
2
3
3
4
5
6
7
8
9
10
11
12
13
14
15
16
4
5
6
7
8
9
10
11
12
13
14
15
16
JSC-093350089
control
JSC-093350089
ISS-grown
0
20
40
60
80
100
120
140
% of production of ATCC 1015
Nigragillin
JSC-093350089
control
JSC-093350089
ISS-grown
0
20
40
60
80
100
120
140
% of production of ATCC 1015
Bicoumanigrin A
JSC-093350089
control
JSC-093350089
ISS-grown
0
20
40
60
80
100
120
140
160
180
200
% of production of ATCC 1015
Pyranonigrin A
*
JSC-093350089
control
JSC-093350089
ISS-grown
0
20
40
60
80
100
120
140
% of production of ATCC 1015
Kotanin
*
JSC-093350089
control
JSC-093350089
ISS-grown
0
20
40
60
80
100
120
140
% of production of ATCC 1015
Nigerazine B
JSC-093350089
control
JSC-093350089
ISS-grown
0
20
40
60
80
100
120
% of production of ATCC 1015
albA pathway SMs
JSC-093350089
control
JSC-093350089
ISS-grown
0
20
40
60
80
100
120
% of production of ATCC 1015
Pestalamide B
A
B
Figure 5.3: Secondary metabolite production of regrown ISS- and ground-grown JSC-093350089 (A) Secondary metabolite
profiles of regrown ISS- and ground-grown JSC-093350089 when grown on GMM. (B) Metabolite quantification, showing
the percent change for each metabolite in relation to regrown ground-grown JSC-093350089; significance was determined
using Welch’s t-test.
- 93 -
5.4. Discussion A. Błachowicz
significant increase in the production of the antioxidant pyranonigrin A [281] and
kotanin was observed when compared to ground controls.
5.4 Discussion
In previous studies, A. niger JSC-093350089 isolated from the ISS was thor-
oughly characterized to assess the molecular phenotype of a fungal strain inhabiting
the ISS [188]. Although these studies provided insight into the characteristics
necessary for fungi to withstand the ISS environment, lack of proper controls did
not allow definite ascribing of molecular changes triggered by the space environ-
ment. Therefore, to further investigate the differences in JSC-093350089 that were
observed when compared to a "terrestrial" strain, the isolate was sent to the ISS in
a planned experiment. Genomic, proteomic, and metabolomic alterations occurring
in ISS-grown samples were analyzed following sample return and compared to
ground-grown counterparts.
Genome analysis of ISS-grown JSC-093350089 revealed the introduction of
SNPs and INDELs in response to space conditions. Interestingly, the majority of
observed non-synonymous SNPs were located within chromosomes VIII and XII,
which suggests that only selected regions of the genome undergo positive selection
to confer selective advantage while adapting to the space environment. This is
in agreement with previous reports of space-induced genetic variations, as ISS-
grown Aspergillus nidulans [262] and spaceflight-grown Staphylococcus aureus [282]
both exhibited genetic mutations that occurred in specific clustered regions of the
genome. Although the functions of many genes containing non-synonymous SNPs
were unknown, several of these genes possessed transposable element and DNA-
binding activity. One such gene was an analogue of transposon I factor and has
RNA-directed DNA polymerase activity, which is consistent with genetic changes
observedintransposableelementgenesinbothA. nidulans [262]andS. aureus [282].
Alterations in transposable element genes likely influence their activity and lead to
the introduction of novel variations within the genome in response to environmental
stress [283]. The results from this study further underscore the significant role
of transposable elements in adaptation to the spacecraft environment. Future
- 94 -
5.4. Discussion A. Błachowicz
studies should investigate into the functions of uncharacterized genes containing
non-synonymous SNPs, as such knowledge may provide key information on how
fungi adapt to space conditions.
Investigation into the proteome of ISS- and ground-grown JSC-093350089
revealed that space conditions induce changes in protein expression when com-
pared to ground controls. Interestingly, several proteins involved in carbohydrate
metabolism displayed altered abundance in response to the space environment.
This observation stays in agreement with previous studies, which reported similar
differences in proteome of JSC-093350089 A. niger [188], Aspergillus fumiga-
tus [219] (Blachowicz unpublished), and A. nidulans [262], upon exposure to space
conditions. Additionally, ISS-grown JSC-093350089 exhibited up-regulation of
starvation-induced glycoside hydrolases, including AamA, and CbhB. This fur-
ther confirms that changes in carbohydrate metabolism, which play a key role in
adapting to scarce nutrient availability, confer selective advantage in the space
environment. Further, differential expression of XlnR-regulated proteins appears
to be a key adaptive response to space conditions, as both this study and the
previous study characterizing the space-induced molecular phenotype of JSC-
093350089 [188] revealed differential expression of various XlnR-regulated proteins.
Activation of XlnR-regulated metabolic pathways leads to use of versatile carbon
sources [280], which may facilitate survival in hostile space environments. A few
proteins up-regulated in ISS-grown JSC-093350089 were involved in stress response,
including the cell wall organization protein EcmA and flavohemoglobin, whose
analogue in Aspergillus flavus has been reported to have a function correlated
with hyphal growth phenotype [284]. These observations further suggest that
modulation of fungal growth and cell organization is an essential response to space
conditions.
Metabolomic analysis of ISS-grown JSC-09335008 showed alterations in sec-
ondary metabolite production in response to the space environment. This ob-
servation was not surprising, as fungi often respond to versatile environmental
conditions by altering the type and yield of produced SMs [59]. Interestingly,
ISS-grown JSC-09335008 showed decreased production of all SMs, which is the
- 95 -
5.4. Discussion A. Błachowicz
opposite production pattern observed during the initial characterization of the
metabolome of JSC-09335008 when compared to a “terrestrial” strain (Romsdahl,
et al., submitted). This discrepancy may be related to the fact that metabolomic
profile of ISS-isolated JSC-09335008 was compared to the well-studied ATCC 1015
strain, rather than a proper control.
Additionally, growth temperature seemed to significantly impact the types of
SMs produced. When both ISS- and ground-grown JSC-09335008 were regrown in
the same conditions as ISS-isolated JSC-09335008 in the previous study (Romsdahl,
et. al., submitted), similar trends in SM production yields were observed. Regrowth
on Earth of the ISS-grown JSC-09335008 strain revealed increased yields of all SMs,
including approximately 60% increased production of the antioxidant pyranoni-
grin A when compared to yields of regrown ground controls. Pyranonigrin A was
previously proposed to have a radioprotective nature, as pyranonigrin A-deficient
JSC-09335008 strain was more sensitive to UV-C exposure than the wild type
JSC-09335008 strain (Romsdahl, et. al., submitted). Interestingly, pyranonigrin
A production was not detected in the ISS- and ground-grown samples following
the experiment on the ISS. Due to these discrepancies in observed SM profiles of
immediately processed and regrown samples, along with the proposed radioprotec-
tive nature of pyranonigrin A (Romsdahl, et. al., submitted), the most important
question to address is whether pyranonigrin A truly provides A. niger protection
while in space. Such protection could have various biotechnological applications
for use pyranonigrin A as a radioprotective agent, including within human space
programs and cancer therapies. Future studies should examine production of pyra-
nonigrin A under varying temperatures aboard the ISS, as well as its protective
nature within the space environment to definitively answer that question.
This study is the first report of the multi-omic response of A. niger to space
conditions during a controlled experiment, which enhances our understanding of its
space -induced phenotype. Such understanding may be translated to development
of protective measures for both astronauts and the spacecraft during future manned
space explorations, as A. niger is ubiquitous fungus and present in many human-
occupied closed habitats [49, 8]. Lastly, a thorough understanding of the space-
- 96 -
5.5. Materials and Methods A. Błachowicz
induced secondary metabolomic alterations of industrially important A. niger may
result in creating a potent producer of compounds of interest during space voyages.
5.5 Materials and Methods
5.5.1 Isolation and identification of A. niger
ProcedurestoisolateandidentifyA. niger collectedfromtheISSweredescribed
previously [188]. In brief, sterile swabs soaked in saline solution were used to
sample the ISS surface and transported to Earth. Particles retrieved from the swab
were spread onto potato dextrose agar (PDA) plates and any growing colonies
were purified, collected and further analyzed. One of the collected isolates was
identified as A. niger via ITS region sequencing, which was subsequently confirmed
via whole genome sequencing (WGS).
5.5.2 Growth conditions
JSC-093350089 was cultivated on glucose minimal medium (GMM) agar plates
(6 g/L NaNO
3
, 0.52 g/L KCl, 0.52 g/L MgSO
4
7H
2
O, 1.52 g/L KH
2
PO
4
, 10 g/L
D-glucose, 15 g/L agar supplemented with 1 mL/L of Hutner’s trace elements)
coveredwithacellophanemembrane. EachoftenpreparedPetriplates(D = 10 cm)
was inoculated with 10
7
spores/plate. Subsequently, plates were sealed with 3M™
Micropore™ Surgical Tape (VWR International, Radnor, PA, USA), and placed
in four BRIC canisters (3 and 2 plates/BRIC). BRIC canisters were divided into
two groups, which were exact mimics, and transferred to 4

C. The first group of
BRIC canisters was sent aboard the ISS and continuously kept at 4

C (20 days)
prior to being transferred to ambient temperature 22

C for 12 days. After that
time BRICs were transferred to and kept at 4

C preceding return to Earth (10
days). Upon arrival to Earth, BRICs were turned over to the science team for
the downstream analyses, which commenced immediately. The second group of
BRIC canisters, treated as controls, was kept on Earth at Kennedy Space Center
(KSC) and mimicked the ISS experiment timeline with roughly 2 hr of delay.
Control samples were shipped from KSC to research team prior to turn over of the
ISS-grown samples.
- 97 -
5.5. Materials and Methods A. Błachowicz
5.5.3 Secondary metabolite (SM) extraction and analysis
Upon return to Earth single plates from both ground- and ISS-kept BRICs were
used to collect agar plugs in triplicate for secondary metabolite (SM) extraction.
Plugs were extracted with 3 mL of MeOH and 1:1 MeOH:DCM each followed by one
hour sonication. Crude extract was evaporated in vacuo to yield a residue that was
then suspended in 1 mL of 20% DMSO/MeOH and 10 μL was examined by high
performance liquid chromatography-photodiode array detection-mass spectrometry
(HPLC-DAD-MS) analysis. HPLC-MS was carried out using ThermoFinnigan
LCQ Advantage ion trap mass spectrometer with an RP C
18
column (Alltech
Prevail C
18
3 mm 2:1100 mm) at a flow rate 125 μL/min. The solvent gradient
for LC/MS was 95% MeCN/H
2
O (solvent B) in 5% MeCN/H
2
O (solvent A) both
containing 0.05% formic acid, as follows: 0% solvent B from 0 to 5 min, 0 to 100%
solvent B from 5 min to 35 min, 100% solvent B from 35 to 40 min, 100 to 0%
solvent B from 40 to 45 min, and re-equilibration with 0% solvent B from 45 to
50 min.
5.5.4 SM statistical analysis
To compare the yields of produced SMs in ISS-grown, ground-grown, and
regrown samples, the area under the electrospray ionization curve (ESI) was
integrated for each compound. SM data collected from three independent biological
replicates of ISS- and ground-grown, and regrown JSC-093350089 were used for
testing statistical significance of production yields of identified SMs by Welch’s
corrected t-test. The data are presented as column charts with corresponding error
bars. Data analysis was conducted using GraphPad Prism version 7.
5.5.5 Protein extraction
Mycelia and spores from GMM agar plates were collected and stored at
80

C prior to protein extraction. Proteins were extracted with the lysis buffer
consisted of 100 mM triethylammonium bicarbonate (TEAB) with 1:100 Halt
Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL) and 200 μg/mL
phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Mycelia and
- 98 -
5.5. Materials and Methods A. Błachowicz
spores were homogenized by bead beating using Precellys 24 homogenizer (Bertin,
Rockville, MD). The lysed fungi were centrifuged at 17,000 g for 15 min and the
protein concentrations in the supernatants were measured by the Bradford assay
(Bio-Rad Laboratories, Inc. Hercules, CA, USA).
5.5.6 Tandem mass tag (TMT) labeling
200 μg proteins from each sample were precipitated in 20% TCA at 4

C.
Protein pellets were washed with ice-cold acetone, and re-suspended in 25 μL
TEAB (100 mM) and 25 μL 2,2,2-trifluoroethanol (TFE). Proteins were reduced
with 1 μL of tris(2-carboxyethyl)phosphine (TCEP, 500 mM), alkylated with
iodoacetamide (IAA, 30 mM) and digested with 2.5 μg/sample of trypsin/lysC
(Promega, Madison, WI, USA). The digested peptides were quantified using the
Pierce Quantitative Colorimetric Peptide Assay (Thermo Scientific, Waltham,
MA, USA). 40 μg of peptides from each specific sample was labeled with the
Thermo Scientific TMTsixplex Isobaric Mass Tagging Kit (JSC-E1 (ground 1) with
TMT
6
-128, JSC-E2 (ground 2) with TMT
6
-130, JSC-S1 (ISS 1) with TMT
6
-129,
JSC-S2 (ISS 2) with TMT
6
-131) according to the manufacturer’s protocol. The
TMT
6
-130 and -131 labels were used as a reference that contained an equal amount
of the peptides from each of the four samples. All labeled-peptide mixtures were
combined into a single tube, mixed, and fractionated using the Thermo Scientific
Pierce High pH Reversed-Phase Peptide Fractionation Kit. Fractions were dried
using a SpeedVac concentrator and re-suspended in 1% (v/v) formic acid prior to
LC-MS/MS analysis.
5.5.7 LC-MS/MS analysis
The samples were analyzed on an Orbitrap Fusion Tribrid mass spectrome-
ter with an EASY-nLC 1000 Liquid Chromatograph, a 75 μm2 cm Acclaim
PepMap100 C
18
trapping column, and a 75 μm25 cm PepMap RSLC C
18
ana-
lytical column, and an Easy-Spray ion source (Thermo Scientific, Waltham, MA,
USA). The peptides were eluted at 45

C with a flow rate of 300 nL/min over
a 110 min gradient, from 3-30% solvent B (100 min), 30-50% solvent B (3 min),
- 99 -
5.5. Materials and Methods A. Błachowicz
50-90% solvent B (2 min), and 90% solvent B (2 min). The solvent A was 0.1%
formic acid in water and the solvent B was 0.1% formic acid in acetonitrile. The
full MS survey scan (m/z 400-1500) was acquired at a resolution of 120,000 and
an automatic gain control (AGC) target of 210
5
in the Orbitrap with the 50 ms
maximum injection time for MS scans. Monoisotopic precursor ions were selected
with charge states 2-7, a10 ppm mass window, and 70 s dynamic exclusion. The
MS
2
scan (m/z 400-2000) was performed using the linear ion trap with the 35%
CID collision energy. The ion trap scan rate was set to “rapid”, with an AGC
target of 410
3
, and a 150 ms maximum injection time. Ten fragment ions from
each MS
2
experiment were subsequently selected for an MS
3
experiment. The
MS
3
scan (m/z 100-500) was performed to generate the TMT reporter ions in the
linear ion trap using HCD at a 55% collision energy, a rapid scan rate and an AGC
target of 5×103, and a maximum injection time of 250 ms.
5.5.8 Proteome data analysis
AllMSdata(MS
1
, MS
2
, andMS
3
)weresearchedusingtheProteomeDiscoverer
(version 2.1.0.81, Thermo Scientific) with the Sequest-HT searching engines against
an Aspergillus niger CBS 513.88 database containing 10549 sequences (NCBI).
The searches were performed with the following parameters: 5 ppm tolerance
for precursor ion masses and 0.6 Da tolerance for fragment ion masses. The
static modification settings included carbamidomethyl of cysteine residues, while
dynamic modifications included oxidation of methionine, TMT6plex modification
of lysine -amino groups and peptide N-termini, and acetyl modification of protein
N-terminus. A false discovery rate (FDR) of 1% was set using a target-decoy
database search. The reporter ions integration tolerance was 0.5 Da and the co-
isolation threshold was 75%. The average signal-to-noise threshold of all reporter
peaks was bigger than 10. The total intensity of a reporter ion for a protein was
calculated based on the sum of all detected reporter ions of associated peptides
from that protein. The ratios between reporter ions and the reference reporter
ions (TMT
6
-130 or TMT
6
-131) were used to estimate the abundance ratio of each
protein.
- 100 -
5.5. Materials and Methods A. Błachowicz
For statistical analysis, the sum of reporter ion intensities for each protein was
Log2 transformed and the biological duplicate measurement for each protein was
averaged. Only the proteins that were identified and quantified in both biological
replicates were used for the analysis. Student t-test was performed to identify
proteins with changed abundance. Proteins with p-value < 0:05 were further
evaluated for increased and decreased abundance using a cut-off value of2 fold
change.
5.5.9 Genomic DNA extraction and whole genome sequencing (WGS)
Mycelia and conidia were collected from ISS- and ground-grown JSC-09335008
GMM agar plates. DNA was extracted using the Power Soil DNA Isolation Kit (Mo
Bio Laboratories, Carlsbad, California, USA) following the manufacturers protocol.
Extracted DNA was checked for quality using Qubit 2.0 Fluorometer and used for
paired-end library preparation with TruSeq Nano DNA Library Preparation Kit
(Illumina, San Diego, California, USA) followed by WGS at the Duke Center for
Genomic and Computational Biology. Samples were sequenced using a HiSeq 4000
Illumina Sequencer generating 101 base long reads.
5.5.10 Genetic variation identification
Illumina sequence reads were trimmed using Trimmomatic v 0.36 (Bolger
et al., 2014) and checked for quality using FastQC v 0.11.5. The genome and
annotation files for A. niger CBS 513.88 [285] were downloaded from the FungiDB
web portal [157]. Reads were mapped to CBS 513.88 the reference genome using
the Burrows-Wheeler Aligner (BWA) software package v 0.7.12 [286], and fur-
ther processed with SAMtools v 1.6 to generate sorted BAM files [287]. SNPs
and INDELs were identified using GATK v 3.7 [288]. Duplicates were marked
using Picard-tools MarkDuplicates to remove PCR artifacts. Sequence reads
containing putative INDELs were realigned using GATK’s IndelRealigner to
generate an updated BAM file. Variants within each sample were called us-
ing GATK’s Haplotype Caller. GATK’s VariantFiltration was used to filter each
VCF file based on stringent cutoffs for quality and coverage {SNPs: QD < 2:0,
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5.5. Materials and Methods A. Błachowicz
MQ < 40:0, QUAL < 100, FS > 60:0, MQRankSum < 12:5, SOR > 4:0,
ReadPosRankSum <8:0; Indels: QD < 2:0, FS > 200:0, MQRankSum <12:5,
SOR > 4, InbreedingCoef f <0:8, ReadPosRankSum <20:0}, so that only
high-quality variants remained.
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Chapter6
Characterization of
Aspergillus niger isolated from the
International Space Station
1
6.1 Abstract
The initial characterization of the Aspergillus niger isolate JSC-093350089,
collected from U.S. segment surfaces of the International Space Station (ISS), is
reported, along with a comparison to the extensively studied strain ATCC 1015.
Whole-genome sequencing of the ISS isolate enabled its phylogenetic placement
within the A. niger/welwitschiae/lacticoffeatus clade and revealed that the genome
of JSC-093350089 is within the observed genetic variance of other sequenced
A. niger strains. The ISS isolate exhibited an increased rate of growth and
pigmentdistribution compared to a terrestrial strain. Analysis of the isolate’s
proteome revealed significant differences in the molecular phenotype of JSC-
093350089, including increased abundance of proteins involved in the A. niger
starvation response, oxidative stress resistance, cell wall modulation, and nutrient
acquisition. Together, these data reveal the existence of a distinct strain of A. niger
on board the ISS and provide insight into the characteristics of melanized fungal
1
Romsdahl, Blachowicz et al., mSystems, 2018; The author helped with the proteome analysis
and preparation of the corresponding figure.
6.2. Introduction A. Błachowicz
species inhabiting spacecraft environments.
Importance
Athoroughunderstandingofhowfungirespondandadapttothevariousstimuli
encountered during spaceflight presents many economic benefits and is imperative
for the health of crew. As A. niger is a predominant ISS isolate frequently detected
in built environments, studies of A. niger strains inhabiting closed systems may
reveal information fundamental to the success of long-duration space missions.
This investigation provides valuable insights into the adaptive mechanisms of
fungi in extreme environments as well as countermeasures to eradicate unfavorable
microbes. Further, it enhances understanding of host-microbe interactions in closed
systems, which can help NASA’s Human Research Program maintain a habitat
healthy for crew during long-term manned space missions.
6.2 Introduction
Throughout the history of human space exploration, filamentous fungi have
traveled with us and are omnipresent on spacecraft [28, 289, 71]. Microorganisms
have been reported to cause biodegradation of structural spacecraft components,
resulting in decreased integrity of spacecraft hardware [289]. Microbial infections
also constitute a major health risk for astronauts, especially in closed environments
where the combined stresses of sleep disruption, microgravity, and high levels of
radiation may further compromise the human immune system [289, 290]. Studies
have suggested that microbial virulence and antimicrobial resistance increase in
response to spacecraft environments [291, 292, 293]. Other reports have associated
the abundance of filamentous fungus in indoor environments with allergies and
invasive infections [294, 295]. Additionally, fungi produce a myriad of bioactive
secondary metabolites (SMs) in response to environmental stressors, and while
many SMs have diverse therapeutic and industrial applications, others are toxins
and can have detrimental effects on human health [296]. As we set our exploration
sights beyond low-Earth orbit, a thorough understanding of how fungi respond
and adapt to the various stimuli encountered during spaceflight is critical to the
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6.2. Introduction A. Błachowicz
success of long-term space travel.
Microorganisms inhabiting the International Space Station (ISS) are exposed
to microgravity and have increased exposure to high-energy radiation as a result of
being outside Earth’s protective atmosphere [297]. In general, it is thought that mi-
crogravity alters biological processes by initially altering the physical forces acting
on the cell and its environment. This results in decreased transfer of extracellular
nutrients and metabolic by-products, causing the cell to be exposed to a completely
different chemical environment[297]. The inside cabin of the ISS is exposed to
a complex radiation environment [298], at levels that are not fungicidal [299],
permitting fungi to thrive. Radiation primarily interacts with biological systems
through the ionization and excitation of electrons in molecules, and its strong mu-
tagenic properties result in an increased rate of biological evolution [297]. Further,
radiation can have many harmful effects on biological systems, which results in the
development of adaptive responses. Fungi inhabiting spacecraft are also forced to
acclimate to reduced nutrient availability, as the National Aeronautics and Space
Administration (NASA) routinely performs stringent microbial monitoring and
remediation on the ISS [8].
Aspergillus niger was reported to be the predominant species isolated in one
ISS microbial monitoring study [8], which is consistent with its frequent detection in
built environments [110]. A. niger is a melanized fungal species that is ubiquitous
in nature and commonly used in biotechnology industries as a production host
for citric acid and enzymes [276]. Despite the recurring detection of A. niger
in spacecraft environments, investigations into its genetic alteration and gene
expression modulation under ISS conditions have not been carried out. Although
A. niger is less pathogenic to humans than other Aspergillus species, such as
A. fumigatus and A. flavus [276], it has been associated with ear infections and can
cause invasive pulmonary aspergillosis in immunocompromised patients [300]. This
enhances the need for studies to understand how A. niger responds and adapts to
the environment of the ISS, where microgravity might play a role in compromising
the human immune system [289, 290]. Additionally, melanized fungi are highly
resistant to ionizing radiation and respond to radiation with enhanced growth
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6.3. Results A. Błachowicz
and upregulation of many proteins [7, 301], some of which may provide important
insight into the adaptive evolutionary mechanism of melanized fungal species.
The objective of this study was to investigate a strain of A. niger isolated
from surfaces of the ISS, with the aim to characterize its molecular phenotype.
Although it has been well established that fungi are ubiquitous on spacecraft [28,
289, 71, 8], very few studies have been conducted to characterize fungi isolated
from the ISS [155]. Given that melanin production in fungi is considered an
evolution-derived trait to confer radiation resistance [7, 302], the present study of
a melanized fungus that has inhabited the ISS may reveal important insights into
the key traits necessary to withstand such environments. Our work investigated
differences of the ISS A. niger isolate from Earth isolates to better understand the
characteristics of strains isolated from the space station built environment. Due to
the significance of secondary metabolic processes in filamentous fungi [57], A. niger
ATCC 1015 was used as a terrestrial reference strain for physiologic and proteomic
analyses because its SM profile has been thoroughly characterized [187], and we
aim to build on this work by investigating SM production in JSC-093350089.
6.3 Results
6.3.1 Identification of A. niger sampled from the ISS
Identification of A. niger sampled from the ISS.Sampling of surfaces on the
ISS during microbial monitoring surveys resulted in the isolation of numerous
bacterial and fungal strains [8]. A strain of A. niger, JSC-093350089, identified
by morphological characteristics and verified by internal transcribed spacer (ITS)
region sequencing, was used for this study. This strain was isolated by swabbing
surface materials on the U.S. segment of the ISS. Due to the nature of this
sampling method, it is impossible to know the exact duration of time that this
strain was on the ISS. The 36.08-Mb genome sequence of JSC-093350089 was
generated using whole-genome paired-end sequencing (WGS), which was further
improved to high-quality assemblies of 223 scaffolds possessing 12,532 coding
sequences and 287 tRNAs. The JSC-093350089 genome was similar in size to
other A. niger genomes, which typically range from 34.0 to 36.5 Mb [303, 285,
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6.3. Results A. Błachowicz
Figure 6.1: Phylogenetic characterization of JSC-093350089 displaying its relative
placement within the A. niger/welwitschiae/lacticoffeatus clade. Figure courtesy of Dr.
J. Romsdahl and Dr. J. E. Stajich.
304]. To further verify the identity of JSC-093350089 and place it into the larger
context of the A. niger/welwitschiae/lacticoffeatus clade, phylogeny was assessed
using maximum likelihood (Fig. 6.1). Of the A. niger strains surveyed, the ISS
isolate displayed the closest phylogenetic relationship to A. niger (phoenicis).
Compared to ATCC 1015, an industrial strain used for citric acid production [303],
and CBS 513.88, an ancestor of the A. niger strains used industrially for enzyme
production [285], it differed by 37,548 and 39,433 variants, respectively.
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6.3. Results A. Błachowicz
Figure 6.2: In vitro growth of JSC-093350089 compared to ATCC 1015. (A) Growth
on GMM at 30

C after 7 days, showing colony morphology and color. (B) Radial
growth at 30

C on GMM. Statistical analyses were performed by multiple t tests,
corrected for multiple comparisons using the Holm-Sidak method.P = 0:00210:0002;
P < 0:0001. Figure courtesy of Dr. J. Romsdahl.
6.3.2 Visual characterization and growth rates of JSC-093350089 in
vitro
The basic physiological phenotype of JSC-093350089 was investigated on
glucose minimal medium (GMM) agar plates. Visual characterization of centrally
inoculated GMM plates revealed differences in pigment distribution and colony
diameter after 7 days of growth (Fig. 6.2A). JSC-093350089 colony size appeared
larger, and pigment had spread to the periphery of the colony in a shorter time
than for ATCC 1015. Assessment of radial growth rates revealed that the ISS
strain grew at a significantly higher rate than ATCC 1015 after 3 days of growth
(Fig. 6.2B).
6.3.3 Overview of proteome analysis
ToinvestigatethedifferencesintheproteomesofJSC-093350089andATCC1015,
total protein was extracted from each strain and subjected to tandem mass tag
(TMT) labeling, followed by liquid chromatography-mass spectrometry (LC-MS)
analysis. All MS data were analyzed using the Proteome Discoverer with the
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6.3. Results A. Błachowicz
Sequest-HT search engine against the A. niger CBS 513.88 protein database
(NCBI). The CBS 513.88 protein database was used because it has been exten-
sively annotated and enabled subsequent functional analysis using the AspGD
Gene Ontology (GO) Slim Mapper tool. The abundance ratios for all proteins
were normalized to ATCC 1015, which resulted in the identification of 218 pro-
teins with increased abundance and 109 proteins with decreased abundance (fold
change (FC) >j2jº, in JSC-093350089, relative to ATCC 1015. Distribution of
AspGD GO Slim terms among differentially expressed proteins is displayed in
Fig. 6.3. Many proteins that exhibited increased abundance in JSC-093350089
were involved with carbohydrate metabolic processes (10.1% of all upregulated
proteins), response to stress (9.6%), organelle organization (9.6%), and transport
(8.7%). Proteins involved in cytoskeleton organization, protein folding, secondary
metabolic processes, and transcription were associated with only increased protein
abundance in JSC-093350089, while proteins involved in cellular homeostasis were
associated with only decreased protein abundance in JSC-093350089. GO Slim
term enrichment analysis was conducted using FungiDB [157], which identified
significantly overrepresented proteins that exhibited increased abundance in the
proteome of JSC-093350089. Significantly overrepresented GO Slim terms included
carbohydrate metabolic processes (4.7% of background genes with this term),
cellular component assembly (5.3%), catabolic processes (4.1%), protein complex
assembly (6.4%), and response to stress (3.2%).
6.3.4 Differential abundance of cell wall modulation proteins
The proteome of JSC-093350089 revealed differential levels of cell wall mod-
ulation proteins (Table 6.1). Conidia of A. niger possess a relatively thick cell
wall made of a network of carbohydrates, including -glucans, chitin, -glucans,
galactomannan, and galactosaminogalactan, with an outer cell wall layer consisting
of complex melanin pigments [305]. The polyketide synthase AlbA (An09g05730),
which is required for the production of 1,8-dihydroxynaphthalene-melanin (DHN-
melanin) in A. niger [187], was over 2-fold more enriched in JSC-093350089 than
in ATCC 1015. The protein abundance of hydrophobin Hyp1 (An07g03340) was
nearly 3-fold higher than in ATCC 1015. RodA, the homologue of Hyp1 in A.
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6.3. Results A. Błachowicz
Figure 6.3: Biological process GO Slim categories of differentially expressed proteins.
Differentially enriched proteins (FC >j2j;P < 0:05) were mapped to terms representing
various biological processes using AspGD Gene Ontology (GO) Slim Mapper. The author
helped with the figure and the proteome analysis.
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6.3. Results A. Błachowicz
nidulans, has been reported to play a role in biofilm formation and efficient de-
construction of cell wall polysaccharides [306]. Its homologue in A. fumigatus was
shown to enhance fungal virulence by masking dectin-1- and dectin-2-mediated
recognition of conidia in vivo [307].
Differential expression was observed for a number of genes encoding glycoside
hydrolases, which were identified using the CAZy database. The starvation-
induced cellobiohydrolase CbhB (An01g11660), which is regulated by XlnR [308],
exhibited levels nearly 3-fold higher in the proteome of JSC-093350089 than in
ATCC 1015 [309]. XlnR is a transcriptional activator that regulates xylanolytic, en-
doglucanase, and cellobiohydrolase gene expression in A. niger [308, 310]. Increased
protein abundance was observed for -d-xylosidase XlnD (An01g09960), which is
also regulated by XlnR [310]. Decreased protein abundance was observed for XlnR-
regulated -galactosidase LacA (An01g12150), which is exclusively expressed on
xyloglucan-derived substrates [311]. Similarly, XlnR-regulated endoglucanase EglC
(An07g08950), which exhibits its greatest activity toward xyloglucan, also displayed
decreased protein abundance [312]. Other starvation-induced cell wall degradation
glycoside hydrolases enriched in JSC-093350089 included -1,3-glucanase AgnB
(An07g08640) and -glucanase Scw4 (An06g01530). Differential abundance of
glycoside hydrolases involved in starch utilization was also observed. Extracellular
acid -amylase AamA (An11g03340), which plays a role in starch degradation
and is regulated by starch degradation regulator AmyR [313], was present in
JSC-093350089 at levels 4-fold higher than that in ATCC 1015. Four of the five
enzymes in the family of GH92, which consists of mannosidases, displayed increased
abundance in the proteome of JSC-093350089. These GH92 proteins included
An08g08370, An13g01260, An14g04240, and An16g02910.
6.3.5 Differential abundance of stress response proteins
Our study also revealed differential abundance of proteins involved in the
stress response of A. niger (Table 6.2). Heat shock proteins, including DnaK-type
molecular chaperone Ssb2 (An16g09260) and An06g01610, were present in JSC-
093350089 at levels 2-fold and 5-fold higher than that of ATCC 1015, respectively.
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6.3. Results A. Błachowicz
Table 6.1: Relative abundance of cell wall modulation proteins
ORF
b
Protein CAZy family Description LogFC
a
An16g02910 GH92 -Mannosidase 3:53
An02g09050 GelG GH72 -1,3-Glucanotransferase 2:99
An14g04240 GH92 -1,2-Mannosidase 2:25
An07g08640 AgnB GH71 -1,3-Glucanase 2:23
An13g01260 GH92 -1,2-Mannosidase 2:15
An11g03340 AamA GH13 Acid -amylase 2:01
An11g06080 GH3 -Glucosidase 1:68
An06g01530 Scw4 GH17 -Glucanase 1:60
An01g11660 CbhB GH7 1,4--Glucan cellobiohydrolase 1:53
An02g13180 BgxB GH55 -1,3-Glucanase 1:48
An07g03340 Hyp1 Spore wall fungal hydrophobin 1:47
An01g09290 TraB GH37 Trehalase 1:27
An09g05730 AlbA Polyketide synthase 1:24
An08g11070 SucA GH32 Invertase 1:23
An08g08370 GH92 -Mannosidase 1:19
An14g04190 GbeA GH13 1,4--Glucan branching enzyme 1:18
An01g09960 XlnD GH3 -d-Xylosidase 1:06
An14g05340 UrghB GH105 Rhamnogalacturonyl hydrolase 1:00
An10g00400 GelA GH72 -1,3-Glucanotransferase 1:04
An16g06800 EglB GH5 Endoglucanase 1:13
An09g03100 AgtA GH13 GPI-anchored -
glucanosyltransferase
c
1:20
An04g06930 AmyC GH13 -Amylase 1:22
An18g03570 BglA GH3 -Glucosidase 1:22
An01g12150 LacA GH35 -Galactosidase 1:40
An02g00610 GH2 -Glucuronidase 1:41
An12g08280 InuE GH32 Exoinulinase 1:50
An11g02100 GH1 -Glucosidase 1:54
An14g01770 GH3 -Glucosidase 1:54
An11g00200 GH3 -Glucosidase 1:69
An07g08950 EglC GH5 Endoglucanase 1:82
An15g03550 GH43 Endoarabinase 1:91
a
Log2 fold change of JSC-093350089 compared to ATCC 1015 (P < 0:05)
b
open reading frame
c
glycosylphosphatidylinositol
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6.4. Discussion A. Błachowicz
Table 6.2: Relative abundance of stress response proteins
ORF
b
Protein Description LogFC
a
An12g10720 Catalase 3:71
An06g01610 Heat shock protein 2:51
An02g07350 LEA domain protein 1:95
An16g04420 Ish1 Stress response protein 1:53
An08g05850 SakA MAP kinase
b
1:50
An18g02900 Svf1 Survival factor 1 1:43
An07g07970 Srk1 Serine/threonine protein kinase 1:21
An16g09260 Ssb2 Heat shock protein 1:09
a
Log2 fold change of JSC-093350089 compared to ATCC 1015 (P < 0:05)
b
mitogen-activated protein
An06g01610 is very similar to late embryogenesis abundant (LEA)-like Hsp12 of
Saccharomyces cerevisiae and has been reported to stabilize the plasma mem-
brane [314]. Increased protein abundance was observed for the serine/threonine
protein kinase Srk1 (An07g07970) and the mitogen-activated protein kinase SakA
(An08g05850), which have been reported to mediate cell cycle arrest and mi-
tochondrial function in response to oxidative stress [315]. Other proteins that
exhibited higher levels in JSC-093350089 included the oxidative stress protein Svf1
(An18g02900) and An02g07350, which encodes a protein homologous to group 3
LEA proteins responsible for mitigating stress-induced damage, such as protecting
seeds from drought [316, 317]. The catalase An12g10720 was present at levels
13-fold higher than that of ATCC 1015. Increased abundance was also observed
for the stress response nuclear envelope protein Ish1, whose expression has been
reported to increase in response to glucose starvation and osmotic stress [318].
6.4 Discussion
In the current study, the molecular phenotype of a strain of A. niger isolated
fromtheISSwascharacterized. Despiteitsfrequentdetectioninbuiltenvironments,
this is the first investigation into the “omic” differences of an ISS A. niger isolate
from an Earth strain. As the frequency and duration of manned space missions
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6.4. Discussion A. Błachowicz
increase, investigations into how fungi respond and adapt to various stimuli
encountered during spaceflight are imperative for the health of crew and present
many economic benefits. Further, such studies provide insight into the adaptive
evolutionary mechanism of melanized fungal species and the biological alterations
of microbes isolated from extreme spaceflight environments.
ThegenomeofJSC-093350089waswithinthegeneticvariationofother A. niger
strains, suggesting that its ability to survive and proliferate in a spacecraft envi-
ronment is not contingent on enhanced genetic variance. This finding is consistent
with a previous report on the genetic variance of ISS Aspergillus isolates [155]. To
further understand the effect of microgravity and enhanced irradiation on fungal
genomics, future studies should investigate the same strain grown under both space
and ground conditions to quantify and identify any mutations that may result
from life on the ISS. Additional sequencing of terrestrial A. niger strains will also
be important to better identify the donor population of the strain and further
isolate the sequence variation that is specific to ISS-derived strains.
One characteristic of the ISS isolate was increased protein abundance of AlbA,
a key biosynthesis enzyme involved in the production of DHN-melanin in A. niger.
While A. niger historically has black conidia due to its high melanin content,
the A. niger albA mutant was reported to display a white or colorless conidial
phenotype [187]. This is consistent with reports that fungi isolated from high-
radiation environments exhibit increased melanin production. One study found
that A. niger strains occupying the south-facing slope of the “Evolution Canyon”
in Israel, which receives 200% to 800% higher solar radiation than the north-facing
slope, produced three times more melanin than did strains isolated from the north-
facing slope [53]. It is reasonable to presume that increased melanin production is
a key adaptive response to the enhanced irradiation environment of the ISS, as
there is considerable evidence that melanized fungi are highly resistant to ionizing
radiation under experimental conditions [7, 319]. In fact, it has been reported
that exposure of melanin to ionizing radiation alters its electronic properties, and
melanized fungal cells exhibit increased growth rates following exposure to ionizing
radiation [56].
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6.4. Discussion A. Błachowicz
Interestingly, the ISS isolate exhibited a higher growth rate than the terrestrial
strain, which is consistent with previous reports of enhanced growth in melanized
fungifollowingradiationexposureandAspergillus andPenicillium speciesrecovered
from the ISS and Mir space stations [7, 155, 56, 77]. Although this finding cannot
be definitively attributed to the isolation environment, it is conceivable that rapid
growth may confer a selective advantage in environments operating under strict
microbial monitoring procedures. The reported increase in colony pigmentation
distribution may point to enhanced melanin production in the ISS isolate, as the
AlbA protein was 2-fold more enriched than in the terrestrial strain. However,
the ability to rapidly spread pigment to the periphery of the colony may offer
additional modes of protection from high levels of radiation present in spacecraft.
The ISS isolate displayed general hallmarks of carbon starvation. During
starvation, A. niger produces a myriad of glycoside hydrolases that facilitate the
release of nutrients from biopolymers and the recycling of cell wall components
to generate energy and building blocks that can be used for maintenance and
conidiogenesis [62, 320]. The observed enrichment of starvation-induced nutrient
acquisitionenzymesmaybetheresultofadaptationtothelow-nutrientenvironment
that exists as a result of stringent microbial monitoring and remediation by
NASA [8]. The same may also be true for the increased abundance of the glycoside
hydrolase AamA. AamA is highly upregulated in growing hyphae at the periphery
of mycelium [321], and following secretion from exploring hyphae, AamA degrades
starch into small molecules that can be taken up by the fungus to serve as nutrients.
The significant enrichment of AamA suggests that JSC-093350089 can utilize starch
encountered during colonization more efficiently than ATCC 1015, which may have
conferred a selective advantage in the low-nutrient spacecraft environment.
During spaceflight, ionizing radiation can generate reactive oxygen species
(ROS)viathehydrolysisofintracellularwater, whichcanresultinoxidativedamage
to DNA, proteins, lipids, and other cell components [322]. Accordingly, catalase
was among the highest-upregulated proteins in the ISS isolate, which degrade
H
2
O
2
and therefore play a major role in curtailing oxidative stress [323, 324].
This is consistent with previous reports that spaceflight induces the expression
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6.4. Discussion A. Błachowicz
of oxidative stress resistance genes in microbes, animals, and astronauts [42, 193,
194, 195, 325, 326, 301], and increased susceptibility to ionizing radiation has been
observed in S. cerevisiae strains lacking cytosolic catalase [327, 328]. Similarly,
increased abundance was also observed for kinases that mediate key biological
processes in response to oxidative stress [315]. The high levels of oxidative stress
response proteins, as found in this study, are consistent with the observed response
of the melanized yeast Wangiella dermatitidis following exposure to ionizing
radiation [301]. On the other hand, increased resistance to oxidative stress may be
a response to microgravity, as low-shear modeled microgravity has induced such a
response in bacteria [329].
This study has revealed the existence of a distinct strain of A. niger on board
the ISS that exhibited differential growth and conidiation patterns compared
to a terrestrial strain. Proteomic analysis revealed significant differences in the
phenotype of JSC-093350089 that included enrichment of proteins involved in
the A. niger starvation response, oxidative stress resistance, cell wall modulation,
and nutrient acquisition. Given the ubiquity of A. niger in nature along with its
genetic diversity among sequenced strains [303] (26), it is not surprising that JSC-
093350089 exhibited a distinct molecular phenotype, and more studies will reveal
if the observed phenotype is widespread for other A. niger strains isolated from
the ISS. Since most of the microgravity-induced response studies were carried out
utilizing opportunistic pathogens of bacteria and yeast, the “omics” characterization
of ISS A. niger, a saprophyte, which exhibited higher melanin content than its
Earth counterparts, could be a model to elucidate molecular mechanisms involved
in microbial adaptation to the ISS environment. Developing countermeasures
to eradicate problematic microorganisms that adapt to unfavorable conditions
would help NASA’s Human Research Program in planning for long-duration
manned missions. Additionally, such analyses will further our understanding
of the molecular pathways that define host-microbe interactions, thus enabling
development of suitable cleaning strategies to maintain the health of habitat and
coliving crew for future missions.
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6.5. Material and Methods A. Błachowicz
6.5 Material and Methods
6.5.1 Isolation and identification of the ISS A. niger isolate
Surface samples were collected from the U.S. segment of the ISS using the
Surface Sampling kit (SSK) (NASA, 2011). Microbes were removed from surfaces
using a swab and sterile saline solution (0.85% sodium chloride) and were trans-
ported to Earth for analyses. Materials retrieved from the swabs were subsequently
inoculated onto potato dextrose agar (PDA) supplemented with chloramphenicol.
The PDA plates were incubated at ambient cabin temperature (28

C to 37

C)
for 5 days. The fungal colonies that exhibited growth were further purified and
stored at80

C in sterile glycerol stock until further analyses. When required,
fungal isolates were revived on PDA medium, and DNA from pure cultures was
extracted (UltraPure DNA kit [Mo Bio, Carlsbad, CA]). An approximately 600-bp
region consisting of ITS 1, 5.8S, and ITS 2 of the isolated fungal DNA was PCR
amplified, using primers ITS1F (5
0
TTG GTC ATT TAG AGG AAG TAA 3
0
) [126]
and Tw13 (5
0
GGT CCG TGT TTC AAG ACG 3
0
) and following the established
protocol [127]. The UNITE database was used to determine the closest similarity
to ITS sequences of fungal type strains [128]. The identity of the ISS isolate was
subsequently confirmed by WGS.
6.5.2 Genome sequencing, assembly, and annotation
Extracted DNA was sent to the Macrogen clinical laboratory (Macrogen
Inc., Rockville, MD, USA) for WGS. Library preparation was carried out using
the Illumina Nextera kit (random fragmentation, adapter ligation, and cluster
generation) and quantified with Quant-iT double-stranded DNA (dsDNA) high-
sensitivity assays. Generated libraries were sequenced with 100 bp paired-end
sequencing protocols on the Illumina HiSeq 2500 platform. Raw data images were
produced utilizing HCS (HiSeq Control Software v2.2.38) for system control, and
base calling (BCL) was done through an integrated primary analysis using Real
Time Analysis software v1.18.61.0. The BCL binary was converted into FASTQ
utilizing Illumina package bcl2fastq (v1.8.4). The NGS QC Toolkit version 2.3 [330]
was used to filter the data for high-quality vector- and adapter-free reads for genome
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6.5. Material and Methods A. Błachowicz
assembly (cutoff read length for high quality, 80%; cutoff quality score, 20), and
22,769,466 vector filter reads were obtained after the quality check. High-quality
vector-filtered reads were used for de novo assembly with the MaSuRCA genome
assembler (k-mer size, 70) [331]. The final assembly consisted of 223 scaffolds with
a total size of 36,079,011 bp (100). The N
50
scaffold length was 543,773 kb,
and the largest scaffold was 1,390.254 kb. There was no random “N” joining of the
contigs to maintain high assembly quality. Quality check of the final assembly was
performed using the quality assessment tool for genome assemblies (QUAST) [332].
The number of N’s detected was less than 12 per 100 kb, which represents very
good assembly.
Genome annotation was performed with funannotate (v1.3.0-beta), which
utilizes a combination of ab initio gene prediction tools [247, 248] and experimental
evidence including proteins and transcriptome sequencing (RNA sequencing) and
a consensus gene calling with EvidenceModeler [251].
6.5.3 Phylogenetic analysis
PhylogeneticanalysisofA. niger strainswasperformedbyidentifyingconserved
protein coding genes in available A. niger genomes and close relatives. These data
wereobtainedbydownloadingpublicsequencedatafromNCBIandtheDepartment
of Energy’s Joint Genome Institute (JGI) Mycocosm. The assemblies for strains
A1, ATCC 10864, An76, FDAARGOS 311, FGSC A1279, H915-1, L2, and SH-2
were downloaded from the NCBI Assembly Archive. The strains FDAARGOS 311
and An76 already had deposited annotations and were downloaded directly. For the
remaining strains, gene prediction with Augustus (v 3.2.2) [247] used the pretrained
model ‘aspergillus_niger_jsc_093350089’ generated from the genome annotation
procedure. This parameter set is deposited in https://github.com/hyphaltip/
fungi-gene-prediction-params. Additional strains with annotation from JGI
were downloaded (ATCC 1015, DSM 1, CBS 513.88, and NRRL 3) along with
related species (A. niger van Tieghem ATCC 13496, A. welwitschiae CBS 139.54b,
A. phoenicis, A. lacticoffeatus CBS 101883, and A. brasiliensis CBS 101740).
Coding sequences were obtained, translated into proteins, and searched for a
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6.5. Material and Methods A. Błachowicz
conserved set of 71 protein coding gene markers, “AFTOL_70,” as part of the
(1000 Fungal Genomes project). These markers were searched using PHYling,
which first searches for conserved markers using HMMsearch followed by extraction
of best hits and concatenated alignment of all the orthologous matches. A back-
translated alignment of coding sequences was produced from the input proteins in
order to resolve the closely related strains in this data set. A phylogenetic tree was
inferred from the coding sequence tree using IQTREE (v1.6.3) first by identifying
a partition scheme with -m TESTMERGE -st CODON parameters followed by a
tree inference using the options -st CODON -bb 1000 -spp Partition.txt to infer
the tree and obtain branch support with ultrafast bootstrapping on the reduced
partition parameters under a codon model in IQTREE [333, 334, 335]. To identify
the number of single nucleotide variations occurring between JSC-093350089 and
both ATCC 1015 and CBS 513.88, variants were called using the Harvest suite’s
Parsnp tool [336].
6.5.4 Growth conditions
JSC-093350089 and ATCC 1015 were cultivated on 10 cm petri dishes contain-
ing 25 mL glucose minimal medium (GMM) agar plates (6 g/L NaNO
3
, 0.52 g/L
KCl, 0.52 g/L MgSO
4
7H
2
O, 1.52 g/L KH
2
PO
4
, 10 g/L d-glucose, 15 g/L
agar supplemented with 1 mL/L of Hutner’s trace elements) with a cellophane
membrane on top, on which the fungus was grown. Unless otherwise specified,
10
7
conidia per petri dish (D = 10 cm) were inoculated into each medium and
incubated at 30

C for 5 days.
6.5.5 Physiological analysis
Growth rates were assessed by centrally inoculating 10
4
conidia on GMM
plates in replicates of 5 and measuring radial growth at the same time each day.
Statistical analyses were performed using multiple t tests and corrected for multiple
comparisons using the Holm-Sidak method. Photos depicting morphological
differences were taken after 7 days.
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6.5. Material and Methods A. Błachowicz
6.5.6 Protein extraction
Mycelia from GMM agar plates were collected and stored at80

C prior to
protein extraction. For protein extraction, the lysis buffer consisted of 100 mM
triethylammonium bicarbonate (TEAB) with 1 Halt protease inhibitor cocktail
(100), with the final concentration of each component being 1 mM AEBSF [4-
(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], 800 nM aprotinin, 50 μM
bestatin, 15 μM E64, 20 μM leupeptin, and 10 μM pepstatin A (Thermo Scientific,
Rockford, IL) and 200 μg/mL phenylmethylsulfonyl fluoride (Sigma-Aldrich, St.
Louis, MO). Mycelia were homogenized directly using Precellys 24 homogenizer
(Bertin, Rockville, MD) in which each sample was processed inside a 2 mL cryotube
with 0.5 mm glass beads three times (at 4

C and 6,500 rpm for 1 min., repeated 3
times with 15 s pauses in between). The lysed fungi were centrifuged at 17,000 G
for 15 min. Protein concentrations in the supernatants were measured by the
Bradford assay with albumin for the standard curve (Bio-Rad Laboratories, Inc.,
Hercules, CA).
6.5.7 Tandem mass tag (TMT) labeling
Two hundred micrograms of proteins from each sample was precipitated in 20%
trichloroacetic acid (TCA) at 4

C. Protein pellets were obtained by centrifugation
(17,000 g), washed with ice-cold acetone, and resuspended in 25 μL TEAB (50 mM
final concentration) and 25 μL 2,2,2-trifluoroethanol (TFE) (50% final concen-
tration). Proteins were reduced by adding 1μL of tris(2-carboxyethyl)phosphine
(TCEP; 500 mM) followed by incubation for 1 hr at 37

C (10 mM final TCEP
concentration). Proteins were alkylated in the presence of iodoacetamide (IAA;
30 mM) in the dark for 1 hr at room temperature. A 2.5 μg per sample quantity
of mass-spectrometry-grade trypsin-LysC (Promega, Madison, WI) was used to
digest the peptides overnight at 37

C.
The digested peptides were quantified using the Pierce quantitative colorimetric
peptide assay (Thermo Scientific). Forty micrograms of peptides from each specific
samplewaslabeledwiththeThermoScientificTMTsixplexisobaricmasstaggingkit
(ATCC 1015-GMM with TMT
6
-128, and JSC-GMM with TMT
6
-129) according
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6.5. Material and Methods A. Błachowicz
to the manufacturer’s protocol. The TMT
6
-130 and -131 labels were used as
a reference that contained an equal amount of the peptides from each of the
four samples. All labeled-peptide mixtures were combined into a single tube,
mixed, and fractionated using the Thermo Scientific Pierce high-pH reversed-phase
peptide fractionation kit. Fractions were dried using a SpeedVac concentrator and
resuspended in 1% formic acid prior to LC-tandem MS (MS/MS) analysis.
6.5.8 LC-MS/MS analysis
The samples were analyzed on an Orbitrap Fusion Tribrid mass spectrometer
withanEasy-nLC1000liquidchromatograph, a75μmby2cmAcclaimPepMap100
C
18
trapping column, a 75 μm by 25 cm PepMap RSLC C
18
analytical column,
and an Easy-Spray ion source (Thermo Scientific). The column temperature was
maintained at 45

C, and the peptides were eluted at a flow rate of 300 nl/min
over a 110-min gradient, from 3 to 30% solvent B (100 min), 30 to 50% solvent B
(3 min), 50 to 90% solvent B (2 min), and 90% solvent B (2 min). Solvent A was
0.1% formic acid in water, and solvent B was 0.1% formic acid in acetonitrile.
The full MS survey scan (m/z 400 to 1,500) was acquired in the Orbitrap at
a resolution of 120,000 and an automatic gain control (AGC) target of 210
5
.
The maximum injection time for MS scans was 50 ms. Monoisotopic precursor
ions were selected with charge states 2 to 7, a10 ppm mass window, and 70 s
dynamic exclusion. The MS
2
scan (m/z 400 to 2,000) was performed using the
linear ion trap with the collision-induced dissociation (CID) collision energy set to
35%. The ion trap scan rate was set to “rapid,” with an AGC target of 410
3
and
a maximum injection time of 150 ms. Ten fragment ions from each MS
2
experiment
were subsequently selected for an MS
3
experiment. The MS
3
scan (m/z 100 to
500) was performed to generate the TMT reporter ions in the linear ion trap using
heated capillary dissociation (HCD) at a collision energy setting of 55%, a rapid
scan rate and an AGC target of 510
3
, and a maximum injection time of 250 ms.
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6.5. Material and Methods A. Błachowicz
6.5.9 Proteome data analysis
All MS spectra were searched using the Proteome Discoverer (version 2.1.0.81;
Thermo Scientific) with the Sequest-HT searching engines against an Aspergillus
niger CBS 513.88 database containing 10,549 sequences (NCBI). The searches were
performed with the following parameters: 5 ppm tolerance for precursor ion masses
and 0.6-Da tolerance for fragment ion masses. The static modification settings
included carbamidomethyl of cysteine residues and dynamic modifications included
oxidation of methionine, TMTsixplex modification of lysine -amino groups and
peptide N termini, and acetyl modification of protein N terminus. A target-decoy
database search was used to set a false-discovery rate (FDR) of 1%. The reporter
ion integration tolerance was 0.5 Da. The coisolation threshold was 75%. The
average signal-to-noise threshold of all reporter peaks was bigger than 10. The
total intensity of a reporter ion for a protein was calculated based on the sum
of all detected reporter ions of associated peptides from that protein. The ratios
between reporter ions and the reference reporter ions (TMT
6
-130 or -131) were
used to estimate the abundance ratio of each protein.
For statistical analysis, the sum of reporter ion intensities for each protein was
log2 transformed and the technical triplicate measurements for each protein were
averaged. Only the proteins that were identified with at least one peptide detected
in each technical replicate, and quantified in all three technical replicates, were
considered for the analysis. Student’s t test was performed to identify proteins that
are differentially expressed. Proteins with p-value 0:05 were further evaluated
for increased or decreased abundance using a cutoff value of log2 fold change of
j1j.
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Chapter7
Metabolomic analysis of Aspergillus
niger isolated from the
International Space Station reveals
the radiation resistance potential of
pyranonigrin A
1
7.1 Abstract
Secondary metabolite (SM) production of Aspergillus niger JSC-093350089,
isolated from the International Space Station (ISS), is reported, along with a
comparison to the experimentally established strain ATCC 1015. The analysis
revealed enhanced production levels of naphtho-
-pyrones and therapeutically rele-
vant SMs, including bicoumanigrin A, aurasperones A and B, and the antioxidant
pyranonigrin A. Using targeted gene deletions, the gene cluster responsible for
pyranonigrin A biosynthesis was identified, which consists of a hybrid nonribo-
somal peptide synthetase/polyketide synthase (NRPS/PKS) adjacent to three
contiguous tailoring enzymes. UV-C sensitivity assays enabled characterization of
1
Romsdahl, Blachowicz et al., Fungal Genetics and Biology, in revision; The author helped
analysing SM profiles of the ISS and ATCC isolates and prepared the corresponding figure. The
author also helped with the initial set up of the UV-C experiments and the figure preparation.
7.2. Introduction A. Błachowicz
pyranonigrin A as a UV resistance agent in the ISS isolate.
7.2 Introduction
Filamentous fungi are ubiquitous in spacecraft environments due to anthro-
pogenic contamination and an inability to completely sterilize the craft and
cargo [71, 289, 28]. Microbial infections are a major health risk for astronauts,
and are exacerbated by the combined stresses of microgravity, sleep disruption,
alterations in food intake, confined living space, and high levels of radiation that
can compromise the immune system [289, 290]. Additionally, several studies have
indicated that spacecraft environments increase microbial virulence and antimicro-
bial resistance [293, 291, 292]. As we make strides towards human interplanetary
exploration, investigations into the characteristics of filamentous fungi that reside
in spacecraft environments are critical for crew health. Additionally, such stud-
ies present diverse industrial and therapeutic opportunities, as fungi produce a
plethora of bioactive secondary metabolites (SMs) in response to external stressors.
These small molecules often kill or inhibit growth of other organisms, enabling
fungi to successfully compete within the complex ecosystem they reside in. While
some SMs are toxic to humans, others have diverse industrial and therapeutic
applications, including antibiotic, anticancer, antioxidant, immunosuppressant,
and cholesterol-lowering activities [296]. The remarkable structural and functional
diversity of fungal SMs arise from the combinatorial and modular nature of their
biosynthesis, in which the SM core backbone is biosynthesized by a core synthase
enzyme, such as a polyketide synthase (PKS), a nonribosomal peptide synthetase
(NRPS), or a hybrid NRPS/PKS, and further diversified by a number of tailoring
enzymes that are clustered together within the genome [337].
In order to understand the characteristics of microbes residing in the Inter-
national Space Station (ISS), National Aeronautics and Space Administration
(NASA) has implemented a robust microbial monitoring system [110]. In one
study, the filamentous fungus Aspergillus niger was reported to be the predom-
inant isolate [8]. A. niger is a melanized fungal species commonly used in the
biotech industry as a production host for citric acid and enzymes [276]. Melanized
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7.2. Introduction A. Błachowicz
fungi are highly resistant to ionizing radiation under experimental conditions [338,
319], and it has been reported that the electronic properties of melanin change
following exposure to ionizing radiation [56]. Additionally, highly-melanized fungal
cells exhibit increased growth compared to non-melanized fungal cells following
exposure to ionizing radiation, suggesting that melanin has the capacity to capture
and utilize energy [56]. Several studies have reported the association of melanin
production with fungal virulence [339, 340, 341], which enhances the need for
studies that assess the characteristics of melanized fungi inhabiting spacecraft
environments.
Naphtho-
-pyrones are the predominant class of SMs produced by A. niger, and
they possess a diverse array of reported biological properties, including anti-HIV,
anti-hyperuricemia, anti-tubercular, antimicrobial, antitumor, and antioxidant
activities [342]. Advances in genome sequencing of A. niger has revealed its
capacity to produce many other SMs in addition to the naphtho-
-pyrones, as
the experimentally established A. niger ATCC 1015 genome harbors 33 PKSs,
15 NRPSs, and 9 NRPS-PKS hybrid genes [343]. Many of these SM biosynthesis
genes are silent or expressed at very low levels in standard laboratory conditions,
which has resulted in a lack of studies on SMs that may have useful industrial and
therapeutic properties. Given that fungi produce an array of SMs in response to
environmental stress, investigations into the SM production of A. niger strains
isolated from space environments may reveal novel mechanisms of radiation re-
sistance in melanized fungal species. Further, such investigations may facilitate
the identification of biosynthetic gene clusters of SMs produced in low levels in
“Earth” strains, but high levels in “space” strains. Additionally, any SMs that
confer radiation resistance can potentially be harnessed as radioprotective drugs,
which can have extensive benefits in space programs and protecting humans from
harmful radiation exposure.
Here, we report the SM production of JSC-093350089, a strain of A. niger
isolated from surfaces of the International Space Station (ISS) and previously
described[188]. OuranalysisrevealedthattheISSisolateproduceshighlevelsofthe
antioxidant pyranonigrin A [281] when compared to the experimentally established
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7.3. Results A. Błachowicz
strainATCC1015. Oxidativestressisimplicatedinmanyhumandiseases, including
cancer, diabetes, cardiovascular, and neurodegenerative diseases, and therefore
antioxidants have significant therapeutic potential [344]. Identification of the genes
involved in SM biosynthesis is crucial for optimization of compound yield and
engineering of second generation molecules. We therefore developed an efficient
genetargetingsysteminJSC-093350089andusedtargetedgenedeletionstoidentify
the biosynthesis genes responsible for pyranonigrin A production. Finally, the
radiation resistance potential of pyranonigrin A was assessed, which demonstrated
its potential use as a radioprotective agent.
7.3 Results
7.3.1 Secondary metabolite analysis of JSC-093350089
SM profiles of JSC-093350089 and ATCC 1015 were examined after growth
on GMM agar medium using high-performance liquid chromatography coupled
with diode-array detection and electrospray ionization tandem mass spectrometry
(HPLC-DAD-MS). SMs were identified based on mass, UV-Vis absorption, and
retention time, which were in good agreement with literature [187]. The identity
of pyranonigrin A was further verified by purchasing the pure compound from
Enzo Life Sciences (not shown). The data revealed that each strain produced a
distinct SM profile, with the production yield of most SMs significantly altered
(Fig. 7.1A). Production yield analysis was carried out for each SM (Fig. 7.1B).
Compared to ATCC 1015, a significant decrease in the production of nigragillin,
an insecticide [345], was observed (P = 0:0001). The most significant difference
was observed with the antioxidant pyranonigrin A [281], which exhibited a 6000%
increase in production in JSC-093350089 (P =0:04), as it was produced at basal
levels in ATCC 1015. Nigerazine B displayed no statistical difference in production
levels (P = 0:06). In the ISS strain, pestalamide B production was approximately
2 times that of ATCC 1015 (P = 0:03), and biocoumanigrin A, which was reported
to have cytotoxic activity against human cancer cell lines, exhibited a production
yield 2.5 times that of ATCC 1015 (P = 0:01). Kotanin production in the ISS
strain was approximately 10 times that of ATCC 1015 (P = 0:03).
- 126 -
7.3. Results A. Błachowicz
The majority of SMs produced were identified as naphtho-
-pyrones, including
aurasperone A, B and C, fonsecinone A, B and C, a fonsecinone C derivative,
and asperpyrone B and C. These SMs, highlighted in green in Fig. 7.1A, are
biosynthesized by the PKS AlbA [187]. The molecular formula of the final SM was
predicted using high-resolution mass spectrometry, and a thorough literature search
revealed that no known A. niger SM matched this formula. This SM was later
determined to also be biosynthesized by the albA pathway when A. niger devoid
of AlbA failed to produce the unknown compound. The combined production
yields of albA pathway SMs were approximately 2.5 times higher in the ISS stain
compared to ATCC 1015 (P = 0:03).
7.3.2 Analysisofthepotentialgeneclustersresponsibleforproduction
of pyranonigrin A in silico
Considering the significant therapeutic and radioresistant potential of pyra-
nonigrin A, we set out to identify the genes involved in its biosynthesis. To
identify the biosynthetic gene cluster responsible for pyranonigrin A production,
we searched for the core backbone synthase gene involved its biosynthesis. The
biosynthetic pathway of pyranonigrin E, a SM very similar to pyranonigrin A, was
recently proposed and pynA (An11g00250) was identified as the PKS-NRPS hybrid
involved in its biosynthesis [346]. We hypothesized that pyranonigrin A is either
biosynthesized by the same cluster responsible for pyranonigrin E production, or
by a different cluster harboring a PKS-NRPS hybrid gene similar to pynA. The
genome of ATCC 1015 possesses 8 PKS-NRPS hybrid genes other than pynA.
BLAST analysis was performed using the Joint Genome Institute (JGI) MycoCosm
database [67] on the 8 remaining PKS-NRPS hybrids to determine which genes
possessed high sequence homology to pynA. The results revealed that An18g00520
possessed high sequence similarity to pynA, with 53.4% sequence identity and
89.8% subject coverage.
- 127 -
7.3. Results A. Błachowicz
Figure 7.1: (A) Secondary metabolite production in JSC-093350089 relative to ATCC
1015 following growth on GMM for 5 days, as detected by DAD total scan. Each
individual metabolite’s production yield is reported as increased, decreased, or no change,
compared to that of ATCC 1015. (B) Quantification of secondary metabolites showing
percent change for each metabolite in JSC-093350089 when compared to ATCC 1015.
Significance was determined using Welch’s t-test. The author helped with the SM analysis
and figure preparation.
- 128 -
7.3. Results A. Błachowicz
7.3.3 DevelopmentofanefficientgenetargetingsysteminJSC-093350089
and identification of the PKS-NRPS hybrid responsible for pyra-
nonigrin A biosynthesis
To determine which of the two putative PKS-NRPS hybrids is involved in the
biosynthesis of pyranonigrin A, a genetic system was developed in JSC-093350089.
The kusA gene was first deleted to decrease the rate of nonhomologous integration
of transforming DNA fragments, thereby improving gene targeting [347]. Next,
the pyrG gene was deleted in the kusA- background to generate CW12003, an aux-
otrophic mutant that requires uracil and uridine supplementation [348]. CW12003
was then used to generate mutant strains CW12004 and CW12005, which had the
pynA gene and An18g00520 genes deleted, respectively. JSC-093350089 and the
two mutant strains were then cultured on GMM, and SMs were extracted following
5 days of growth and subjected to HPLC-DAD-MS analysis. Observation of SM
traces as detected by UV-Vis total scan and mass spectroscopy in positive ion
mode [M+H]
+
mz = 224 revealed an increase in production of pyranonigrin A in
pynA- (CW12004) and the complete elimination of pyranonigrin A in An18g00520-
(CW12005), revealing that An18g00520 is responsible for the production of pyra-
nonigrin A. This finding was recently confirmed in Penicillium thymicola and
reported while our work was being completed [349].
7.3.4 IdentificationofpyranonigrinAbiosynthesisgeneclusterbound-
aries
Next, we aimed to identify additional genes involved in pyranonigrin A biosyn-
thesis and any detectable intermediates. To accomplish this, we identified genes
surrounding pyrA (Fig. 7.2A and Table 7.1) as the genes involved in fungal SM
biosynthesis are usually clustered in the genome [57]. Interestingly, when we com-
pared the genes surrounding pyrA in A. niger to their homologs in P. thymicola
using the JGI MycoCosm database [67], we noticed that the distribution of genes
surrounding pyrA differed between the P. thymicola and A. niger genomes [349].
For example, pyrD, a hydrolase predicted to be involved in pyranonigrin A biosyn-
thesis in P. thymicola, is adjacent to pyrC in the P. thymicola genome and has
only two genes separating it from pyrA. However, its homolog within the A. niger
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7.3. Results A. Błachowicz
Table 7.1: Putative function of genes within the pyranonigrin A biosynthetic gene cluster
and their homologs in Penicillium thymicola.
Gene ORF P. thymicola Identity (%) Putative function
homolog

An18g00470 180933 (pyrD) 148/256 (58%) Hydrolase
An18g00480 104760 386/554 (70%) Cyclohexamide resistance protein
pyrC An18g00490 104735 190/269 (71%) FAD binding monooxygenase
pyrB An18g00500 104701 296/453 (65%) Cytochrome P450
pyrE An18g00510 168730 326/454 (72%) FAD binding oxidoreductase
pyrA An18g00520 168734 2371/3881 (61%) PKS-NRPS hybrid
An18g00530 Hypothetical protein

JGI Protein ID
genome, An18g00470, is adjacent to An18g00480 and has four genes separating it
from pyrA. Similarly, although An18g00510 is adjacent to pyrA in the A. niger
genome, its P. thymicola homolog has three genes separating it from pyrA in the
P. thymicola genome, and was predicted to not be involved in pyranonigrin A
biosynthesis [349]. To investigate these observations, we generated a gene deletion
library to identify the genes involved in pyranonigrin A biosynthesis.
A. niger produces large quantities of naphtho-
-pyrone SMs [187], which we
suspected may hinder our ability to detect any intermediate compounds in JSC-
093350089 tailoring enzyme deletant strains. To circumvent this, we first generated
CW12006, a JSC-093350089 mutant strain deficient in AlbA, and therefore also
deficient in naphtho-
-pyrone production. The AfpyrG gene was then recycled
to enable the generation of additional deletion mutations in CW12007. Next,
we used CW12007 to individually delete 5 genes surrounding pyrA and generate
a pyranonigrin A biosynthetic gene cluster mutant library with albA- genetic
background. The deletant strains were cultured in pyranonigrin A-producing
conditionsandtheirSMproductionwasanalyzedusingHPLC-DAD-MS(Fig.7.2B).
Deletion strains CW12009, CW12010, and CW12011, which had An18g00490,
An18g00500, and An18g00510 deleted, respectively, resulted in the complete
elimination of pyranonigrin A, which confirmed their involvement in its biosynthesis.
- 130 -
7.3. Results A. Błachowicz
DAD (Total scan) MS (Total ion current)
10 15 20 25 30 35 40 10 15 20 25 30 35 40
Time (min) Time (min)
WT
albA-
An18g00480-, albA-
pygB-, albA-
pygC-, albA-
pygD-, albA-
pygA-, albA-
An18g00530-, albA-
WT
albA-
An18g00480-, albA-
pygB-, albA-
pygC-, albA-
pygD-, albA-
pygA-, albA-
An18g00530-, albA-
An18g00480
pygB pygC pygD
pygA An18g00530
5 kb
A
B
Figure 7.2: (A) Secondary metabolite production in JSC-093350089 relative to ATCC
1015 following growth on GMM for 5 days, as detected by DAD total scan. Each
individual metabolite’s production yield is reported as increased, decreased, or no change,
compared to that of ATCC 1015. (B) Quantification of secondary metabolites showing
percent change for each metabolite in JSC-093350089 when compared to ATCC 1015.
Significance was determined using Welch’s t-test. Figure courtesy of Dr. J. Romsdahl.
- 131 -
7.4. Discussion A. Błachowicz
Deletion strains CW12008 and CW12013, which had An18g00480 and An18g00530
deleted, respectively, resulted in unchanged SM profiles, which indicated that these
two genes are not involved in pyranonigrin A biosynthesis and are outside the
gene cluster border. These results suggest that the pyranonigrin A biosynthetic
gene cluster consists of pyrA, pyrB, pyrC, and An18g00510, which we designated
as pyrE (Fig. 7.2A and Table 7.1). These finding are in contrast to the recent
study conducted in P. thymicola, which proposed that pyrD, rather than pyrE, is
involved in pyranonigrin A biosynthesis [349].
7.3.5 Assessment of the UV resistance potential of pyranonigrin A
We hypothesized that the enhanced production levels of pyranonigrin A in
the ISS isolate played a role in protecting the strain from the high levels of
radiation present in the spacecraft. This hypothesis was evaluated by comparing
the UV sensitivity of pyranonigrin A-producing JSC-093350089 to pyranonigrin A-
deficient JSC-093350089. It has been reported that kusA deletion significantly
enhances the sensitivity of A. niger to UV exposure [347]. Therefore, to investigate
whether pyranonigrin A confers UV resistance to A. niger, the kusA gene was
first reintegrated into the pyrA- deletion strain (CW12005) to generate CW12015.
Next, the JSC-093350089 WT and pyrA- strains were exposed to varying doses of
UV-C radiation ranging from 5–25 mJ/cm
2
in triplicate. The results indicated that
pyranonigrin A deficiency significantly reduces the viability of UV-exposed strains
at doses greater than 15 mJ/cm
2
(Fig. 7.3). The effect became more pronounced
as the radiation dose increased, with an approximate viability reduction of 34%
observed at 15 mJ/cm
2
(P = 0:005), 43% observed at 20 mJ/cm
2
(P = 0:005), and
68% observed at 25 mJ/cm
2
(P = 0:0002).
7.4 Discussion
Although the persistence of A. niger in spacecraft [8, 110] has been well-
documented, few studies have investigated how spacecraft conditions alter its SM
production. Metabolomic characterization of A. niger strains that have inhabited
spacecraft can provide valuable information about SM-based adaptation mecha-
- 132 -
7.4. Discussion A. Błachowicz
0 5 10 15 20 25
0
20
40
60
80
100
120
UVC Dose [mJ/cm
2
]
Percent Viability
WT vs pygA-
WT
pygA-
**
*
*
0 5 10 15 20 25
0
20
40
60
80
100
120
UVC Dose [mJ/cm
2
]
Percent Viability
WT vs pygA-
WT
pygA-
**
*
*
WT
pygA-
Figure 7.3: Percent survival following exposure to varying doses of UV-C radiation for
JSC-093350089 WT and JSC-093350089 pyrA- (CW12005). The author helped with the
initial set up of the UV-C experiments and the figure preparation.
nisms of fungi capable of surviving such environments and provides many economic
benefits. Despite a substantial effort to map fungal SMs to their biosynthesis genes,
most clusters remain unlinked to their final product. In many cases, this is due to
low production levels of many metabolites. There is therefore significant potential
in analyzing SM production in fungal species isolated from various extreme envi-
ronments, as such conditions may naturally optimize production yields of useful
SMs, and reduce the cost of laborious laboratory-based optimization.
Metabolomic analysis of the A. niger laboratory strain and ISS isolate revealed
significant differences in SM production levels. This finding is not surprising given
thatfungalSMproductionishighlydependentonenvironmentalconditions. Fungal
SMs are often produced to confer selective advantage through habitat defense,
competitor inhibition, chemical signaling, nutrient acquisition, or other self-defense
mechanisms [59]. It remains uncertain whether the observed SM profile differences
are the direct result of exposure to unfavorable ISS conditions, such as microgravity,
enhanced radiation, and low nutrient availability. Still, this investigation provides
valuable insight into the metabolomic fingerprint of melanized fungal species
capable of withstanding the ISS environment. Further, it reveals the existence
of a strain capable of producing enhanced levels of therapeutically relevant SMs,
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7.4. Discussion A. Błachowicz
including the antioxidant pyranonigrin A [281], the human cancer cytotoxic agent
bicoumanigrin A [350], the antimicrobial aurasperone A [351], and the antifungal
and antioxidant aurasperone B [342].
A cumulative increase in production wasobservedfor naphtho-
-pyrones, which
are produced by the same PKS responsible for 1,8-dihydroxynaphthalene-melanin
(DHN-melanin) biosynthesis. This stays in agreement with the observed enrichment
of the AlbA in the proteome of JSC-093350089, relative to ATCC 1015 [188].
Such observations suggest that the ISS isolate produces enhanced levels of DHN-
melanin, andisconsistentwithpreviousreportsthatfungiinhabitinghigh-radiation
environments produce enhanced levels of melanin [53]. Melanin has significant
radioprotective properties and appears to play a role in energy transduction, as
melanizedfungiexhibitincreasedratesofgrowthfollowingexposuretoradiation[56,
7].
Perhaps the most significant observation was enhanced production of the
antioxidant pyranonigrin A in JSC-093350089 relative to ATCC 1015, which only
produces the SM at basal levels. On the ISS, radiation capable of penetrating the
spacecraft generates reactive oxygen species (ROS) within biological systems [352].
Oxidative stress occurs when ROS overwhelm an organism’s antioxidant defense
mechanisms, resulting in the generation of oxidative damage among DNA, proteins,
lipids, and other vital cell components [352, 322]. Antioxidants have enormous
therapeuticpotential, astheyneutralizetheharmfuleffectsofROS,whichcancause
or exacerbate a range of human diseases, including cancer, diabetes, cardiovascular,
and neurodegenerative diseases [353]. Examination of metabolic reserves of fungi
isolated from enhanced radiation environments therefore provides a means of
identifying fungal strains that produce optimized levels of specific therapeutics, as
illustrated by this study.
It is reasonable to presume that enhanced production of oxidative stress
resistance agents is a key adaptive characteristic of fungi inhabiting high-radiation
environments, as this has been observed among other species [193, 354]. This
may explain the enhanced production levels of pyranonigrin A in the ISS isolate,
and is supported by the UV resistance study, which suggests that pyranonigrin A
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7.4. Discussion A. Błachowicz
plays a significant role in conferring radiation resistance in A. niger. This finding
illustrates the potential for pyranonigrin A to be utilized as a radiation protective
agent for other organisms present on spacecraft, such as plants or humans. One
example is through the generation of transgenic plants capable of biosynthesizing
pyranonigrin A, which may minimize harmful mutations in plant DNA that can
cause tissue damage and genetic modifications in seeds [355]. Additionally, such
radioprotective compounds can potentially have enormous applications for humans
in space programs or cancer therapies [352].
Although fungi have immense capacity to produce therapeutically and indus-
trially relevant SMs, such as pyranonigrin A [296], most SM clusters are silent
under standard laboratory conditions, hindering their full potential. Genetic modi-
fication techniques provide a powerful way to activate silent cluster and optimize
production levels of specific SMs of interest [58]. However, these techniques usually
require knowledge of the specific genes involved in the biosynthesis of the molecule
of interest. Therefore, to facilitate product yield optimization and the generation
of potentially useful second generation compounds, we generated a gene cluster
deletion library and identified the genes responsible for pyranonigrin A biosynthesis.
Future studies should aim to optimize pyranongrin A production levels through
genetic engineering techniques, as such investigations may play a substantial role
in reducing production costs and expand compound availability for future testing
and development.
In summary, this study harnessed the altered SM profile of an ISS A. niger
isolate to facilitate identification of the pyranonigrin A biosynthetic gene cluster.
The potential for pyranonigrin A to be utilized by space programs or chemotherapy
recipients as a radiation-resistant therapy was assessed. These findings illustrate
the economic potential associated with investigating the metabolite production
of microbes isolated from extreme environments and provide a means for the
exploitation of pyranonigrin A in various applications.
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7.5. Materials and Methods A. Błachowicz
7.5 Materials and Methods
7.5.1 Secondary metabolite extraction and analysis
JSC-093350089 and ATCC 1015 were cultivated at 28

C on GMM agar plates,
starting with 10
7
spores per Petri dish (D = 10 cm). After 5 days, agar was
chopped into small pieces and extracted with 25 mL methanol (MeOH), followed
by 25 mL 1:1 MeOH-dichloromethane, each with 1 hr of sonication and filtration.
The extract was evaporated in vacuo and re-dissolved in 2 mL of 20% dimethyl
sulfoxide in MeOH and a portion (10μL) was examined by high performance liquid
chromatography-photodiode array detection-mass spectroscopy (HPLC-DAD-MS)
analysis. HPLC-MS was carried out using a ThermoFinnigan LCQ Advantage ion
trap mass spectrometer with a reverse-phase C
18
column (3 μm; 2.1 by 100 μm;
Alltech Prevail) at a flow rate of 125 μL/min. The solvent gradient for HPLC-
DAD-MS was 95% MeCN/H
2
O (solvent B) in 5% MeCN/H
2
O (solvent A) both
containing 0.05% formic acid, as follows: 0% solvent B from 0 to 5 min, 0 to
100% solvent B from 5 to 35 min, 100 to 0% solvent B from 40 to 45 min, and
re-equilibration with 0% solve B from 45 to 50 min.
7.5.2 Strains and molecular manipulations
Deletion cassettes were generated using the double joint PCR technique [356].
DNA insertions into the A. niger genome were performed using protoplasts and
standard PEG transformation. To develop an efficient gene targeting system in
JSC-093350089, the kusA gene was first deleted by replacing it with the hygromycin
resistance marker (hph). The two amplified flanking sequences and the hygromycin
phosphortransferase gene (hph) marker cassette amplified from pCB1003 (Fungal
Genetics Stock Center) were fused together into one construct by fusion PCR using
nested primers, and the mutation was selected for by growth on media containing
100 μL/mL hygromycin. Diagnostic PCR was performed on the deletant strain
using external primers (P1 and P6) from the first round of PCR. The difference in
size between the gene replaced by the selection marker and the native gene allowed
us to determine whether the transformants carried the correct gene replacement.
Next, an auxotrophic mutant in the kusA- background was generated by
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7.5. Materials and Methods A. Błachowicz
deleting the pyrG gene. Two 1500 base pair fragments upstream and downstream
of pyrG were amplified and fused together. The mutation was selected for by
growth on media supplemented with 1.5 mg/mL of 5-fluoroorotic acid (5-FOA), as
only cells lacking the pyrG gene can survive when 5-FOA is present. The correct
transformants were identified by performing diagnostic PCR on the deletant strain
using external primers (P1 and P6) from the first round of PCR. All other deletant
strains were generated by replacing each target gene with the A. fumigatus pyrG
gene (AfpyrG). Double deletion mutants were generated by recycling the AfpyrG
gene in CW12006. This involved amplifying two 1500 base pair fragments
upstream and downstream from the alba::AfpyrG region of JSC-093350089 albA
genome, which were then fused together. The mutation was selected for by growth
on media supplemented with 1.5 mg/mL of 5-fluoroorotic acid (5-FOA). Correct
transformants were identified using diagnostic PCR with external and internal
primers (P1 and AfpyrG rev; AfpyrG Fw and P6).
To reintegrate the kusA gene into the genome of CW12005, the AfpyrG gene
used to initially delete pyrA was deleted, which generated CW12014. The kusA
genewasamplifiedfromJSC-093350089gDNAtoinclude 1500basepairfragment
upstream and 500 bp fragment downstream from kusA. This fragment was then
fused to the AfpyrG gene and the original 3’ region amplified for initial kusA
deletion. The kusA was reintegrated into the genome of CW12014 to generate
CW12015. Correct transformants were identified using diagnostic PCR with
external primers (P1 and P6).
7.5.3 Radiation resistance analysis
Radiation resistance was assessed using JSC-093350089 WT and CW12015.
Both strains were cultivated at 28C on GMM agar plates by seeding 10
7
spores
per Petri dish (D = 10 cm). Spores were collected after 5 days of growth and
counted. An equal amount of spores were resuspended in 5 mL of GMM agar and
poured onto Petri dishes consisting of 20 mL GMM agar. Mycelia-containing plates
were exposed to varying doses of UV-C radiation in triplicate using a CL-1000
Ultraviolet Crosslinker (UVP, Inc.).
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Chapter8
International Space Station
conditions alter genomics,
proteomics, and metabolomics in
Aspergillus nidulans
1
8.1 Abstract
The first global genomic, proteomic, and secondary metabolomic characteri-
zation of the filamentous fungus Aspergillus nidulans following growth onboard
the International Space Station (ISS) is reported. The investigation included the
A. nidulans wild-type and three mutant strains, two of which were genetically
engineered to enhance secondary metabolite production. Whole genome sequencing
revealed that ISS conditions altered the A. nidulans genome in specific regions. In
strain CW12001, which features overexpression of the secondary metabolite global
regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential
expression of proteins involved in stress response, carbohydrate metabolic pro-
cesses, and secondary metabolite biosynthesis was also observed. ISS conditions
significantly decreased prenyl xanthone production in the wild-type strain and
1
Romsdahl, Blachowicz et al., Applied Microbiology and Biotechnology, 2018; The author
helped with sample processing for genomic, metabolomic and proteomic analyses and preparation
of the figures presenting proteomic and metabolomic results.
8.2. Introduction A. Błachowicz
increased asperthecin production in LO1362 and CW12001, which are deficient in
a major DNA repair mechanism. These data provide valuable insights into the
adaptation mechanism of A. nidulans to spacecraft environments.
8.2 Introduction
It has been well documented that fungal populations persist in extreme condi-
tions, such as various temperatures and pH [5, 2, 4, 3, 1], desiccation [6], ionizing
radiation [7, 357], Mars-like conditions [358, 359, 360], and the spacecraft environ-
ment [9, 8]. Through adaptation, fungi have developed the capacity to sense and
respond to external stimuli, enabling their survival in a wide variety of ecological
niches [2, 9, 7, 1, 8]. These omnipresent microorganisms can be both beneficial
and detrimental to human health, as fungi produce a myriad of secondary metabo-
lites (SMs) in response to environmental stressors with activities ranging from
therapeutic to toxic [150, 296, 149]. SMs have had a tremendous impact on human
health, as the majority of small molecule drugs introduced between 1981 and 2010
were either SMs, SM derivatives, SM mimics, or possessed a SM pharmacophore.
In fact, approximately 49% of all anticancer drugs are SMs or were inspired by
SMs [296]. Fungi are also potent producers of enzymes and therefore have various
industrial applications [361, 362]. We are on the cusp of significant advances in
human interplanetary space exploration, as the National Aeronautics and Space
Administration (NASA) aims to send humans back to the Moon and subsequently
to Mars in the 2030s. In entering this new era of human spaceflight, a thorough
understanding of how fungi respond and adapt to the various stimuli encountered
while in space will play an important role in ensuring a healthy living environment
for crew members. Further, such studies enable the evaluation of fungi as drug
production hosts during these exploration class missions, as fungi currently play
an indispensable role in pharmaceutical biotechnology on Earth.
Fungi residing onboard the International Space Station (ISS) are exposed
to microgravity and increased levels of radiation due to being outside of the
Earth’s protective atmosphere [297]. Microgravity is thought to decrease the
transfer of extracellular nutrients and metabolic by-products, which may alter
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8.2. Introduction A. Błachowicz
the chemical environment that the cell is exposed to [297, 363]. Radiation alters
biological processes by acting as a promoter of mutagenesis, which may result in
an increased rate of biological evolution and lead to the development of adaptive
responses [297]. Although it has been established that fungi are ubiquitous
in spacecraft environments [289, 71, 28, 79, 8], our understanding of how fungi
respond and adapt to the various conditions encountered during spaceflight remains
in its infancy [72]. The objective of this study was to investigate the changes
encountered in various aspects of fungal “omics” under ISS conditions using the well-
characterized organism Aspergillus nidulans. Of 30 distinct fungal species retrieved
from ISS habitat surfaces during one microbial monitoring study, Aspergillus was
the dominant genus, featuring a diverse population of 13 species, with A. nidulans
being one of four fungal species isolated from both surfaces and air onboard the
ISS [71]. Of particular interest is how the space environment alters secondary
metabolism, as fungal SM production is highly variable and dependent on external
stimuli [364]. A. nidulans is an extensively studied model organism and in the
last decade many of the genes and regulatory networks involved in SM formation
have been characterized [365]. This new information enabled a comprehensive
investigation into genomic, proteomic, and metabolomic alterations in response to
the space environment.
Herein, we report the multi-omic characterization of wild-type (WT) A. nidu-
lans FGSC A4 and three mutant strains, displayed in Supplemental Table S1,
following 4 and 7 days of growth onboard the ISS and compared to ground counter-
parts (Fig. 8.1). The first mutant strain, LO1362, is the A. nidulans nkuA deletion
strain, which is a homolog of the human KU70 gene. These genes are crucial for
nonhomologous end joining of DNA double strand break repair, and therefore
deletion of nkuA disrupts a major DNA repair mechanism in A. nidulans [366].
The second mutant strain, LO8158, is deficient in mcrA, which is a negative
regulator of at least 10 SM gene clusters [367]. Deletion of this gene stimulates SM
production while impairing fungal growth. The final mutant strain used in this
study was an A. nidulans laeA overexpression mutant, CW12001. LaeA is a global
positive regulator of secondary metabolism, and therefore overexpression of laeA
increases the production levels of a number of SMs [368]. ISS- and Earth-grown
- 140 -
8.3. Results A. Błachowicz
Figure 8.1: Schematic overview of the A. nidulans ISS experiment. The A. nidulans
wild-type (FGSC A4) and three mutant strains (LO1362, LO8158, and CW12001) were
seeded onto GMM agar OmniTray plates and integrated into PHAB systems. Samples
were transported to the ISS at 4

C, where they remained for approximately 26 or
23 days until being subjected to growth in SABL systems at 37

C for either 4 or
7 days, respectively. Earth-grown PHABs were simultaneously transferred to on-ground
containers mimicking ISS SABL systems. Following growth, all samples were subjected
to 4

C and ISS-grown samples were transported to Earth. All samples were subjected
to genomic, proteomic, and metabolomic analyses. Figure courtesy of Dr. J. Romsdahl.
strains were subjected to genomic, proteomic, and metabolomic analyses with the
aim of identifying genetic and molecular alterations that occur in fungi in response
to the space environment, as such findings could pave the way to new discoveries
that benefit human spaceflight and people on Earth.
8.3 Results
8.3.1 Genome variation among ISS-grown samples
To identify genomic alterations occurring in A. nidulans strains in response
to ISS conditions, whole genome paired-end sequencing (WGS) was performed
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8.3. Results A. Błachowicz
on ISS-grown samples (4- and 7-day) and ground-grown control strains (7-day).
Reads were aligned to the FGSC A4 reference genome and any SNP present in
ground controls was removed from each strain’s sample set, which resulted in the
removal of 208, 194, 231, and 224 SNPs from FGSC A4, LO1362, LO8158, and
CW12001, respectively. This revealed 129, 136, 108, and 106 SNPs and 36, 39,
41, and 31 INDELs in ISS-grown FGSC A4, LO1362, LO8158, and CW12001,
respectively, that occurred in ISS-grown samples when compared to ground controls,
the features of which are displayed in Table 8.1. The total number of missense
mutationsobservedinISS-grownstrainsrangedfrom9to15foreachstrain’ssample
set. These mutations were observed within only five protein-coding genes, with the
same mutation often present in multiple samples (Table 8.2). A total of 13 unique
missense base mutations were observed within AN5254, which encodes a protein
containing domains predicted to be involved in RNA binding and RNA-directed
DNA polymerase activity. Two unique mutations that resulted in premature stop
codonwerealsoobservedwithinAN5254, oneofwhichoccurredinboththe4-and7-
day LO8158 and CW12001 ISS-grown samples. The remaining missense mutations
occurred within AN0532, AN0535, AN0537, and AN0538, which are within a few
genes away from one another in the genome. AN0532 encodes a predicted DDE1
transposable element gene, while the products of AN05235, AN0537, and AN0538
are uncharacterized. In both CW12001 ISS-grown samples, identical mutations
that resulted in the loss of the original stop codon were observed within the laeA
(AN0807) gene. The same frameshift stop-gain mutation independently occurred
in all space-grown LO1362 and LO8158 samples within the mnpA gene (AN10311),
which encodes a hyphal cell wall mannoprotein that may influence the surface
structure [369].
For all strains, most SNPs (>77%) and INDELs (>89%) occurred in intergenic
regions. Most intergenic SNPs were clustered nearby several specific genes and
did not appear to be strain specific (data not shown). Among all strains, many
intergenic SNPs occurred near genes involved in transcription and translation,
including the putative C6 transcription factor AN4972, the transcription elon-
gation factor AN11131, the U3 small nucleolar ribonucleoprotein AN4298, the
S-adenosylmethionine-dependent methyltransferase AN10829 with a predicted
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8.3. Results A. Błachowicz
Table 8.1: Features of SNPs and INDELs
Strain ID
FGSC A4 LO1362 LO8158 W12001
No. of SNPs 129 137 108 106
Intergenic 111 116 84 88
Missense 10 11 15 9
Synonymous 4 3 6 3
Intron 0 4 2 2
UTR 1 2 0 2
Stop gained 2 1 1 1
Stop lost 0 0 0 1
No.of INDELs 36 38 41 31
Intergenic 36 34 37 29
Frameshift 0 2 1 1
UTR 0 2 3 1
Stop gained 0 1 1 0
role in translational read-through, and AN6968 which is predicted to have RNA-
directed DNA polymerase activity. Intergenic SNPs were also clustered nearby
the putative alanine-tRNA ligase AN9419, the putative C4 sterol methyl oxidase
AN6973 which has a predicted role in sterol metabolism, and AN9410 which has a
predicted role in lipid metabolic processes. Intergenic SNPs were also observed
near AN0538 and AN0539, which are within the uncharacterized cluster of genes
that were reported above to possess high numbers of missense mutations. Most
of the remaining intergenic SNPs occurred near AN6972, AN7848, AN10328, and
AN11577, the products of which remain uncharacterized.
8.3.2 Proteomic profiling of ISS-grown A. nidulans
To investigate alterations in the proteome of A. nidulans strains following
growth on the ISS, total protein was extracted from two biological replicates of
each ISS-grown sample and Earth-grown counterpart. All samples were subjected
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8.3. Results A. Błachowicz
Table 8.2: Comparative analysis of nonsynonymous SNPs occurring during spaceflight
Gene Base mutation Mutation type FGSC A4 LO1362 LO8158 CW12001
4D 7D 4D 7D 4D 7D 4D 7D
AN5254 ChrV_A3367369G Missense + + + +
ChrV_C3367409T Missense + +
ChrV_A3367453G Missense +
ChrV_C3367733T Missense +
ChrV_G3367916A Missense + +
ChrV_T3367958C Missense + +
ChrV_C3367973T Missense + +
ChrV_C3368005T Missense + + + +
ChrV_C3368023T Stop gained + + + +
ChrV_T3368023C Stop gained +
ChrV_A3368024G Missense + + + +
ChrV_G3368024A Missense +
ChrV_T3368096C Missense + +
ChrV_T3368312C Missense + +
ChrV_C3368312T Missense +
AN0807 ChrVIII_G2423110A Stop lost + +
AN0538 ChrVIII_A3254138C Missense +
ChrVIII_T3254236C Missense +
ChrVIII_C3254236T Missense + +
AN0537 ChrVIII_A3255566G Missense +
ChrVIII_C3255576T Missense +
ChrVIII_T3255576C Missense +
ChrVIII_G3255581A Missense +
ChrVIII_A3255581G Missense +
ChrVIII_T3255781C Missense + + + +
ChrVIII_C3255975A Splice region + +
ChrVIII_G3256068A Missense +
ChrVIII_A3256068G Missense +
AN0535 ChrVIII_A3259230G Stop gained + + +
ChrVIII_A3259256G Missense + + +
ChrVIII_C3259257T Missense + + +
ChrVIII_T3259346C Missense +
ChrVIII_C3259346T Missense + +
ChrVIII_G3259467C Missense + + +
ChrVIII_G3259508C Missense + +
ChrVIII_C3259563T Missense +
AN0532 ChrV_C3267200T Missense +
ChrV_T3267230C Missense + + + +
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8.3. Results A. Błachowicz
to TMT labeling and LC-MS analysis. The resulting MS data were analyzed using
Proteome Discoverer with the Sequest-HT search engine against the A. nidulans
FGSCA4proteindatabase(NCBI).TheabundanceratiosforallISS-grownsamples
were normalized to their Earth-grown counterparts, which led to the identification
of up- and downregulated proteins (fold change (FC) >j2jº;P < 0:05º in response
to the ISS environment (Fig. 2a). Interestingly, only two proteins, QutC (AN1140),
which is involved in quinic acid utilization, and AN2704, a putative aryl-alcohol
oxidase-related protein, were upregulated in the A. nidulans WT strain. The
number of proteins upregulated and downregulated in the three ISS-grown mutant
strains ranged from 4 to 28 and 2 to 77, respectively, of 10,525 predicted proteins
in A. nidulans FGSC A4 in total. Distribution of AspGD Gene Ontology (GO)
Slim terms [370] among the differentially expressed proteins for ISS-grown mutant
strains is displayed in Fig. 8.2b–d. The GO Slim categories that possessed the
highest number of differentially expressed proteins in LO1362 were stress response
and carbohydrate metabolic processes. Similarly, in both LO8158 and CW12001,
most differentially expressed proteins were involved in carbohydrate metabolism.
Our study revealed differential abundance of proteins involved in the A. nidu-
lans stress response following growth on the ISS (Table 8.3). The heat shock
protein Hsp20 (AN10507) was highly affected by ISS conditions, displaying a 2-
and 4-fold increase in protein abundance in LO1362 and CW12001, respectively,
and a 2-fold decrease in protein abundance in LO8158. Induction of Hsp20 has
been reported following exposure to osmotic stress in A. nidulans [371]. Conversely,
the osmotic stress response protein CipB (AN7895) was downregulated in LO1362,
and the chaperone/heat shock protein Awh11 (AN3725) was downregulated in
LO1362 and LO8158. Differential abundance of proteins involved in oxidative
stress response was observed among all ISS-grown strains relative to Earth-grown
counterparts. The glutathione S-transferase GstB (AN6024), which has been
reported to be significantly induced in response to menadione-induced oxidative
stress [372], was upregulated over 2-fold in LO8158 but downregulated 1.7- and
1.8-fold in LO1362 and CW12001, respectively, after 7 days of growth on the
ISS. Two catalases, CatA (AN8637) and AN8553, exhibited decreased protein
abundance in LO8158 and increased protein abundance in CW12001. Similarly,
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8.3. Results A. Błachowicz
Figure 8.2: Overview of proteomic analysis. a. Number of up- and downregulated
proteins in ISS-grown strains (FC >j2j, P < 0:05) compared to ground-grown counter-
parts. b–d. Biological process GO Slim categories of differentially expressed proteins.
Differentially expressed proteins in b. LO1362, c. LO8158, and d. CW12001 were
mapped to terms representing various biological processes using AspGD Gene Ontology
(GO) Slim Mapper. The author helped with the proteome analysis and figure preparation.
both the nitrosative stress response protein AN2470 and the menadione stress-
induced protein AN5564 were downregulated approximately 3-fold in LO8158 and
upregulated approximately 1.7-fold in CW12001. Upregulation was observed for
phosphatidylinositol phospholipase C AN3636 whose ortholog plays a major role
in responding to nutrient deprivation in Candida albicans [373]. Additionally, the
GPI-anchored protein EcmA (AN4390), whose ortholog plays a major role in cell
wall integrity, morphogenesis, and virulence, was upregulated 2-fold in LO8158
after 7 days of growth on the ISS [374].
Among all strains, proteins involved in secondary metabolism were differentially
expressed in response to ISS conditions (Table 8.3). The polyketide synthase AptA
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8.3. Results A. Błachowicz
Table 8.3: Differentially expressed proteins by strain and biological process
Biological ORF Protein FGSC A4 LO1362 LO8158 CW12001
process 4D 7D 4D 7D 4D 7D 4D 7D
Response AN2470 0:04 0:01 0:72 0:51 0:42 1:56 0:24 0:82
to stress AN3636 0:10 1:02 0:09 0:42
AN4891 Asf1 0:42 1:05 0:33 0:22
AN5564 0:15 0:08 0:04 0:14 0:07 1:49 0:12 0:75
AN3725 Awh11 0:04 0:16 1:27 0:68 0:43 1:07 0:22 0:02
AN8637 CatA 0:21 0:24 0:64 0:41 0:35 1:65 0:31 0:90
AN8553 0:24 0:36 0:30 0:03 0:04 1:53 0:00 0:68
AN7895 CipB 0:02 0:09 0:65 1:21 0:25 0:72 0:39 0:53
AN4390 EcmA 0:13 0:24 0:38 0:17 0:30 1:23 0:02 0:72
AN1216 GppA 0:02 0:11 1:07 0:33 0:15 0:69 0:33 0:38
AN6024 GstB 0:04 0:10 0:81 0:75 0:11 1:26 0:19 0:85
AN10507 Hsp20 0:60 0:69 1:03 0:99 0:48 1:38 0:99 2:20
AN5217 PilA 0:30 0:01 0:31 0:41 0:00 1:04 0:05 0:75
Secondary AN6000 AptA 0:02 0:90 0:24 0:42 0:25 0:61 0:15 1:06
metabolism AN0147 MdpD 0:11 1:24 0:04 0:37
AN10038 MdpJ 0:23 0:89 0:19 1:06
AN7911 OrsB 0:24 0:01 0:66 0:72 1:01 0:39 0:03 0:61
AN7812 StcN 0:05 0:07 0:69 0:74 0:19 0:96 0:18 1:15
AN7817 0:43 0:37 0:32 0:32 1:24 0:07 0:22 0:33
Carbohydrate AN0567 0:08 0:23 0:51 0:62 0:24 1:37 0:41 0:65
metabolism AN10124 0:37 0:44 0:71 0:72 1:14 0:32 0:15 0:13
AN1715 0:18 0:16 0:52 0:39 0:20 1:49 0:26 0:92
AN2334 0:05 0:08 0:01 0:49 0:05 1:13 0:11 0:69
AN6035 0:20 0:72 0:68 0:55 1:58 0:17 0:85
AN8068 0:32 0:25 0:91 0:79 0:11 1:53 0:05 1:03
AN9443 0:55 0:32 1:14 0:58
AN1277 AbfC 0:20 0:04 0:76 0:72 0:22 1:09 0:05 0:71
AN5634 AcuD 0:17 0:03 0:19 0:09 0:02 1:57 0:30 0:91
AN1918 AcuF 0:22 0:26 0:03 0:16 0:01 1:38 0:17 0:90
AN7345 AgdC 0:09 0:13 0:38 0:52 1:63 0:14 0:49 0:10
AN7396 BglM 0:19 0:04 0:72 0:56 0:29 1:33 0:21 0:46
AN0494 CbhB 0:55 0:07 1:02 1:13 0:52 0:43 0:22 1:25
AN6792 GfdB 0:03 0:02 0:46 0:21 0:26 1:35 0:07 1:03
AN0756 LacA 0:09 0:31 0:16 0:30 0:56 1:09 0:15 1:05
AN3368 MndB 0:13 0:55 0:74 0:82 1:00 0:38 0:44 0:07
AN7349 MutA 0:28 0:16 0:50 0:13 1:26 0:51 0:43 0:22
AN7135 RglA 0:33 0:35 0:68 0:60 0:32 1:48 0:06 1:08
AN5061 XgeB 0:22 1:12 0:07 0:03
AN1818 XlnC 0:11 0:11 0:85 0:82 0:08 1:01 0:32 0:88
AN7401 XlnE 0:12 1:10 0:36 0:11
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8.3. Results A. Błachowicz
(AN6000), which is required for biosynthesis of the spore pigment asperthecin [375],
was upregulated over 2-fold in CW12001 and nearly 2-fold in FGSC A4 strain after
7 days of growth on the ISS. LO1362 also exhibited upregulation of the monooxyge-
nase MdpD (AN0147) after 7 days of growth. The MdpD protein is the product of
a gene in the prenyl xanthone gene cluster, which is responsible for the production
of monodictyphenone, emericellin, shamixanthone, and epishamixanthone [376,
377]. Interestingly, the opposite was observed in CW12001, as MdpJ (AN10038),
which is the product of another gene in the prenyl xanthone gene cluster, exhibited
decreased protein abundance in ISS-grown samples after 4 days. A similar trend
was observed for proteins involved in the biosynthesis of the potent carcinogenic
mycotoxin sterigmatocystin (ST) [378, 379]. ST gene cluster products AN7817
and StcN (AN7812) exhibited increased protein abundance in LO8158, while StcN
was downregulated in CW12001, following growth in ISS conditions.
The proteome of ISS-grown A. nidulans samples also revealed differential levels
of proteins involved in carbohydrate metabolism when compared to Earth-grown
controls (Table 8.3). Many glycoside hydrolases involved in carbohydrate degrada-
tion processes were upregulated more than 2-fold in ISS-grown LO8158 samples,
including beta-1,4-endoglucanase AN8068 and beta-glucosidase BglM (AN7396),
both of which are involved in cellulose degradation; alpha-arabinofuranosidase
AbfC (AN1277), involved in pectin degradation; endo-1,4-beta-xylanase XlnC
(AN1818), involved in xylan degradation; and beta-galactosidase LacA (AN0756),
involved in xyloglucan, xylan, pectin, and galactomannan degradation. These
glycoside hydrolases also exhibited decreased protein abundance in CW12001
when compared to ground counterparts. A similar trend was observed with alco-
hol oxidase AN0567, beta-glycosidase AN10124, ketose-1,6-bisphosphate aldolase
AN2334, dehydratase AN6035, and rhamnogalacturonan lyase RglA (AN7135),
which were upregulated at least 2-fold in ISS-grown LO8158 samples and downreg-
ulated at least 1.5-fold in CW12001. Conversely, mannose-6-phosphate isomerase
AN1715, isocitrate lyase AcuD (AN5634), phosphoenolpyruvate carboxykinase
AcuF (AN1918), and NAD+ dependent glycerol 3-phosphate dehydrogenase GfdB
(AN6792) were downregulated at least 2.5-fold in ISS-grown LO8158 samples and
upregulated at least 1.8-fold in CW12001 samples.
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8.4. Secondary metabolome alterations in ISS-grown A. nidulans A. Błachowicz
8.4 SecondarymetabolomealterationsinISS-grownA.nidu-
lans
Alterations in SM production of A. nidulans in response to ISS conditions
were assessed by extracting SMs from three biological replicates of each ISS- and
Earth-grown counterpart, which were analyzed using HPLC-DAD-MS. All SMs
were identified based on mass, UV absorption, and retention time, which led
to the identification of austinol and dehydroaustinol [380], terrequinone [381],
sterigmatocystin and its intermediate [382], nidulanin A and its analogues [383],
emericellamides [384], prenyl xanthones [377], and asperthecin [343] (Fig. 8.4).
Relative differences in SM production levels of ISS-grown samples and Earth-
grown counterparts were quantified by integrating the area under each SM’s EIC
trace (Fig. 8.5). ISS conditions induced asperthecin production in LO1362 and
CW12001 after 7 days of growth, with production levels increased by over 300
and 150%, respectively (Fig. 8.3). Production levels of prenyl xanthones decreased
approximately 5-fold in FGSC A4 ISS-grown samples (Fig. 8.5f). In LO8158
samples, emericellamide and terrequinone production decreased (Fig. 8.5b, e),
while nidulanin and sterigmatocystin production increased in ISS-grown samples
(Fig 8.5c, d).
8.5 Discussion
Although the persistence of fungi within space vessels is well documented and
unavoidable [77, 71, 8], little is understood about how fungi respond and adapt
to spacecraft conditions, such as microgravity and enhanced radiation. To date,
most microbiological studies conducted in such environments have focused on
changes occurring within bacteria or the microbiome as a whole [293, 292, 8, 38,
363]. Additionally, despite the various therapeutic and industrial applications
of SMs, few studies have analyzed the global influence of space conditions on
fungal secondary metabolism, as previous investigations have often focused on the
production of a single SM [385, 386, 30]. Therefore, with the duration of space
missions expected to increase, a major goal of this study was to investigate space-
induced alterations in fungal “omics” to identify specific adaptation biomarkers
- 149 -
8.5. Discussion A. Błachowicz
nkuAΔ 4d
nkuAΔ 7d
oe:LaeA 4d
oe:LaeA 7d
0
100
200
300
400
Asperthecin (no cellophane)
% of production of control
Control
Space
*
**
HO
OH O
O OH
OH
OH
OH
nkuAΔ 4d
nkuAΔ 7d
oe:LaeA 4d
oe:LaeA 7d
0
100
200
300
400
Asperthecin (no cellophane)
% of production of control
Control
Space
*
** Control
ISS-grown
LO1362 control
LO1362 ISS-grown
CW12001 control
CW12001 ISS-grown
a b
c
100
200
300
400
Percent of ground control
10 15 20 25 30 35 40 45 50
Time (min)
Figure 8.3: a. LC-MS profiles depicting asperthecin production after 7 days of growth
on the ISS, as detected by UV total scan. b. Quantification of asperthecin production
showing percent change for ISS-grown samples relative to Earth-grown counterparts.
Significance was determined using Welch’s t test. c. Chemical structure of asperthecin.
The author helped with the SM analysis and figure preparation.
and acquire insight into potential benefits of fungi that may not be discernable
using traditional methodology.
This study revealed that the spacecraft environment alters the A. nidulans
genome in specific regions. Five protein-coding genes displayed signatures of
positive selection in the form of a high ratio of nonsynonymous to synonymous
SNPs across all ISS-grown samples. These data stand in agreement with a study
that investigated genomic alterations occurring in the bacterium Staphylococcus
aureus during spaceflight, in which missense mutations were clustered and oc-
curred within only nine protein-coding genes [282]. High numbers of intergenic
mutations were clustered near genes encoding transcriptional and translational
machinery. One gene with several nonsynonymous and two unique stop-gain muta-
tions encodes a putative retrotransposon, suggesting that its suppression confers
selective advantage during growth in spacecraft environments. Other missense
and intergenic mutations were clustered within and around a specific region of
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8.5. Discussion A. Błachowicz
Figure 8.4: LC-MS profile of SMs identified in A. nidulans strains following growth on
GMM for 7 days at 37

C, as detected by UV total scan. The author helped with the SM
analysis and figure preparation.
the genome (AN0532–AN0538), suggesting that it underwent positive selection
and therefore plays a role in adapting to the space environment. One of these
genes also encoded an uncharacterized transposable element gene, underscoring
the significance of alterations in transposable element activity in response to
growth in ISS conditions. These findings are consistent with the results from
the aforementioned S. aureus study, as variations were also observed within a
putative transposase [282]. Interestingly, transposable element activity has been
associated with stress response due to the novel variation it introduces into the
genome [283]. Future work should focus on characterizing the activities of the rest
of these genes, as many of their functions remain unknown. Such knowledge may
provide information key to elucidating the genetic adaptation mechanisms of fungi
residing in spacecraft environments.
Correlations linking genomic and proteomic data were observed with the
two strains genetically engineered to increase SM production. Exposure to the
space environment appeared to curtail enhanced SM production in the laeA
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8.5. Discussion A. Błachowicz
Figure 8.5: Secondary metabolite quantification showing percent change for ISS-grown
samples in relation to Earth-grown counterparts for a. austinol and dehydroaustinol,
b. terrequinone, c. sterigmatocystin, d. nidulanins, e. emericellamides, and f. prenyl
xanthones. Significance was determined using Welch’s t-test. The author helped with
the SM analysis and figure preparation.
overexpression strain through the introduction of a point mutation that resulted in
loss of the laeA stop codon, thereby activating nonstop decay degradation of laeA
mRNA [387, 388]. The ISS-induced downregulation of laeA was also observed in
the proteome of those samples, which exhibited an expression profile opposite that
of the mcrA deletion strain for many proteins. Proteins involved in SM biosynthesis
pathways regulated by laeA exhibited decreased and increased abundance in ISS-
grown CW12001 and LO8158, respectively. The extensively studied protein LaeA
forms a nuclear complex with VeA and VelB that coordinates secondary metabolism
regulation with fungal development [389]. LaeA is often referred to as a global
- 152 -
8.5. Discussion A. Błachowicz
regulator of secondary metabolism [390] and has been reported to also influence
proteins involved in carbohydrate metabolism and oxidative stress response [391].
Accordingly, several proteins involved in carbohydrate and antioxidant metabolic
processes exhibited opposite protein expression profiles in LO8158 and CW12001.
Regulation by laeA has been reported for some of these proteins in Aspergillus
flavus, including endo-1,4-beta-xylanase, beta-glycoside, endo-beta-1,4-glucanase,
and oxidative stress response proteins CatA and GstB [391]. These findings
verify the extensive regulatory role of laeA and highlight the complexity involved
in identifying the laeA-controlled processes responsible for conferring selective
advantage of the observed laeA stop lost mutation. This is compounded by
reports that laeA also alters chromatin remodeling, cell growth and metabolism,
conidiogenesis, conidial-chain elongation, sporulation, pigmentation, and colony
hydrophobicity in various fungal species [392, 393, 391, 59]. It is also possible
that curtailing laeA overexpression is favored in ISS conditions to reduce energy
expenditures in a stressful environment.
ISSconditionssignificantlyincreasedproductionofasperthecin,ananthraquinone
pigment, in LO1362 and CW12001. Both mutants are deficient in NkuA pro-
duction, which facilitates nonhomologous end-joining (NHEJ) DNA repair, the
favored DNA double strand break repair pathway in filamentous fungi [366]. We
had anticipated that NkuA-deficient strains would be particularly interesting in
flight studies due to impairment of a preferred DNA repair pathway in a muta-
genic, high-radiation environment. Additionally, deletion of the nkuA homolog has
been reported to increase sensitivity toward gamma irradiation in other fungal
species [347]. Although we cannot discriminate the specific ISS condition that
induced asperthecin production in LO1362, we hypothesize that it served as an
alternative protective mechanism from the high levels of radiation present on the
ISS. Our observation stays in agreement with other reports suggesting that pigment
production is a key adaptive response of fungi exposed to similar environments [394,
53, 7]. It is therefore possible that asperthecin can be used to protect other forms
of life present on the spacecraft.
Cultivating plants for food in space will be crucial for the success of future
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8.5. Discussion A. Błachowicz
space missions. However, space radiation can generate mutations in plant DNA,
including base substitutions, deletions, and chromosomal alterations, which can
result in genetic changes in seeds or tissue damage [395, 355]. The transformation
of asperthecin biosynthesis genes into plants may potentially minimize plant
DNA damage and optimize plant and astronaut health. Future studies should be
conductedbothonthegroundandontheISStoverifythishypothesis. Interestingly,
space conditions did not increase asperthecin production in LO8158, which also
possesses the nkuA- genetic background. Global regulation of SM production was
altered to increase SM production in both LO8158 and CW12001, but the genetic
alteration was reversed through a stop lost point mutation only in CW12001. It
is therefore possible that alternative metabolomic protective mechanisms were
sufficient in LO8158, and therefore, asperthecin production was not enhanced in
ISS conditions.
Only a small proportion of proteins were differentially expressed in ISS-grown
A. nidulans samples, which emphasizes the potential and safety of A. nidulans as a
therapeutic production host during outer space missions. This finding may not hold
true across the Aspergillus genus, as increased virulence has been reported in As-
pergillus fumigatus strains isolated from the ISS [155]. Currently, if pharmaceutical
stocks in space are depleted, a small, unmanned spacecraft is launched to restock
crew supplies. In the era of long-term space travel, the duration of future space
missions is expected to drastically increase. The inability to deliver required drugs
to astronauts in a timely manner could result in serious complications. Through
heterologous expression of specific genes, A. nidulans introduces the ability to
biosynthesize a wide range of pharmaceutical drugs within a week, which could
significantly improve astronauts’ safety during long-term manned space missions.
In summary, this work has revealed the multi-omic response of the well-
characterized model filamentous fungus A. nidulans to spacecraft conditions.
These findings illustrate the potential of asperthecin to confer radiation resistance
and of A. nidulans to be utilized as a small molecule production host in space.
Further, specific genetic mutations involved in the adaptive mechanism of fungi in
space environments were identified. Such knowledge may be valuable to NASA’s
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8.6. Materials and Methods A. Błachowicz
Space Biology Program in planning for future outer space explorations.
8.6 Materials and Methods
8.6.1 Strains, media, and growth conditions
The WT A. nidulans FGSC A4 strain was obtained from the Fungal Genetics
Stock Center [396]. LO1362 and LO8158 were obtained from previous studies [367].
The laeA overexpression strain (CW12001) was generated using the constitutive
gpdA promoter according to standard protocol [397]. Protoplast production,
construction of fusion PCR products, and transformation were carried out as
described previously [398, 399]. Primers used are listed in Table S2 and correct
transformants were verified using diagnostic PCR.
All strains were seeded onto Nunc OmniTray plates (Thermo Fisher Scientific,
Waltham, MA, USA) containing 46 mL of solid GMM media (6 g/L NaNO
3
,
0.52 g/L KCl, 0.52 g/L MgSO
4
7H
2
O, 1.52 g/L KH
2
PO
4
, 10 g/L D-glucose,
15 g/L agar supplemented with 1 mL/L of Hutner’s trace elements). Seeded
Nunc OmniTrays were loaded into Plate Habitat (PHAB) systems (BioServe Space
Technologies, Boulder, CO, USA), with six OmniTray plates in each PHAB, and
immediately transferred to 4

C. The PHAB system is a growth platform for
biological materials that allows for gas exchange. Each PHAB was equipped with a
temperature logger (HOBO) that accurately measured the temperature throughout
the duration of the experiment. PHABs containing cultures bound for the ISS were
transferred to 4

C cold bags approximately 28 hr prior to launch and transported
to the ISS on the SpaceX CRS-8 (Space Exploration Technologies) mission that
launched on April 8, 2016. On the ISS, PHABs were loaded into Space Automated
Bioproduct Lab (SABL) systems, where the 7- and 4-day samples remained at 4

C
for approximately 23 and 26 days, respectively. Ground culture PHABs were near-
synchronously transferred to on-ground containers mimicking ISS SABL systems.
To initiate growth, ISS and ground cultures were subjected to 37

C, where they
remained for either 4 or 7 days, which are optimal growth conditions for SM
production in A. nidulans. Following growth, all samples were subjected to 4

C,
where they remained until ISS-grown agar plates were transported back to Earth
- 155 -
8.6. Materials and Methods A. Błachowicz
on May 11, 2016. A more detailed explanation of space flight hardware, science
preparation and loading, the flight operation timeline, and ISS environmental
parameters can be found in the Supplemental Methods. All subsequent analyses
of ISS- and ground-grown fungal samples, including genomic, proteomic, and
metabolomic characterization, were conducted on Earth.
8.6.2 Genomic DNA extraction, library preparation, and genome se-
quencing
Mycelia were collected from Earth-grown (7-day) and space-grown (4- and
7-day) GMM agar OmniTrays for all strains (FGSC A4, LO1362, LO8158, and
CW12001), frozen with liquid nitrogen, and ground using a mortar and pestle.
DNA was extracted using the Mo Bio PowerMax Soil DNA Isolation Kit (Mo
Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Library preparation and whole genome sequencing were performed at the Duke
Center for Genomic and Computational Biology (Duke University Medical Center,
Durham, NC, USA). DNA quality and quantity were checked using the Agilent 2100
Bioanalyzer DNA assay (Agilent Technologies, Santa Clara, CA, USA) and Qubit
2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The library
was prepared for paired-end sequencing using the TruSeq Nano DNA Library
Preparation Kit (Illumina, San Diego, CA, USA). Samples were sequenced using a
HiSeq 4000 Illumina Sequencer and 101 base read lengths were generated.
8.6.3 Genetic mutation identification
Illumina sequence reads were trimmed using Trimmomatic v 0.36 [400] and
quality was checked using FastQC v 0.11.7. The genome and annotation files for
A. nidulans FGSC A4 [401] were downloaded from the FungiDB web portal [157].
Reads were mapped to the FGSC A4 reference genome using the Burrows-Wheeler
Aligner(BWA)softwarepackagev0.7.12[286]andfurtherprocessedwithSAMtools
v 1.6 to generate sorted BAM files [129]. Single nucleotide polymorphisms (SNPs)
and insertion/deletion mutations (INDELs) were identified using GATK v 3.7 [288].
Duplicates were marked using Picard tools Picard-tools MarkDuplicates to remove
PCR artifacts. Sequence reads containing putative INDELs were realigned using
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8.6. Materials and Methods A. Błachowicz
GATK’s IndelRealigner to generate an updated BAM file. Variants within each
sample were called using GATK’s Haplotype Caller, and the resulting Variant Call
Format (VCF) files were combined using GATK’s Genotype GVCFs so that there
was one VCF file for each strain (four in total). GATK’s VariantFiltration was used
to filter each VCF file based on stringent cutoffs for quality and coverage {SNPs:
QD < 2:0, MQ < 40:0, QUAL < 100, FS > 60:0, MQRankSum <12:5, SOR >
4:0, ReadPosRankSum <8:0; INDELs: QD < 2:0, FS > 200:0, MQRankSum <
12:5, SOR > 4, InbreedingCoef f <0:8, ReadPosRankSum <20:0}, so that
only high-quality variants remained.
8.6.4 Protein extraction
Mycelia were collected from Earth-grown (7-day) and space-grown (4- and
7-day) GMM agar OmniTrays for all strains (FGSC A4, LO1362, LO8158, and
CW12001), frozen with liquid nitrogen, and ground using a mortar and pestle.
For protein extraction, the lysis buffer consisted of 100 mM triethylammonium
bicarbonate (TEAB) with 1 Halt Protease Inhibitor Cocktail (100) (Thermo
Fisher Scientific, Waltham, MA, USA) and 1 mM phenylmethylsulfonyl fluoride
(Sigma-Aldrich, St. Louis, MO, USA). The frozen ground mycelia were transferred
and subjected to a Precellys 24 homogenizer (Bertin Instruments, Rockville, MD,
USA) in which each sample was processed inside a 2 mL cryotube with 1.0 mm
glass beads for three times (at 4

C, 6500 rpm, 1 min, repeated three times with
15 s pauses in between). The lysed cells were centrifuged at 17,000 G for 15 min.
Protein concentrations in the supernatants were measured using the Bradford
assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
8.6.5 Tandem mass tag labeling
About200μgproteinsfromeachsamplewereprecipitatedin20%trichloroacetic
acid (TCA) at 4

C. Protein pellets were obtained by centrifugation (17,000 G),
washed with ice-cold acetone, and resuspended in 25 μL TEAB (100 mM) and
25 μL 2,2,2-trifluoroethanol (TFE). Proteins were reduced by adding 1 μL of
tris(2-carboxyethyl) phosphine (TCEP, 500 mM) and incubated for 1 hr at 37

C
- 157 -
8.6. Materials and Methods A. Błachowicz
(10 mM final TCEP concentration). Proteins were alkylated in the presence of
iodoacetamide (IAA, 30 mM) in the dark for 1 hr at room temperature, and 2.5 μg
per sample of mass spec grade trypsin/lysC (Promega, Madison, WI, USA) was
used to digest the peptides overnight at 37

C.
The digested peptides were quantified using the Pierce Quantitative Colori-
metric Peptide Assay (Thermo Fisher Scientific, Waltham, MA, USA). Forty
micrograms of peptides from each sample were labeled with the Thermo Scientific
Tandem Mass Tag 6-plex (TMT
6
) Isobaric Mass Tagging Kit according to the
manufacturer’s protocol. The TMT
6
-130 label was used as either the 4- or 7-day
strains’ reference that contained 5 μg of peptides from each of the eight strains.
The TMT
6
-131 label was used as a total mixture reference that contained 2.5 μg
of peptides from each of the 16 strains. All six labeled peptide mixtures were
combined into a single tube, mixed, and fractionated using the Pierce High pH
Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Waltham,
MA, USA). While this kit usually uses only eight fractions with step elution of up
to 50% acetonitrile, we added a ninth fraction eluting at 100% acetonitrile. Nine
fractionated samples were dried using a SpeedVac concentrator and resuspended
in 1% formic acid prior to LC-MS/MS analysis.
8.6.6 LC-MS/MS analysis
The samples were analyzed on an Orbitrap Fusion Tribrid mass spectrometer
with an EASY-nLC 1000 Liquid Chromatograph, a 75 μm by 2 cm Acclaim
PepMap100 C
18
trapping column, a 75μm by 25 cm PepMap RSLC C
18
analytical
column, and an Easy-Spray ion source (Thermo Fisher Scientific, Waltham, MA,
USA). The column temperature was maintained at 45

C and the peptides were
eluted at a flow rate of 300 nL/min over a 110 min gradient, from 3 to 30%
solvent B (100 min), 30–50% solvent B (3 min), 50–90% solvent B (2 min), and
90% solvent B (2 min). Solvent A was 0.1% formic acid in water and solvent B
was 0.1% formic acid in acetonitrile.
The full MS survey scan (m/z 400 to 1,500) was acquired in the Orbitrap at
a resolution of 120,000 and an automatic gain control (AGC) target of 210
5
.
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8.6. Materials and Methods A. Błachowicz
The maximum injection time for MS scans was 50 ms. Monoisotopic precursor
ions were selected with charge states 2-7, with a10 ppm mass window, and 70 s
dynamic exclusion. The MS
2
scan (m/z 400 to 2,000) was performed using the
linear ion trap with the collision-induced dissociation (CID) collision energy set to
35%. The ion trap scan rate was set to “rapid,” with an AGC target of 410
3
and
a maximum injection time of 150 ms. Ten fragment ions from each MS
2
experiment
were subsequently selected for an MS
3
experiment. The MS
3
scan (m/z 100 to
500) was performed to generate the TMT reporter ions in the linear ion trap using
heated capillary dissociation (HCD) at a collision energy setting of 55%, a rapid
scan rate and an AGC target of 510
3
, and a maximum injection time of 250 ms.
8.6.7 Quantitative proteomics analysis
All MS/MS spectra were analyzed using the Proteome Discoverer v 2.2.0.388
(Thermo Fisher Scientific, Waltham, MA, USA) with the Sequest-HT searching
engines against an Aspergillus nidulans FGSC A4 database containing 10,525
protein group sequences based on the annotated genome (NCBI, BioProject PR-
JEA40559, Assembly GCA_000011425.1, Release date 2009 September 24). The
search was performed with the following parameters: 2 maximum missed cleavage
sites, 6 minimum peptide length, 5 ppm tolerance for precursor ion masses, and
0.6 Da tolerance for fragment ion masses. The static modification settings included
carbamidomethyl of cysteine residues and dynamic modifications included oxida-
tion of methionine, TMT6plex modification of lysine -amino groups and peptide
N-termini, and acetyl modification of protein N-terminus. A false discovery rate
(FDR) of 1% for peptides and proteins was obtained using a target-decoy database
search. The reporter ion integration tolerance was 0.5 Da, while the co-isolation
threshold was 75%. The average signal-to-noise threshold of all reported peaks was
greater than 10. The quantitative abundance of each protein was determined from
the total intensity of the detected reporter ions. The ratios between the reporter
and the reference reporter ion (TMT
6
-131) were used to estimate the abundance
ratio of each protein.
For the statistical analysis, technical triplicate measurements for each protein
- 159 -
8.6. Materials and Methods A. Błachowicz
were averaged. Only proteins that were identified and quantified with at least one
peptide detected in all three technical replicates were considered for the analysis.
The normalization across two biological sample sets in eight TMT experiments was
carried out according to [402] with modifications. Briefly, the data from the eight
TMT experiments were first corrected for small systematic differences resulting
from sample loading variations and labeling efficiency by normalizing the reporter
ion totals for each channel. The trimmed mean of M values (TMM) normalization
corrected the compositional bias by aligning the median of the distribution of
abundance intensities between samples [403]. Internal reference scaling was used
to adjust eight TMT data sets onto the same intensity scale. The normalized data
was then averaged and log2 transformed. One-way ANOVA was preformed to
identify proteins that were differentially expressed among strains in either 4 or 7
days, respectively (P value 0.05). The identified proteins were also evaluated for
up- and downregulation by setting a2-fold change cutoff.
8.6.8 Secondary metabolite extraction and analysis
Organic compounds were extracted by taking three plugs of fungal-grown agar
andextractingwith3mLmethanol(MeOH),followedby3mL1:1MeOH–dichloromethane,
each with 1 hr of sonication and filtration. The extract was evaporated in vacuo us-
ing a rotary evaporator, redissolved in 250 μL of 20% dimethyl sulfoxide in MeOH,
and a portion (10 μL) was examined by high-performance liquid chromatography-
photodiode array detection-mass spectroscopy (HPLC-DAD-MS) analysis. HPLC-
DAD-MS was carried out using a ThermoFinnigan LCQ Advantage ion trap mass
spectrometer with a reverse-phase C
18
column (3μm; 2:1100μm; Alltech Prevail)
at a flow rate of 125 μL/min. The solvent gradient for HPLC-DAD-MS was 95%
MeCN/H
2
O (solvent B) in 5% MeCN/H
2
O (solvent A) both containing 0.05%
formic acid, as follows: 0% solvent B from 0 to 5 min, 0 to 100% solvent B from 5
to 35 min, 100 to 0% solvent B from 40 to 45 min, and re-equilibration with 0%
solvent B from 45 to 50 min. For quantification, positive-ion electrospray ionization
(ESI) was used for the detection of austinol, dehydroaustinol, sterigmatocystin,
nidulanin A and analogues, emericellamides A and C–F, emericellin, shamixan-
thone, and epishamixanthone. Negative-ion ESI was used for the detection of
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8.6. Materials and Methods A. Błachowicz
asperthecin, terrequinone, and sterigmatocystin intermediate. Relative production
levels were quantified by integrating the area under each SM’s ESI trace.
- 161 -
Chapter9
Conclusions and Perspectives
Mankind has been hugely benefitting from the richness of the fungal kingdom.
For years various businesses, including pharmaceutical, textile and food industries
have been utilizing bioactive compounds produced by fungi to their advantage.
Discoveries of penicillin [143], lovastatin [145] or cyclosporine [265] revolutionized
medical field, while utilizing the A. niger [268], A. pullulans [404, 405] and
other filamentous fungi for biotechnological production of enzymes contributes to
generating enormous economic gains [406]. Despite many known applications, the
true potential embedded in versatile fungal kingdom remains mostly unknown.
There is therefore, a strong incentive to further study and characterize fungal
isolates.
The efforts to unravel unknown fungal world have been undoubtedly facilitated
by the advancement in the next generation sequencing techniques. Increase in the
number of fully sequenced fungal genomes promoted novel compound discovery, as
insights into genomic sequence allow for secondary metabolite (SM) gene cluster
prediction [67]. Further, the whole genome sequencing (WGS) enables comparative
studies between fungal isolates grown or isolated from distinct environments.
Such an application of the WGS has led to an entirely new area of research that
may increase our understanding of how fungi adapt to and cope with varying
environmental conditions. We can study both genetic diversity of fungal strains
belonging to one species and the structure and dynamic of the whole microbial
A. Błachowicz
population within the selected environmental niche.
An increasing body of research revealed that fungi are crucial constituent of
indoor microflora, including simulated closed habitats [49] and the International
Space Station [8]. As humankind is again invigorated by the idea of manned
outer space explorations there is an unmet need for the studies characterizing
both human-microbial interactions in confined spaces and microbial molecular
adaptation mechanisms to space conditions. Fungi are especially intriguing in such
studies, as they can be both useful for mankind and threatening to human health
and habitat during space voyages.
To advance our understanding of how fungi respond and adapt to space condi-
tions, including microgravity and enhanced irradiation our team applied multi-omic
approach. Proteomic, metabolomic and genomic analyses carried out on several
fungi isolated from the ISS or grown aboard the ISS in controlled experiments,
showed enormous potential of such holistic scientific approach. Collective data
from omic analyses presented in this thesis revealed unique alterations in fungal
genome, proteome and metabolome due to space environment, which only rein-
forced our perception of fungal plasticity and adaptability to extreme conditions.
Undoubtedly, the presented results are just the tip of the iceberg in the emerging
research into fungal adaptive responses to the ISS conditions. Future research
should further validate the observed phenotypic changes and determine whether
the observed alterations are strain or species specific.
It remains imperative to study both, fungi, which unknowingly followed people
and cargo aboard the ISS and the subjects of strictly planned experiments, as they
will address different questions associated with space explorations. Analyses of
the environmental samples collected during the microbial observatory of the ISS
will feature characterization of human-fungal dynamics in confined spaces, while
strictly controlled experiments involving fungi will contribute to understanding the
fungal adaptive responses and will facilitate demonstrating fungal biotechnological
potential. Advancements in DNA-based techniques should be further utilized
to test the presence of fungal virulence factors and genes associated with stress
response, DNA repair, carbon metabolism and antimicrobial resistance in complex,
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A. Błachowicz
environmental samples. Such analyses may reveal the impact of fungal microflora
on human health and operational mission success. Lastly, transcriptomic and
epigenetic analyses should be implemented and optimized to test for immediate
fungal responses to distinct space conditions.
In conclusion, I believe that the research presentedin this thesis is an excellent
base for future studies delving into fungal molecular adaptation mechanisms.
Further multi-omic analyses of fungal adaptive responses may lead to unraveling
the possible biotechnological applications in the space industry, detecting and
preventing potential health- and habitat-related hazards, and discovering novel
compounds, which may benefit humans. A collective effort should be directed
towards utilizing enormous fungal potential in the future space explorations.
- 164 -
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Abstract (if available)

Abstract Filamentous fungi are dual organisms that can be both useful for mankind and threatening to human habitats and health. These omnipresent extremotolerant microorganisms are associated with a range of hostile environments and the human body. Immense adaptability to a variety of conditions enables fungi to thrive in what seems like inhospitable niches, including man-made closed habitats. One such habitat is the International Space Station (ISS), which is a research platform under strict microbiological scrutiny. Recent advances in the next generation sequencing technologies have transformed the scientists’ approach to study fungal adaptation mechanisms. Insights into genome, proteome and metabolome allow for holistic assessment of changes in the molecular suite of fungi exposed to extreme conditions. In this work multi-omic approach was applied to study response of various filamentous fungi to space environment. We investigated both, unintentional fungal “hitchhikers” that unknowingly followed people and cargo aboard the ISS Aspergillus fumigatus (Chapter 2 and 3) and Aspergillus niger (Chapter 6 and 7), and fungi sent to the ISS in strictly controlled experiments, including several Chernobyl isolated fungi, ISS-isolated A. niger (Chapter 4 and 5), and Aspergillus nidulans (Chapter 8). ❧ Aspergillus fumigatus is a ubiquitous in nature saprophytic fungus that may pose a health hazard to immunocompromised individuals. Upon isolation from the ISS, during microbial observatory study, two A. fumigatus isolates were characterized to investigate space conditions-induced phenotype when compared to well-studied Af293 and clinical isolate CEA10. Initial analyses showed that genomic diversity of both ISS-isolates was within the genetic variance of 95 environmental and clinical isolates, however both ISS isolates showed increased virulence in larval zebrafish model of invasive aspergillosis (Chapter 2). Observed increase in virulence prompted further molecular analyses of ISS isolates. Proteome characterization revealed up-regulation of proteins involved in carbohydrate metabolism, secondary metabolism and stress responses in both ISS isolates when compared to clinical isolates. Among increased in abundance proteins were TpcK, TpcF and TpcA involved in trypacidin biosynthesis, Asp-hemolysin, AcuE and PdcA involved in glyoxylate cycle and ethanol fermentation, respectively (Chapter 3). ❧ A. niger, ubiquitous in built and natural environments fungus, which has known biotechnological applications was among ISS-isolated and further characterized strains. Proteomic analysis of ISS-isolated A. niger revealed altered abundance of proteins involved in carbohydrate metabolism, cell wall modulation, and oxidative stress response when compared to well-studied ATCC 1015. Similarly to A. fumigatus whole genome analysis revealed genetic variance within the diversity observed among other species in the A. niger/ welwitschiae/lacticoffeatus clade (Chapter 6). Further, secondary metabolite (SM) analysis showed significant increase in the yields of produced compounds, especially antioxidant Pyranonigrin A. Gene targeted deletion was used to determine gene cluster responsible for Pyranonigrin A production. Lastly, radiation protective potential of Pyranonigrin A against UV-C was tested (Chapter 7). ❧ To study fungal adaptation to space conditions in controlled experiments several Chernobyl- and ISS-isolated fungi were selected and characterized prior to space flight. Assessment of UV-C and simulated Mars conditions resistance revealed strain-dependent survival. Further, upon exposure to SMC two surviving strains A. fumigatus and Cladosporium cladosporoides showed induced production of SM and differential expression of proteins involved in translation, carbohydrate metabolism and energy conversion processes when compared to unexposed control strains (Chapter 4). Genomic characterization of ISS-grown A. niger revealed accumulation of SNPs within specific regions of the genome, while observed INDELs were distributed across all chromosomes. Proteome analysis showed differential expression of proteins involved in carbohydrate metabolism, and stress response, while SM characterization revealed that SM production is altered by space conditions (Chapter 5). Lastly, when fungal model organism A. nidulans and three mutant strains were grown aboard the ISS and compared to ground counterparts alterations in specific genome regions were observed. Additionally, proteins involved in carbohydrate metabolism and stress response were differentially expressed among the ISS-grown strains and alterations in SM yields were revealed (Chapter 8). ❧ This thesis underscores the significance of multi-omic characterization in comprehension of the fungal response to distinct environmental conditions. Such in depth analyses are imperative for elucidating plausible mechanisms of enhanced virulence of A. fumigatus and may unveil biotechnological applications of fungi during space explorations. Further, understanding of possible molecular alterations triggered by irradiation is crucial for the success of long-term manned space flights to ensure both astronauts’ health and maintenance of the closed habitat.

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Asset Metadata

Creator Blachowicz, Adriana (author)

Core Title Multi-omic data mining to elucidate molecular adaptation mechanisms of filamentous fungi exposed to space environment

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 04/30/2019

Defense Date 03/19/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Fungi,genomics,International Space Station,metabolomics,OAI-PMH Harvest,proteomics

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Clay C. C. (committee chair), Haworth, Ian (committee member), Okamoto, Curtis (committee member)

Creator Email ada.blach@gmail.com,blachowi@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-161967

Unique identifier UC11660649

Identifier etd-Blachowicz-7370.pdf (filename),usctheses-c89-161967 (legacy record id)

Legacy Identifier etd-Blachowicz-7370.pdf

Dmrecord 161967

Document Type Dissertation

Format application/pdf (imt)

Rights Blachowicz, Adriana

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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