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Targeting trehalose catalytic shift as a novel strategy to control the emergence of multidrug-resistant tuberculosis
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Targeting trehalose catalytic shift as a novel strategy to control the emergence of multidrug-resistant tuberculosis
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Copyright 2024 Stephanie Dihardjo
Targeting Trehalose Catalytic Shift as a Novel Strategy to Control the Emergence of
Multidrug-resistant Tuberculosis
By
Stephanie Dihardjo
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2024
ii
Table of Contents
List of Figures................................................................................................................................iii
Abstract.......................................................................................................................................... iv
Chapter 1: Introduction................................................................................................................... 1
Chapter 2: Results and Discussion.................................................................................................. 7
Generation of RIF-resistant strain............................................................................................... 7
RIF-resistant strain metabolomics .............................................................................................. 8
TreS expression level increased in RIF-resistant strains ............................................................ 9
RIF-resistant strain resistance to antimicrobial drugs............................................................... 10
Generation of CRISPRi TreS knock-down strain..................................................................... 10
RIF-resistant strain has greater TreS activity............................................................................ 11
Generation of INH-resistant strain............................................................................................ 12
INH-resistant strain susceptibility to antimicrobial drugs........................................................ 13
Altered cell wall integrity in INH-resistant strain..................................................................... 14
Greater cellular uptake in INH-resistant strain ......................................................................... 15
RIF-resistant strain and INH-resistant strain Metabolomics .................................................... 16
Chapter 3: Materials and Methods................................................................................................ 17
Tables............................................................................................................................................ 24
Figures........................................................................................................................................... 25
References..................................................................................................................................... 39
iii
List of Figures
Figure 1. Generation of RIF-resistant strain .................................................................................25
Figure 2. RIF-resistant strain is resistant to RIF...........................................................................26
Figure 3. Targeted metabolomics of RIF-resistant strains............................................................27
Figure 4. TreS expression level increased in RIF-resistant strains. .............................................28
Figure 5. RIF-resistant strain resistance to other antimicrobial drugs..........................................29
Figure 6. Generation of CRISPRi TreS knock-down strain..........................................................30
Figure 7. RIF-resistant strain has greater TreS activity ................................................................31
Figure 8. Suppression of treS gene cause susceptibility to other antimicrobial drugs..................32
Figure 9. INH-resistant strain is more susceptible to other antimicrobial drugs...........................33
Figure 10. INH-resistant strains are more susceptible to BDQ and RIF.......................................34
Figure 11. INH-resistant strain has lower abundance of mycolic acid .........................................35
Figure 12. INH-resistant strains have lower abundance in three types of mycolic acids..............36
Figure 13. INH-resistant strains have higher cellular uptake........................................................37
Figure 14. Metabolomics of RIF-resistant strain and INH-resistant strain...................................38
iv
Abstract
Tuberculosis (TB) is an infectious disease that is cause by Mycobacterium tuberculosis
(Mtb). TB remains as one of the top leading infectious killers worldwide and a major contributor
of antimicrobial resistance. Effective treatment for TB is destabilized by the presence of
multidrug-resistant (MDR) TB as there has been no effective treatment method available to treat
MDR TB. MDR TB is Mtb strains that are resistant to two first-line TB antibiotics, which are
isoniazid (INH) and rifampicin (RIF). Understanding the mechanisms underlying the emergence
of MDR and exploring new treatment approaches is crucial in combating this growing public
health concern. Mtb that can survive under various stress conditions is known as latent
tuberculosis wherein Mtb resides in a state of persister cells. Persister cells are bacterial cells that
have acquired a tolerance to various stress conditions and remain viable. From previous findings,
Mtb trehalose metabolism could trigger the formation of transient drug tolerant fraction and this
metabolic change can also lead to massive development of drug-resistant mutants [20]. The RIFresistant strain has shown an increased in trehalose catalytic shift activity and exhibited crossresistance to other antimicrobial drugs. For instances, the RIF-resistant strain exhibit resistance
to INH and bedaquiline (BDQ). In a contrary, the INH-resistant strain had become more
susceptible to other antimicrobial drugs due to alterations in their cell wall integrity and
subsequent induction of antibiotic uptake. Due to the increase in their trehalose catalytic shift,
the RIF-resistant strain serves as a main source to develop into MDR TB strain. Developing TB
treatments by targeting the trehalose catalytic shift in Mtb could be a breakthrough to prevent the
emergence of MDR TB.
v
1
Chapter 1: Introduction
Tuberculosis (TB) remains as one of the top leading infectious killers in the world with
1.3 million deaths and an estimate of 10.6 million people infected with TB in 2022 [53].
According to World Health Organization, TB is also a major contributor of antimicrobial
resistance with an estimate of 410000 people developed multidrug-resistant or rifampicinresistant TB globally in 2022 [53]. The emergence of multidrug-resistant (MDR) TB is thought
to be due to “persister” bacteria that are refractory to antibiotic treatment [56]. Understanding the
biology of persister cell and mechanistic foundations behind the emergence of MDR TB could
presents a viable strategy for preventing MDR TB.
Tuberculosis is an infectious disease that is cause by Mycobacterium tuberculosis (Mtb).
Mtb is an acid – fast bacilli that have a shape of curved -rod with a thick waxy cell wall. This
thick waxy cell wall has its unique characteristics that help with its pathogenicity, which are
acid-fastness, extreme hydrophobicity, resistance to drying, acidity/alkalinity, and many
antibiotics, as well as distinctive immunostimulatory properties [34]. Mtb is a slow growing
bacterium that usually multiply within the phagocytic cells. Mtb can bind to the phagocytic
receptor to enter the macrophage and evade the host immune response. Once it enters the
macrophage, Mtb can modulate itself to survive in a latent phase and withstand stress conditions
by preserving its energy [37].
Tuberculosis is transmitted through air-borne, and it primarily affects the lungs, but it can
also affect the other body parts. For instances, it can also affect bones, spines, brain, lymph
glands, and other parts of the body [46]. There are two stages of tuberculosis, which are active
TB and latent TB. Active TB is when the bacteria is still actively growing. In this stage, one is
infectious and may show symptoms of tuberculosis. The latent TB is the stage where the bacteria
2
is not actively growing or in a non-replicating state. One with latent tuberculosis is not infectious
and may not show any symptoms of tuberculosis. In this stage, it harbors Mtb infected in a
persister state, which is in dormant and non-replicating state. Persister cells can remain viable
after a prolong exposure of stress condition and it can stay dormant until its reactivated. Stress
condition includes nutrition starvation and exposure to antimicrobial drugs. The Mtb persister
cell is genetically identical with Mtb but it has developed tolerance towards stress condition, in
this instance, antimicrobial drugs. The presence of Mtb persister cell has been one of the
challenges in treating tuberculosis.
Treatment for tuberculosis is known to be complex as it usually includes multiple
antimicrobial drugs and it usually last for a long period of time. Rifampin (RIF) and Isoniazid
(INH) are the most common first-line antimicrobial drugs used to treat active TB. The CDC
recommended TB treatment regimens that comprise three preferred rifamycin-based regimens
and two alternative monotherapy regimens with daily isoniazid [43]. The three rifamycin-based
regimens include 3 months of once-weekly isoniazid plus rifapentine, 4 months of daily
rifampin, and 3 months of daily isoniazid plus rifampin. The two alternative monotherapy
regimens are daily isoniazid for 6 or 9 months. The rifamycin-based regimen is preferable than
the isoniazid monotherapy regimen because it has lower toxicity risk. The longer treatment
duration with INH is associated with adverse effects of hepatoxicity and neuropathy [38]. Also,
due to the longer period for the INH monotherapy regimen takes, it is more likely for the patient
to not complete the whole regimen. The longer treatment is avoided to prevent the development
of MDR TB.
Both RIF and INH have different mechanism of action on Mtb. INH is a prodrug that
require activation by the enzyme catalase-peroxidase KatG, which is coded by the katG gene.
3
The activated form of isoniazid has evidence to have the mode of action in inhibiting the
biosynthesis of cell wall mycolic acids [31]. With the compromised cell wall, bacteria will be
more susceptible to reactive oxygen radicals and other environmental factors. On the other hand,
RIF targets the mycobacterial RNA polymerase by binding to the beta subunit of the RNA
polymerase [10]. Hence, it is resulting in the inhibition of elongation of mRNA and eventually
will interfere with the transcription process. With different mode of action antimicrobial drugs,
an adequate combination of effective drugs can reduce the probability of failure, relapse, and
selection of resistant [41].
There are multiple factors that may contribute to the development of MDR in
tuberculosis. The first factor would be incomplete TB medication for the whole period of
treatment regimen. This mainly arise from patient’s non-compliance to the TB treatment regimen
due to long and complicated regimen. Most treatment regimen for TB take months to complete.
The long period of treatment is due to of Mycobacterium tuberculosis have slow metabolic
processes and long generation time [1]. Even though the treatment takes months to complete,
people usually starting to get better after a couple of weeks of the treatment. Additionally, there
are some adverse effects that might arise from taking the medication. Some of the antibiotics
taken for tuberculosis treatment regimen need to be excreted by the kidney, which may be the
cause of the adverse effect. Due to the long period of tuberculosis treatment regimen and the
adverse effects that might arise from it, most people would not complete the whole tuberculosis
treatment regimen and it may lead to the development of tuberculosis drug-resistant strain. The
second factor would be improper use of antibiotics in the tuberculosis treatment regimen.
Tuberculosis treatment regimen usually consists of multiple antibiotics which includes a
combination of different antimicrobial drugs. Every Tuberculosis treatment regimen includes at
4
least one drug from the first-line drugs of tuberculosis drugs. The first-line antimicrobial drugs
for TB includes Rifampin, Isoniazid, Pyrazinamide, and Ethambutol. Treatment with only one
drug would lead to the development of tuberculosis drug-resistant strain as it is constantly expose
to the same drug. The combination of different drugs would help to targets different site of M.
tuberculosis that may inhibit bacteria from growing. Therefore, improper tuberculosis treatment
regimen could also lead to the development of tuberculosis drug-resistant strain. Lastly,
weakened immune system could also be a factor that may contribute to drug resistant in
tuberculosis. Bacteria can grow more actively in lowered immune system. Thus, increasing the
chance for the bacteria to develop into drug resistant bacteria.
According to WHO, there are four different types of drug resistance in Tuberculosis,
which are: mono-resistance, poly-resistance, multidrug resistance (MDR), and extensive drug
resistance (XDR) [55]. The types of drug resistance tuberculosis are categorize based on the
number of drugs its resistant to. Mono-resistance is the resistance to one antimicrobial drug,
while poly-resistance is the resistance to more than one antimicrobial drug other than INH and
RIF. The mono-resistance and poly-resistance to tuberculosis are usually treated with other drugs
that it’s not resistant to. The MDR, a resistance to at least both isoniazid and rifampicin, is little
more complicated to treat. MDR is the accumulation of mutation in individual target gene. The
treatment for MDR is to prevent it from escalating into an XDR, which is a resistance to any
fluoroquinolone, and at least one of the three second-line injectable drugs in addition to
multidrug resistance.
The Mtb persister cell may have the potential to develop into drug-resistant TB. Mtb
persister cells tolerance is generally attributed to minimally active cellular processes that prevent
antibiotic-induced damage, which has led to the supposition that persister offspring give rise to
5
antibiotic-resistant mutants at comparable rates to normal cells [4]. This finding suggests that
there is a correlation between metabolism and drug tolerance. The Mtb persister cells could
survive under stress conditions due to the alteration in their metabolism. Previously, our
laboratory has recently reported that the Mtb persister cells use trehalose as an internal carbon to
biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus
maintaining levels of ATP and antioxidants [20]. Trehalose is abundant in mycobacterium cell
envelope and known as trehalose monomycolate (TMM) and trehalose dimycolate (TDM). Both
TMM and TDM serve as components in mycobacterium cell wall, as it is shown in figure 11A.
In Mtb persister cells, trehalose is converted to maltose and glucose-6-phosphate by the catalytic
activity of trehalose synthase (TreS). Glucose-6-phosphate is the first intermediate in glycolysis
pathway to support Mtb persister carbon central metabolism. Hence, the TreS knock-out Mtb
strain is unable to form persister and became more susceptible to antimicrobial drugs [20]. This
finding suggests that without the increased in trehalose catalytic shift, bacteria became more
susceptible to antimicrobial drugs and have less chance in developing into drug-resistant
bacteria.
Persistence of the persister cells could also be directly linked with altered mutation rates
and increased probabilities of mutating into antibiotic-resistant genotype [52]. According to this
finding, the persister cells may develop into drug-resistant strains. Hence, the drug-resistant
strain would also be metabolically alter, which is utilizing the trehalose catalytic shift as an
adaptive strategy. In this study, we are hypothesizing that Mtb persister cells and Mtb drugresistant cells shared the same characteristics, which is an increased in trehalose catalytic shift,
that results in increase tolerance to antimicrobial drugs. We have found that RIF-resistant strain
has an increase trehalose catalytic shift; therefore, it is more resistant to other antimicrobial
6
drugs. On the other hand, the INH-resistant strain is more susceptible to other antimicrobial
drugs due to its altered cell wall integrity and greater cellular uptake. Both RIF-resistant strain
and INH-resistant strain are metabolically different. These findings suggest that by targeting the
trehalose catalytic shift, it could be a new approach in discovering new treatments to combat
tuberculosis. In this study, Mycobacterium smegamatis (Msm) is used as a surrogate model of
Mtb as it has faster generation time, and it can be used in BSL2 settings.
7
Chapter 2: Results and Discussion
Generation of RIF-resistant strain
The Rifampicin-resistant strains were artificially generated by exposing the drug
susceptible strain (Naïve) of Msm in high concentration of Rifampicin (RIF) (Figure 1A). Naïve
were exposed to high concentration of RIF (100µg/mL) on 7H10 agar medium. Colonies
observed from this assay is counted and used to calculate the acquisition rate of RIF. As shown
in figure 1B, the rate acquisition of RIF is lower in CRISPRi treS knock-out (iTres) strain. It can
be deduced that by suppressing the expression of treS gene, it lowers the mutation rate. Hence,
the suppression of treS gene lowers mutation rate and have less chances for the development of
drug-resistant strain.
Surviving ten colonies were selected and re-inoculated in 7H9 liquid media to make stock
cultures. These colonies are the RIF-resistant strains and numbered as 1-10 (R1-R10). To
confirm the drug tolerance of the strain, a spot assay of RIF-resistant strain and Naïve were
performed. The OD were matched for all strains and inoculated on 7H10 agar medium with
concentration of 32µg/mL RIF and without RIF as a control. All the RIF-resistant strains were
able to grow under the 32µg/mL RIF treatment while Naïve was not able to grow (Figure 2).
This result confirms the drug tolerance of the RIF-resistant strains and it’s not reversible.
Next, the sequencing analysis of the region in rpoB gene is performed on 10 RIF-resistant
strains. Out of the ten RIF-resistant strains, only R1 and R2 have a mutation in the 81 bp
Rifampin Resistant Determination Region (RRDR) or rpoB gene (Table 1). Most mutations in
rifampicin resistance tuberculosis were determined to be restricted to an 81-bp core region of the
rpoB gene and are dominated by single nucleotide changes, resulting in single amino acid
substitutions [10]. R1 and R2 had developed resistance to RIF due to a mutation within the rpoB
8
gene, which resulted in no beta subunit of the RNA polymerase coded and RIF could not bind to
it. However, the whole genome sequencing for the other strains were not performed, so mutation
in another region could not be identified.
RIF-resistant strain metabolomics
The mutation of rpoB gene can only be found in R1 - R2 and not in R3-R10 but all RIF
drug-resistant strains are resistant to RIF. In persister cells, trehalose fuels the central carbon
metabolism [20]. As it previously mentioned, persister cell is linked with drug-resistant cell, an
untargeted metabolomics analysis was performed to see if there are any metabolism changes
within RIF-resistant strain; hence, how they become resistance to RIF. Metabolomics profile of
339 metabolites were extracted from Naïve and R1 grown on filter cultures in sodium butyrate
with/without trehalose on 7H10 agar media. The carbon source in the media is replaced with
trehalose to see how the bacterial cells are allocating the trehalose in its metabolism. To visualize
the result, the normalized metabolites abundance was analyzed by using the MetaboAnalyst. As
shown in figure 14., Naïve and R1 are metabolically different.
Based on the untargeted metabolomics of RIF-resistant strains, there are some pathways
that are up-regulated in RIF-resistant strains in comparison to Naïve. For instances, the pentose
phosphate pathway is up-regulated in RIF-resistant strain compared to Naïve. In the pentose
phosphate pathway, trehalose and glucose are used to maximize the NADPH production. Hence,
targeted metabolomics of RIF-resistant strains was performed to further analyzed it. Trehalose
synthase (TreS) converts trehalose to maltose, and this is reversible. Then, maltose can be
converted to glucose-6-phosphate (G6P), which can be used to fuel the central carbon
metabolism as shown in figure 3A. The abundance of trehalose is similar between the naïve cell
9
and flux cell (RIF-resistant strain) with the naïve cell has slightly higher abundance of trehalose.
This is due to trehalose is provided as the carbon source in the media. The abundance of
trehalose is slightly lower in RIF-resistant strain as they are using it to support the central carbon
metabolism due to the carbon source has been replaced. The TreS converts trehalose to G6P.
Hence, the abundance of G6P is higher in RIF-resistant strain compared to naïve cells. These
G6P is used to provide energy for the bacteria, and it is shown in the increased in other
metabolites, such as sedoheptulose-7-phosphate (S7P), fructose biphosphate (FBP),
glyceraldehyde-3-phosphate, and phosphoenolpyruvate (PEP) (figure 3B). From this abundance
of metabolites, it can be suggested that the RIF drug-resistant strain have an increased in its
trehalose catalytic shift.
TreS expression level increased in RIF-resistant strains
As it stated in previous study, TreS is responsible for the trehalose-catalytic shift in
persister-like bacilli [20]. TreS enzyme converts trehalose to maltose, then to glucose-6-
phosphate, to support the central carbon metabolism. Metabolites associated with trehalose
catalytic shift is up-regulated in the RIF drug-resistant strains. To confirm if TreS is responsible
for it, the mRNA expression for the TreS gene, which is responsible for the expression of TreS,
is measured in Naïve and RIF-resistant strain. The expression level of TreS is 3 times higher in
RIF-resistant strains compared to Naïve as it is shown in figure 4. This indicates that the
increased of trehalose-catalytic shift in RIF-resistant strain is due to elevated expression level of
TreS.
10
RIF-resistant strain resistance to antimicrobial drugs
The RIF-resistant strains seem to demonstrate resistance to RIF. The trehalose-catalytic
shift mitigates antibiotic effects [20]. With the elevated trehalose-catalytic shift in RIF-resistant
strain, RIF-resistant may also exhibiting resistance to the other antimicrobial drugs. A spot assay
plates of Naïve and R1 were performed on 7H10 agar medium with different antibiotics, in this
case INH and BDQ. Naïve and R1 were also inoculated on 7H10 agar plates without antibiotics
to act as a control. Even though R1 has never been exposed to INH and BDQ, R1 seems to be
more resistant to both INH and BDQ compared to Naïve (Figure 5). The RIF-resistant strain
seems to develop a cross-resistance to INH and BDQ. The elevated trehalose-catalytic shift in
RIF-resistant strain effects it to develop a cross-resistance to other antibiotics.
Generation of CRISPRi TreS knock-down strain
To confirm if the elevated trehalose-catalytic shift in RIF-resistant strain is due to TreS,
the inducible CRISPR-based knock down system was used to generate the Msm iTres strain
(figure 6A) [32]. Plasmids containing dcas9 and the target gene sgRNA induced by tetracycline
were used to target the specific gene in the DNA sequence. Knock-down of the upstream gene
occurred only when the sgRNAs were targeted within or very close to the 3’ end of the upstream
gene coding sequence [32]. Hence, it would sterically hinder the RNA polymerase from
accessing the target gene and will block mRNA transcription. The iTres strain were selected by
using kanamycin, bacterial cultures were regrown to make stock culture. Prior to using the strain,
200ng/mL of anhydrotetracycline (ATc) need to be added into bacterial cultures at least 4 hours
before; mRNA expression level of TreS was measured. The suppression of the TreS is induced
11
by ATc. As it is shown in figure 6B., the trehalose synthase knock-down strain without ATc has
significantly higher TreS activity compared to trehalose knock-down strain treated with
200µg/mL of ATc with a P-value of 0.0003. The generation of iTres strain has successfully
suppress the expression level of TreS.
RIF-resistant strain has greater TreS activity
The drug tolerance in RIF-resistant strain is proposed to be due to the increase in TreS
activity. A new method was developed to monitor the TreS activity by using a trehalose-based
fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission
(RMR-tre) [3]. The RMR-tre will be added into bacterial culture and act as dye. It will bind to
TDM, that will represent TreS activity. Fluorescence-activated cell sorting (FACS) analysis is
used to further analyzed the TreS activity within the strains. The RIF-resistant strain exhibited a
high level of TreS of 42.5% with two peaks (figure 7). To confirm that the high labelling of
RMR-tre level correspond to the TreS activity, this method is also performed on iTres strain.
Naïve and iTres strain exhibited low TreS activity level of 2.35% and 1.23%, respectively. The
low TreS activity in iTres strain indicate that the high labelling of RMR-tre corresponds to TreS
activity. Since the expression level of TreS in iTres strain has been suppressed, it has low TreS
activity similarly to Naïve.
To confirm that the drug resistance of RIF-resistant strain is due to trehalose catalytic
shift, the iTres strain will be tested its resistance with antibiotic as well. In figure 8A, drug
sensitivity assay of Naïve, R1, and iTres strain with INH and BDQ were performed. Under
treatment of INH, R1 is the most resistant to INH with IC50 value of 0.9831, Naïve with IC50
value of 0.8354, and iTres strain with IC50 value of 0.8636. Similarly, under BDQ treatment, R1
12
is the most resistant to BDQ with IC50 value of 0.02036, Naïve with IC50 value of 0.01730, and
iTres strain with IC50 value of 0.01719. These results were confirmed with spot assay plates as it
is shown in figure 8B. R1 colonies on the spot assay is more visible and bigger in sizes on both
INH and BDQ treated plates. Naïve and iTres strain colonies have similar sizes and visibility,
which is smaller than R1. These results are consistent with the FACS analysis data in figure 7.
The suppression of TreS resulted in the iTres strain to be more susceptible to antibiotics,
similarly to Naïve. Hence, the drug resistance in RIF-resistant strain is due to the increased in
trehalose catalytic shift.
Generation of INH-resistant strain
Since the RIF-resistant strain have cross-resistance to the other antibiotics, the INHresistant strain may have cross-resistance to the other antibiotics. The INH-resistant is artificially
generated by exposing Naïve of Msm to high concentration of INH. Naïve was exposed to
200µg/mL of isoniazid on 7H10 agar medium (Figure 1A). Similarly, colonies observed from
this assay is counted and used to calculate the acquisition rate of INH. As shown in figure 1C,
the rate acquisition of INH is lower in iTres strain. Hence, the suppression of treS gene lowers
mutation rate and less chance for the development of drug-resistant strain.
Surviving colonies were selected and re-inoculated in 7H9 liquid media to make stock
cultures. These colonies would be termed as INH drug-resistant strain and numbered as 1-2 (I1 –
I2). Spot assay plates of Naïve, I1, and I2 were performed on 7H10 agar media to confirm the
drug tolerance. The OD were matched for all strains and inoculated on 7H10 agar medium with
concentration of 4.8µg/mL INH and without INH as a control. I1 and I2 colonies were more
visible and bigger in sizes compared to Naïve (figure 9). This indicates that I1 and I2 are more
13
resistant under INH treatment compared to Naïve. This result confirms the drug tolerance of the
INH-resistant strains and it’s not reversible.
INH is a prodrug that require activation by the enzyme catalase-peroxidase KatG, which
is coded by katG gene. However, it is suggested that the antibacterial properties of INH do not
rely on katG of Mtb [18]. Hence, no sequencing analysis was performed on INH drug-resistant
strain.
INH-resistant strain susceptibility to antimicrobial drugs
Spot assay plates of Naïve, I1, and I2 were performed to confirm the cross-resistance of
INH-resistant strain with the other antibiotics. Naïve, I1, and I2 were spotted on 7H10 agar
media supplemented with 32µg/mL of RIF and 0.018µg/mL of BDQ. Both I1 and I2 appeared to
be more sensitive to RIF and BDQ in comparison with naïve as it is shown in figure 9. Under the
treatment of RIF, I1 and I2 were not able to grow. I1 and I2 were able to grow under the
treatment of BDQ but their colony size is significantly smaller than naïve. I1 and I2
susceptibility to BDQ was further confirm through a drug sensitivity test and colony forming unit
assay. The result of the drug sensitivity is naïve have the highest IC50 value of 1.017, I1 with
IC50 value of 1.005, and I2 with IC50 value of 1.004 (Figure 10A). Naïve have the highest IC50
value out of all three strains, therefore, Naïve is more resistant to BDQ. The colony forming unit
assay of naïve, I1, and I2 were performed with different concentration of BDQ. A significant
difference between Naïve, I1, and I2 can be seen under the treatment of 0.00625µg/mL of BDQ
(Figure 10B). There are differences in number of colonies and colony sizes. The number of
colonies was further analyzed to calculate the area under the curve (AUC). In figure 10C, it is
shown that Naïve have an AUC value of 0.1316, I1 have an AUC value of 0.07171, and I2 have
14
an AUC value of 0.07133. The higher the AUC value means the more resistant the strain is under
the treatment of BDQ. Moreover, the diameter of the colonies was measured. The colony sizes of
Naïve is significantly bigger compared to I1 and I2 (Figure 10D).
The colony forming unit assay was also performed on Naïve, I1, and I2 under different
concentration of RIF. Naïve has more colonies observed with bigger colonies sizes compared to
I1 and I2 (figure 10E). The AUC value is calculated from the colonies observed with Naïve has
an AUC value of 788.6, I1 has an AUC value of 421.9, and I2 has an AUC value of 347.3 (figure
10F). From these results, Naïve has shown to be more resistant to RIF compared to I1 and I2.
Furthermore, these results indicate that Naïve is more resistant to BDQ and RIF in comparison to
I1 and I2. Hence, the INH-resistant strain is more susceptible to other antibiotics compared to
Naïve.
Altered cell wall integrity in INH-resistant strain
The activated form of INH can inhibit the biosynthesis of cell wall mycolic acids in Mtb.
Mycolic acids form an efficient permeability barrier and can modulate host innate immune
responses [28]. Mycolic acid is part of the cell wall of Mtb that is important for its pathogenicity
or survival. The exposure to high INH might have affect the cell wall integrity of INH drugresistant strain, which might be one of the reasons for it to be more susceptible to other
antibiotics. The total lipid of Naïve, I1, and I2 were extracted to see the abundance of mycolic
acid. The extracted total lipid is visualized by using thin layer chromatography (TLC) in figure
11. According to figure 11, the intensity of the mycolic acid abundance for Naïve is the most
intense. This indicate that I1 and I2 have less mycolic acid abundance in their cell wall. There
are three types of mycolic acid in Mtb, which are alpha-mycolic acid, methoxy-mycolic acid, and
keto-mycolic acid. Additionally, the mycolic acid of Naïve, I1, and I2 were extracted and
15
visualized by using TLC. There are three distinguish band lines on the TLC that indicate the
three different types of mycolic acid. The line of bands on the top is alpha-mycolic acid, second
line of the bands is the methoxy-mycolic acid, and the third line of bands is the keto-mycolic
acid as indicated in figure 12. The alpha-mycolic acid is the most abundant mycolic acid, which
resulted it has the most intensified bands out of all the three types of mycolic acid. The intensity
of the bands was analyzed and used to construct the graph. Naïve has the most abundance for all
three types of mycolic acid compared to I1 and I2. This result is consistent with the result from
figure 11 as Naïve has the most mycolic acid abundance. As Naïve have higher mycolic acids
abundance than I1 and I2, Naïve is more resistant to antibiotics as mycolic acid serves as a
physical barrier that help with its survival.
Greater cellular uptake in INH-resistant strain
Another factor that can be accounted for the resistance of bacteria would be its cellular
uptake, in this case, its antibiotic uptake. A decrease in drug permeability contributes to
phenotypic drug resistance of dormant Mtb [35]. An increase of antibiotic uptake would result in
bacteria to be more vulnerable to antibiotics. Mycobacterial cell wall has a high lipid content that
act as a major barrier to the penetration of antimicrobial agents [19]. With the integrity of cell
wall decreases, the penetrability of the cell wall increases. The RIF uptake of Naïve, I1, and I2
were measured and shown in figure 13A. During the initial time point, I1 has the most rapid RIF
uptake, I2 has moderate rate of RIF uptake, and Naïve has the slowest rate of RIF uptake. After
prolong exposure, the RIF uptake seems to be similar. To confirm this result, the ethidium
bromide (EtBr) uptake is performed on Naïve, I1, and I2 as it is shown in figure 13B. Consistent
with the RIF uptake test, Naïve have lower EtBr uptake compared to I1 and I2. I1 seems to have
16
greater antibiotics uptake. Both I1 and I2 have greater antibiotics uptake, which may contribute
to its susceptibility to the other antibiotics.
RIF-resistant strain and INH-resistant strain Metabolomics
Metabolomics profile of 339 metabolites were also perform on INH-resistant strain.
Through figure 14, we can see that Naïve, R1, and I1 are metabolically different from each other.
These differences might have contributed to its drug tolerance. These data support on how the
three strains show different resistance to antibiotics. Further analysis needs to be performed.
17
Chapter 3: Materials and Methods
Bacterial strains and culture conditions
Drug susceptible Mycobacterium smegmatis (Naïve), rifampicin-resistant strain
Mycobacterium smegmatis, isoniazid-resistant strain Mycobacterium smegmatis, and trehalose
synthase knock-down strain Mycobacterium smegmatis (iTres) were used in this research. Drug
susceptible Mycobacterium smegmatis is bacteria that have never been exposed to antibiotics.
Both rifampicin-resistant strain and isoniazid-resistant strain Mycobacterium smegmatis were the
surviving bacteria isolated after antibiotic exposure. All bacterial strains used in this research
were cultured at 37°C in Middlebrook 7H9 liquid medium supplemented with 0.5% bovine
serum albumin, 0.085% NaCl, 0.2% dextrose, 0.2% glycerol, and 0.04% tween 80. Bacterial
strains were plated on Middlebrook 7H10 agar medium supplemented with 0.5% bovine serum
albumin, 0.085% NaCl, and 0.2% dextrose.
Metabolomics
Drug susceptible Mycobacterium smegmatis (Naïve), rifampicin-resistant strain
Mycobacterium smegmatis, isoniazid-resistant strain Mycobacterium smegmatis, and trehalose
synthase knock-down strain Mycobacterium smegmatis (iTres) were grown on either 7H9 liquid
media or 7H10 agar media. Bacterial cells were either treated with specific antimicrobial drugs
or grown on specific nutrient supplemented media and taken at specific time points as indicated.
Bacterial cells were re-suspended in acetonitrile:methanol:H2O (40:40:20, v:v:v) and
mechanically lysed by using the tissue homogenizer (Precellys Evolution, Bertin Technologies)
with 0.1mm zirconia beads 7 times for 2 minutes at 6000 rpm. Then, samples were filtered
through a 0.22µm spin-X column. An equal amount of the samples and 4% formic acid in
18
acetonitrile (1:1, v:v) were transferred into metabolomics tube. Metabolites in samples were
identified by using the 1290 infinity II liquid chromatography system (Agilent) coupled with
6230 time-of-flight mass spectrometers (Agilent). Metabolites abundances were normalized to
cell biomass by using BCA protein assay kit (Thermo Fischer Scientific).
Generation of RIF-resistant strain
Drug susceptible Mycobacterium smegmatis (Naïve) was collected at mid-logarithmic
phase growth. Bacterial culture Naïve was inoculated on 7H10 agar media containing 0.5%
bovine serum albumin, 0.085% NaCl, 0.2% dextrose, and 100µg/mL of Rifampicin. Bacterial
culture Naïve was also inoculated on 7H10 agar media containing 0.5% bovine serum albumin,
0.085% NaCl, and 0.2% dextrose as a control.
Generation of INH-resistant strain
Drug susceptible Mycobacterium smegmatis (Naïve) was collected at mid-logarithmic
phase growth. Bacterial culture Naïve was inoculated on 7H10 agar media containing 0.5%
bovine serum albumin, 0.085% NaCl, 0.2% dextrose, and 200µg/mL of Isoniazid. Bacterial
culture Naïve was also inoculated on 7H10 agar media containing 0.5% bovine serum albumin,
0.085% NaCl, and 0.2% dextrose as a control.
mRNA Quantification
Bacterial cells were cultured in 7H9 liquid media until it reaches OD 1.0. Bacterial cells
were harvested, re-suspended in TRI-reagent (Sigma-Aldrich), and transferred into a tube filled
with 0.1mm zirconia beads. Bacterial cells were mechanically lysed by using the tissue
19
homogenizer (Precellys Evolution, Bertin Technologies) twice for 10 minutes at 6000 rpm. The
RNA miniprep kit (Zymo Research) was used to isolate the RNA from the bacterial cells. Then,
the RNA is used to generate cDNA by using the iScript CDNA synthesis kit (BioRad) in a
thermal cycler (Vapo.Protect Mastercycler Pro. Eppendorf). The quantitative real-time
polymerase chain reaction (qRT-PCR) was performed to quantify the cDNA by using the light
cycler (LightCycler96, Roche). The iQ SYBR green supermix (BioRad) is used as a dye that
bind to the DNA sequence.
Plasmid construction
The single guide RNA (sgRNA) was designed to target the 3’ end of the non-template
strand of the target genes. The PLJR962 Msm CRISPRi backbone plasmid was amplified in E.
coli and selected by using kanamycin. The backbone was digested with BsmBI restriction
enzymes and gel purified. The oligo primers were annealed into the digested backbone.
Cell Transformation
Competent cell of Msm was prepared by culturing Msm in 7H9 liguid media until it
reaches OD 1.0. The cell is harvested and re-suspense in 10% glycerol solution. The plasmid is
added into the competent cell by using electroporation. The transformed liquid cultures are
incubated and grown to mid-log phase growth. The liquid cultures are inoculated on 7H10 agar
media supplemented with 50 ng/mL of kanamycin. Colonies observed were inoculated into 7H9
liquid media to make stock culture.
20
FACS Analysis
Drug susceptible Mycobacterium smegmatis (Naïve), rifampicin-resistant strain
Mycobacterium smegmatis, and trehalose synthase knock-down strain Mycobacterium smegmatis
(iTres) were grown on 7H9 liquid media and collected at mid-logarithmic phase growth.
Bacterial cultures were OD-matched and stained with 10µM of RMR-tre for one hour. Samples
were analyzed by using fluorescence-activated cell sorting FACS analysis.
Spot assay
All bacterial cultures used were collected at mid-logarithmic phase growth and ODmatched. The knock-down strain (CRISPRi) was treated with anhydrotetracycline (ATc) at least
4 hours before used. Serial dilutions of bacterial cultures were performed and 2µL of each
bacterial culture dilutions were inoculated on 7H10 agar medium with different concentration of
antibiotics. Bacterial culture dilutions were also inoculated on 7H10 agar medium with no
antibiotic to serve as a control. Images of the plates were observed and taken by using ChemiDoc
MP Imaging System (BioRad).
Drug sensitivity assay
All bacterial cultures used were collected at mid-logarithmic phase growth and matched
to OD 0.02. The knock-down strain (CRISPRi) was treated with anhydrotetracycline (ATc) at
least 4 hours before used. Different concentration of antibiotics (100µL) was transferred into
each well of tissue culture plate, 96 well, flat bottom with low evaporation lid. Bacterial cultures
(100µL) were transferred onto each well and incubated at 37°C for 3-5 days. The OD of the
21
plates was read by using the Molecular Devices FilterMax F5 Multi-Mode Microplate Reader.
Results were analyzed and graphed.
Colony forming unit assay
All bacterial cultures used were collected at mid-logarithmic phase growth and ODmatched. The knock-down strain (CRISPRi) was treated with anhydrotetracycline (ATc) at least
4 hours before used. Serial dilutions of bacterial cultures were performed. 10 µL of each
bacterial culture last dilutions were inoculated and let it drop vertically on 7H10 agar medium
with different concentration of antibiotics. Bacterial colonies observed were counted and images
of the plates were observed and taken by using ChemiDoc MP Imaging System (BioRad).
Mycolic acid extraction and thin-layer chromatography
Drug susceptible Mycobacterium smegmatis (Naïve), rifampicin-resistant strain
Mycobacterium smegmatis, and isoniazid-resistant strain Mycobacterium smegmatis were
cultured in 100mL of 7H9 liquid medium supplemented with 0.5% bovine serum albumin,
0.085% NaCl, 0.2% dextrose, 0.2% glycerol, and 0.04% tween 80 until it reached OD 1.0 in
shaking condition. Bacterial cultures were harvested and dried under nitrogen gas. The bacterial
pellet was resuspended with tetrabutyl ammonium hydroxide (TBAH) and incubated overnight at
100ºC. Then, CH2Cl2, CH3I, and water were added and incubated for 1 hour. Samples were
centrifuged, the lower organic phase is collected, and dried under the nitrogen gas. Diethyl ether
is added into the dried residue, sonicated, and dried under the nitrogen gas. The dried residue
was resuspended with CH2Cl2. An equal amount was spotted onto thin-layer chromatography
(TLC) plates. TLC separated mycolic acid by using hexane/ethyl acetate (19:1, v/v) solvent
22
system. The spots on TLC were visualized with 5% ethanolic molydophosphoric acid. Images of
TLC plates were observed and taken by using ChemiDoc MP Imaging System (BioRad).
Total lipid isolation and thin-layer chromatography
Drug susceptible Mycobacterium smegmatis (Naïve), rifampicin-resistant strain
Mycobacterium smegmatis, and isoniazid-resistant strain Mycobacterium smegmatis were
cultured in 100mL of 7H9 liquid medium supplemented with 0.5% bovine serum albumin,
0.085% NaCl, 0.2% dextrose, 0.2% glycerol, and 0.04% tween 80 until it reached OD 1.0 in
shaking condition. Bacterial cultures were harvested and dried under nitrogen gas. The bacterial
pellet was resuspended with chloroform-methanol (2:1) and incubated overnight. The
chloroform-extracted lipids were washed with acetone and dried under nitrogen gas. An equal
amount was spotted onto thin-layer chromatography (TLC) plates. TLC separated the TDM and
mycolic acid by using chloroform/methanol/water (90:10:1, v/v/v) solvent system. The spots on
TLC were visualized with 5% ethanolic molydophosphoric acid. Images of TLC plates were
observed and taken by using ChemiDoc MP Imaging System (BioRad).
Rifampicin uptake
Drug susceptible strain Mycobacterium smegmatis (Naïve) and isoniazid-resistant strain
Mycobacterium smegmatis were cultured in 7H9 liquid media supplemented with 0.5% bovine
serum albumin, 0.085% NaCl, 0.2% dextrose, 0.2% glycerol, and 0.04% tween 80 until it
matched the OD 1.0. Bacterial cells were exposed to 0.78µg/mL of rifampicin (RIF) and
incubated at 37°C. Samples were taken at 2 hours, 4 hours, 24 hours, and 48 hours. Rifampicin
from the samples were identified by using metabolomics.
23
Ethidium bromide uptake
Drug susceptible strain Mycobacterium smegmatis (Naïve) and isoniazid-resistant strain
Mycobacterium smegmatis were cultured in 7H9 liquid media supplemented with 0.5% bovine
serum albumin, 0.085% NaCl, 0.2% dextrose, 0.2% glycerol, and 0.04% tween 80. Bacterial
cultures were matched to OD 0.5 and diluted in phosphate buffer solution (PBS). The diluted
bacterial cultures were transferred into black, flat-bottom 96-well plates followed by 10%
glucose solution in PBS and 4µg/mL of ethidium bromide (EtBr) solution. The plate was
exposed to fluorescence at 535 nm excitation with 595-nm emission and measured kinetically for
1 hour at room temperature. The EtBr uptake is measured by determining the slope of a linear
regression of the fluorescence expression measured from EtBr accumulation versus time.
24
Tables
Table 1. Sequencing analysis of RDRR region in rpoB gene in RIF-resistant strains.
Isolate (s) Amino Acid Position Nucleotide Change* Amino Acid Change
R1 452 CTG à CCG Leu à Pro
R2 452 CTG à CCG Leu à Pro
R3 NC
R4 NC
R5 NC
R6 NC
R7 NC
R8 NC
R9 NC
R10 NC
*NC: No Change
25
Figures
Figure 1. Generation of RIF-resistant strain
(A) A schematic to show the generation of RIF-resistant strain and INH-resistant strain by using
fluctuation assay. Naïve was cultured on 7H10 agar medium with and without 100µg/mL of RIF.
The colonies that grew on 7H10 agar medium with 100µg/mL of RIF is the RIF-resistant strain.
(B) & (C) Result from the fluctuation assay was used to calculate the mutation rate of CRISPRi
treS knock-out (treSMS KO) strain under treatment or RIF and INH. The suppression of treS gene
resulted in lower rate of acquisition of RIF and INH. Hence, lower mutation rate in treSMS KO
compared to Naïve.
A
B C
26
Figure 2. RIF-resistant strain is resistant to RIF.
To confirm the strains, spot assay plates of naïve and RIF-resistant strains (R1 – R10) were
performed on 7H10 agar medium with and without concentration of 32µg/mL RIF. Naïve is
susceptible under RIF treatment, while the R1-R10 are resistant.
27
Figure 3. Targeted metabolomics of RIF-resistant strains
(A) A schematic presentation of the metabolites involves in the utilization of trehalose as a
carbon source to support the central carbon metabolism. (B) Metabolomics profile of targeted
metabolites were extracted from Naïve and Flux (RIF-resistant Strain) grown on filter cultures in
10mM of sodium butyrate with/without 20mM of trehalose on 7H10 agar media supplemented
with 0.5% bovine serum albumin. The relative abundance of trehalose is greater in Naïve
compared to RIF-resistant strain. The relative abundance of Glucose-6-phosphate (G6P),
Fructose biphosphate (FBP), Sedoheptulose-7-Phosphate (S7P), Glyceraldehyde-3-Phosphate
(Glyceraldehyde 3P), and Phosphoenolpyruvate (PEP) are higher in Naïve compared to the RIFresistant strain. The RIF-resistant strain is utilizing the trehalose as a carbon source to support its
carbon central metabolism.
A B
28
Figure 4. TreS expression level increased in RIF-resistant strains.
Naïve and Flux (RIF-resistant strain) were grown on filter cultures in sodium butyrate
with/without trehalose on 7H10 agar media supplemented with 0.5% bovine serum albumin.
Cultures were extracted and the mRNA expression level of trehalose synthase (TreS) was
performed on Naïve and RIF-resistant strain. The expression level of TreS enzyme in RIFresistant strain is 3 folds higher compared to the Naïve.
NAIVE
FLUX
0.0
1.0
2.0
3.0
4.0
5.0
treS mRNA
F
old
c
h
a
n
g
e
29
Figure 5. RIF-resistant strain resistance to other antimicrobial drugs.
Spot assay plates of Naïve and R1 strains were performed on 7H10 agar medium with different
concentration of antibiotics and decreasing dilution factors (from left to right). Concentration of
antibiotics used are stated under the plate’s picture. R1 is more resistant to the other
antimicrobial drugs even though it’s never been exposed to it.
30
Figure 6. Generation of CRISPRi TreS knock-down strain.
(A) A schematic to show the CRISPRi system knock down mechanism. Plasmids containing the
dcas9 and the target gene sgRNA induced by tetracycline. These plasmids are design to target
specific gene in the DNA sequence by constructed into the backbone of the plasmid. Hence, it is
sterically hinder the RNAP from accessing the target gene and will block mRNA transcription.
Note: Tetracycline induced promoter (Ptet), catalytically dead cas9 gene (dcas9), catalytically
dead cas9 protein (dCas9), single guide RNA (sgRNA), RNA polymerase (RNAP), protospacer
adjacent motif (PAM), non-template strand (NT), template strand (T).
(B) Relative mRNA level of trehalose synthase (TreS) of trehalose synthase knock-down strains.
The suppression of the TreS gene is induced by anhydrotetracycline (ATc). The trehalose
synthase knock-down strain without ATc has significantly higher TreS activity compared to
trehalose knock-down strain treated with 200µg/mL of ATc with a P-value of 0.0003.
0 ATc
200 ATc
0.0
0.5
1.0
1.5
treSMS
Relative mRNA level
0.0003
A B
31
Figure 7. RIF-resistant strain has greater TreS activity.
The treS enzyme activity in Naive, RIF-resistant strain, and CRISPRi treS KD strain (iTres) were
observed by utilizing the fluorescence-activated cell sorting (FACS) analysis. All three strains
were inoculated with RMR-trehalose and analyzed it’s treS enzyme activity. The RIF-resistant
strain has higher TreS activity with TreS activity level of 42.5% compared with the drug
sensitive strain with TreS activity level of 2.35%. The iTres has its treS enzyme gene suppressed
and has TreS activity level of 1.23%. The TreS enzyme responsible for the high TreS activity
level in RIF drug-resistant strain.
32
Figure 8. Suppression of treS gene cause susceptibility to other antimicrobial drugs.
(A) Naïve, R1, and R1 CRISPR + ATC (iTres) were matched to OD 0.02 in 7H9 liquid media
and treated with different concentration of INH and BDQ. R1 is more resistant under INH and
BDQ treatment with IC50 value of 0.9831 and 0.02036, respectively. Naïve and iTres have
similar IC50 value and are more susceptible to INH and BDQ. (B) Naïve, R1, and iTres were
matched to OD 0.1 in 7H9 liquid media and inoculated on 7H10 agar media with different
concentration of INH and BDQ. R1 is more resistant under treatment of INH and BDQ compared
to Naïve and iTres. R1 colonies is also bigger in sizes and more visible.
A
B
33
Figure 9. INH-resistant strain is more susceptible to other antimicrobial drugs.
Spot assay plates of Naïve bacilli (Naïve), INH-resistant strain #1 (I1), and INH-resistant strain
#2 (I2) were performed on 7H10 agar media with different concentration of antimicrobial drugs.
Specific antimicrobial drugs used are stated under the plate’s image. I1 and I2 are more resistant
under 4.8µg/mL of INH treatment compared to the naïve. However, I1 and I2 are more
susceptible under 32 µg/mL of RIF and 0.018µg/mL of BDQ.
34
Figure 10. INH-resistant strains are more susceptible to BDQ and RIF.
(A) A drug sensitivity test was performed on Naïve, I1, and I2. Naïve, I1, and I2 were matched
to OD 0.02 in 7H9 liquid media and treated with different concentration of BDQ. Naïve is more
resistant to BDQ with the highest IC50 value of 1.017, while the IC50 value for I1 is 1.005 and
I2 is 1.004. (B) Colony forming unit assay was performed on Naïve, I1, and I2 with treatment of
BDQ. (C) Colonies observed were counted and used to generate the area under curve (AUC).
Naïve has an AUC value of 0.1316, I1 has an AUC value of 0.07171, and I2 has an AUC value
of 0.07133. Naïve has the highest AUC value and is more resistant under treatment of BDQ. (D)
There is also difference in colony sizes. Naïve colonies size is significantly bigger compared to
I1 and I2. The diameter of the colonies was measured and used to generate the graph. *P <
0.0001, **P < 0.0001, calculated by using one-way ANOVA. (E) Colony forming unit assay was
performed on Naïve, I1, and I2 with different concentration of RIF. (F) Colonies observed were
counted and used to generate the AUC. Naïve has an AUC value of 788.6, I1 has an AUC value
of 421.9, and I2 has an AUC value of 347.3. Naïve has the highest AUC value and is more
resistant under treatment of RIF.
A
B C D
E F
35
Figure 11. INH-resistant strain has lower abundance of mycolic acid.
(A) A schematic of the Mtb cell wall. The Mtb cell wall consists of variable surface glycolipids,
mycolic acids, arabinogalactan, and peptidoglycan. Variable surface glycolipids include TDM,
TMM, PDIM, GPL, etc. (B) The total lipid of Naïve, I1, and I2 were extracted and visualized by
using thin layer chromatography (TLC). The bands on the TLC plates correspond to the
abundance of Mycolic acid. Naïve has higher intensity of the mycolic acid abundance compared
to I1 and I2.
A B
36
Figure 12. INH-resistant strains have lower abundance in three types of mycolic acids.
The mycolic acid of Naïve, I1, and I2 were extracted and visualized by using TLC. The bands on
the TLC plates indicate the 3 types of mycolic acids, which are alpha-mycolic acid, methoxymycolic acid, and keto-mycolic acid. Naïve have higher abundance in all three types of mycolic
acid compared to I1 and I2. This suggests that I1 and I2 have its mycolic acid abundance altered
post exposure of the INH.
37
Figure 13. INH-resistant strains have higher cellular uptake.
(A) The RIF uptake of Naïve, I1, and I2 was measured. All three strains were exposed to
0.78µg/mL of RIF for over the course of 2 days. Samples were taken at different time point to
observe the RIF uptake and were analyzed my using the metabolomics. During the initial time
point, both I1 and I2 have significantly higher RIF uptake compared to Naïve. (B) The ethidium
bromide (EtBr) uptake of Naïve, I1, and I2 was measured. All three strains were exposed to
4µg/mL of EtBr and measured kinetically for 50 minutes. The EtBr uptake were determined by
the slope of linear regression of the fluorescence expression measured from EtBr accumulation
versus time. Naïve have lower EtBr uptake compared to I1 and I2.
A B
38
Figure 14. Metabolomics of RIF-resistant strain and INH-resistant strain.
(A) Metabolomics profile of 339 metabolites extracted from Naïve, R1, and I1 grown on filter
cultures in sodium butyrate with/without trehalose on 7H10 agar media supplemented with 0.5%
bovine serum albumin. (B) PCA score plot of Naïve (blue), R1 (green), and I1 (red) were grown
on filter cultures in sodium butyrate with/without trehalose on 7H10 agar media supplemented
with 0.5% bovine serum albumin in 2D. (C) PCA score plot of Naïve (blue), R1 (green), and I1
(red) were grown on filter cultures in sodium butyrate with/without trehalose on 7H10 agar
media supplemented with 0.5% bovine serum albumin in 3D.
Naïve
I1
R1
Z
A
B
A
C
39
References
1. Abraham, A. O., Nasiru, A. U., Abdulazeez, A. K., Seun, O. O., & Ogonna, D. W. (2020,
February 26). Mechanism of drug resistance in mycobacterium tuberculosis. American
Journal of Biomedical Science & Journal.
https://biomedgrid.com/fulltext/volume7/mechanism-of-drug-resistance-inmycobacterium.001181.php
2. Babu Sait, M. R., Koliwer-Brandl, H., Stewart, J. A., Swarts, B. M., Jacobsen, M.,
Ioerger, T. R., & Kalscheuer, R. (2022, February 8). PPE51 mediates uptake of trehalose
across the mycomembrane of mycobacterium tuberculosis. Nature News.
https://www.nature.com/articles/s41598-022-06109-7
3. Banahene, N., Gepford, D. M., Biegas, K. J., Swanson, D. H., Hsu, Y.-P., Murphy, B. A.,
Taylor, Z. E., Lepori, I., Siegrist, M. S., Obregon-Henao, A., Van Nieuwenhze, M. S., &
Swarts, B. M. (2022, December 7). A far-red molecular rotor fluorogenic trehalose
probe for live mycobacteria detection and drug-susceptibility testing. Angewandte
Chemie (International ed. in English). https://pubmed.ncbi.nlm.nih.gov/36346622/
4. Barrett, T. C., Mok, W. W. K., Murawski, A. M., & Brynildsen, M. P. (2019, March 12).
Enhanced antibiotic resistance development from fluoroquinolone persisters after a
single exposure to antibiotic. Nature News. https://www.nature.com/articles/s41467-019-
09058-4
5. Bendre, Ameya D., et al. “Tuberculosis: Past, Present and Future of the Treatment and
Drug Discovery Research.” Current Research in Pharmacology and Drug Discovery,
vol. 2, 2021, p. 100037., doi:10.1016/j.crphar.2021.100037.
6. Centers for Disease Control and Prevention. (2022, October 13). Drug-resistant TB.
Centers for Disease Control and Prevention.
https://www.cdc.gov/tb/topic/drtb/default.htm
40
7. Chang, C.-H., Chen, Y.-F., Wu, V.-C., Shu, C.-C., Lee, C.-H., Wang, J.-Y., Lee, L.-N., &
Yu, C.-J. (2014, January 13). Acute kidney injury due to anti-tuberculosis drugs: A fiveyear experience in an aging population - BMC infectious diseases. BioMed Central.
https://bmcinfectdis.biomedcentral.com/articles/10.1186/1471-2334-14-23
8. Dartois, V. A., & Rubin, E. J. (2022, April 27). Anti-tuberculosis treatment strategies and
drug development: Challenges and priorities. Nature News.
https://www.nature.com/articles/s41579-022-00731-y
9. Diagnosing and treating tuberculosis. American Lung Association. (n.d.).
https://www.lung.org/lung-health-diseases/lung-disease-lookup/tuberculosis/treating-andmanaging#:~:text=The%20most%20common%20treatment%20for,longer%20than%20ot
her%20bacterial%20infections.
10. Dookie, N., Rambaran, S., Padayatchi, N., Mahomed, S., & Naidoo, K. (2018, May 1).
Evolution of drug resistance in mycobacterium tuberculosis: A review on the molecular
determinants of resistance and implications for personalized care. The Journal of
antimicrobial chemotherapy. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909630/
11. Dupont, C. M., & Kremer, L. (2014, October 20). Extraction and Purification of
Mycobacterial Mycolic Acids. bio-protocol. https://bio-protocol.org/pdf/bioprotocol1265.pdf
12. Eoh, H., Liu, R., Lim, J., Lee, J. J., & Sell, P. (2022). Central carbon metabolism
remodeling as a mechanism to develop drug tolerance and drug resistance in
mycobacterium tuberculosis. Frontiers in Cellular and Infection Microbiology, 12.
https://doi.org/10.3389/fcimb.2022.958240
13. Hameed, S., Sharma, S., & Fatima, Z. (2020). Techniques to understand mycobacterial
lipids and use of lipid-based Nanoformulations for tuberculosis management.
NanoBioMedicine, 433–451. https://doi.org/10.1007/978-981-32-9898-9_18
41
14. Jackson, M. (2014, August 7). The mycobacterial cell envelope-lipids. Cold Spring
Harbor perspectives in medicine.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200213/
15. Jang, J. G., & Chung, J. H. (2020, October). Diagnosis and treatment of multidrugresistant tuberculosis. Yeungnam University journal of medicine.
https://pubmed.ncbi.nlm.nih.gov/32883054/
16. Kalscheuer, R., & Koliwer-Brandl, H. (2014, June 2). Genetics of mycobacterial
trehalose metabolism. Microbiology spectrum.
https://pubmed.ncbi.nlm.nih.gov/26103976/
17. Kastrinsky, D. B., McBride, N. S., Backus, K. M., LeBlanc, J. J., & Barry III, C. E.
(2010, March 17). Mycolic acid/cyclopropane fatty acid/fatty acid biosynthesis and
health relations. Comprehensive Natural Products II.
https://www.sciencedirect.com/science/article/pii/B9780080453828000290
18. Khan, S. R., Manialawy, Y., & Siraki, A. G. (2019). Isoniazid and host immune system
interactions: A proposal for a novel comprehensive mode of action. British Journal of
Pharmacology, 176(24), 4599–4608. https://doi.org/10.1111/bph.14867
19. Lambert, P. A. (2002). Cellular impermeability and uptake of biocides and antibiotics in
gram-positive bacteria and Mycobacteria. Journal of Applied Microbiology, 92.
https://doi.org/10.1046/j.1365-2672.92.5s1.7.x
20. Lee, J. J., Lee, S.-K., Song, N., Nathan, T. O., Swarts, B. M., Eum, S.-Y., Ehrt, S., Cho,
S.-N., & Eoh, H. (2019, July 2). Transient drug-tolerance and permanent drug-resistance
rely on the trehalose-catalytic shift in mycobacterium tuberculosis. Nature News.
https://www.nature.com/articles/s41467-019-10975-7
42
21. Lee, Seung Heon. “Tuberculosis Infection and Latent Tuberculosis.” Tuberculosis and
Respiratory Diseases, U.S. National Library of Medicine, Oct. 2016,
www.ncbi.nlm.nih.gov/pmc/articles/PMC5077723/.
22. Liebenberg, D., Gordhan, B. G., & Kana, B. D. (2022, September 23). Drug resistant
tuberculosis: Implications for transmission, diagnosis, and disease management.
Frontiers in cellular and infection microbiology.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9538507/
23. Marrakchi, H., Lanéelle, M.-A., & Daffé, M. (2014). Mycolic acids: Structures,
biosynthesis, and beyond. Chemistry & Biology, 21(1), 67–85.
https://doi.org/10.1016/j.chembiol.2013.11.011
24. Murphy, K. C., Nelson, S. J., Nambi, S., Papavinasasundaram, K., Baer, C. E., &
Sassetti, C. M. (2018). Orbit: A new paradigm for genetic engineering of mycobacterial
chromosomes. mBio, 9(6). https://doi.org/10.1128/mbio.01467-18
25. Nahid, P., Mase, S. R., Migliori, G. B., Sotgiu, G., Bothamley, G. H., Brozek, J. L.,
Cattamanchi, A., Cegielski, J. P., Chen, L., Daley, C. L., Dalton, T. L., Duarte, R.,
Fregonese, F., Horsburgh, C. R., Ahmad Khan, F., Kheir, F., Lan, Z., Lardizabal, A.,
Lauzardo, M., … Seaworth, B. (2019, November 15). Treatment of drug-resistant
tuberculosis. an official ATS/CDC/ERS/IDSA Clinical Practice guideline. American
journal of respiratory and critical care medicine.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857485/
26. Padda, I. S., & Reddy, K. M. (2023, June 3). Antitubercular medications. StatPearls
[Internet]. https://www.ncbi.nlm.nih.gov/books/NBK557666/
27. Pohane, A. A., Carr, C. R., Garhyan, J., Swarts, B. M., & Siegrist, M. S. (2021, January
19). Trehalose recycling promotes energy-efficient biosynthesis of the mycobacterial cell
envelope. mBio. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845637/
43
28. Portevin, D., Sukumar, S., Coscolla, M., Shui, G., Li, B., Guan, X. L., Bendt, A. K.,
Young, D., Gagneux, S., & Wenk, M. R. (2014). Lipidomics and genomics
of mycobacterium tuberculosis reveal lineage‐specific trends in mycolic
acid biosynthesis. MicrobiologyOpen, 3(6), 823–835. https://doi.org/10.1002/mbo3.193
29. Quigley, J., & Lewis, K. (n.d.). Noise in a metabolic pathway leads to persister formation
in mycobacterium tuberculosis. Microbiology spectrum.
https://pubmed.ncbi.nlm.nih.gov/36194154/#:~:text=antibiotic%2Dtolerant%20state.-,M.
,These%20cells%20are%20termed%20persisters.
30. Rahlwes, K. C., Dias, B. R. S., Campos, P. C., Alvarez-Arguedas, S., & Shiloh, M. U.
(2023). Pathogenicity and virulence of mycobacterium tuberculosis. Virulence,
14(1). https://doi.org/10.1080/21505594.2022.2150449
31. Rattan, A., Kalia, A., & Ahmad, N. (1998). Multidrug-resistant mycobacterium
tuberculosis: Molecular perspectives. Emerging infectious diseases.
https://pubmed.ncbi.nlm.nih.gov/9621190/
32. Rock, J. M., Hopkins, F. F., Chavez, A., Diallo, M., Chase, M. R., Gerrick, E. R.,
Pritchard, J. R., Church, G. M., Rubin, E. J., Sassetti, C. M., Schnappinger, D., &
Fortune, S. M. (2017, February 6). Programmable transcriptional repression in
mycobacteria using an orthogonal CRISPR interference platform. Nature microbiology.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302332/
33. Russell, D. G. (n.d.). Mycobacterium tuberculosis: Here Today, and here Tomorrow.
Nature News. https://www.nature.com/articles/35085034
34. Sakamoto, K. (2012, January 18). The Pathology of Mycobacterium tuberculosis
infection . Sage Journals.
https://journals.sagepub.com/doi/full/10.1177/0300985811429313
44
35. Sarathy, J., Dartois, V., Dick, T., & Gengenbacher, M. (2013). Reduced drug uptake in
phenotypically resistant nutrient-starved nonreplicating mycobacterium tuberculosis.
Antimicrobial Agents and Chemotherapy, 57(4), 1648–1653.
https://doi.org/10.1128/aac.02202-12
36. Sebastian, J., Swaminath, S., Nair, R. R., Jakkala, K., Pradhan, A., & Ajitkumar, P.
(2017, January 24). De novo emergence of genetically resistant mutants of
mycobacterium tuberculosis from the persistence phase cells formed against
antituberculosis drugs in vitro. Antimicrobial agents and chemotherapy.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278719/
37. Sengupta, S., Sengupta, A., Hussain, A., Sarma, J., Banerjee, A., Pandey, S., Tripathi, D.,
Kumar, A., & Peddireddy, V. (2022, September 30). Modulation of host pathways by
mycobacterium tuberculosis for survival. Bacterial Survival in the Hostile Environment.
https://www.sciencedirect.com/science/article/pii/B9780323918060000035
38. Shahid, N. U. A., Naguit, N., Jakkoju, R., Laeeq, S., Reghefaoui, T., Zahoor, H., Yook, J.
H., Rizwan, M., & Mohammed, L. (2022, May 17). Use of isoniazid monotherapy in
comparison to rifamycin-based regimen for the treatment of patients with latent
tuberculosis: A systematic review. Cureus.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9205649/
39. Singh, A., Gupta, A. K., Katiyar, A. S., & Sharma, D. (2022, September 30). Mechanisms
of biofilm-based antibiotic resistance and tolerance in mycobacterium tuberculosis.
Bacterial Survival in the Hostile Environment.
https://www.sciencedirect.com/science/article/pii/B9780323918060000011
40. Smet, K. A. L. D., Weston, A., Brown, I. N., Young, D. B., & Robertson, B. D. (2000,
January 1). Three pathways for trehalose biosynthesis in mycobacteria.
microbiologyresearch.org.
https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-146-1-
199#tab2
45
41. Sotgiu, G., D’ambrosio, L., & Migliori, G. B. (2015). Tuberculosis Treatment and Drug
Regimens. Cold Spring Harb Perspect Med. .
https://doi.org/10.1101/cshperspect.a017822
42. Sterling , T. R., Zenner, D., Cohn , D. L., Reves, R., Ahmed, A., Menzies, D.,
Horsburgh , R., Crane, C. M., Burgos, M., LoBue, P., Winston, C. A., & Belknap , R.
(2020, February 13). Guidelines for the treatment of latent tuberculosis infection:
Recommendations from the National Tuberculosis Controllers Association and CDC,
2020. Centers for Disease Control and Prevention.
https://www.cdc.gov/mmwr/volumes/69/rr/rr6901a1.htm?s_cid=rr6901a1_w
43. Suresh, A. B., Rosani, A., Patel, P., & Wadhwa, R. (2023, November 12). Rifampin.
StatPearls [Internet].
https://www.ncbi.nlm.nih.gov/books/NBK557488/#:~:text=Rifampin%2C%20also%20k
nown%20as%20rifampicin,and%20gram%2Dpositive%20bacterial%20infections.
44. Thanna, S., & Sucheck, S. J. (2016, October 16). Targeting the trehalose utilization
pathways of mycobacterium tuberculosis. MedChemComm.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770839/#:~:text=Trehalose%20is%20a
n%20essential%20metabolite,and%20osmotic%20stress%20in%20bacteria.
45. Torrey, H. L., Keren, I., Via, L. E., Lee, J. S., & Lewis, K. (n.d.). High persister mutants
in mycobacterium tuberculosis. PLOS ONE.
https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0155127
46. Tuberculosis (TB). Cedars Sinai. (n.d.). https://www.cedars-sinai.org/healthlibrary/diseases-and-conditions/t/tuberculosistb.html#:~:text=There%20are%203%20stages%20of,its%20spread%20to%20other%20p
eople.
47. Tuberculosis Drugs and Mechanisms of Action . National Institute of Allergy and
Infectious Diseases. (2016, April 19).
46
48. “Tuberculosis (TB).” Centers for Disease Control and Prevention, Centers for Disease
Control and Prevention, 22 Mar. 2023, www.cdc.gov/tb/default.htm.
49. Vilchèze, Catherine, and William R Jacobs. “Resistance to Isoniazid and Ethionamide in
Mycobacterium Tuberculosis: Genes, Mutations, and Causalities.” Microbiology
Spectrum, U.S. National Library of Medicine, Aug. 2014,
www.ncbi.nlm.nih.gov/pmc/articles/PMC6636829/.
50. von Both, U., Berk, M., Agapow, P.-M., Wright, J. D., Git, A., Hamilton, M. S., Goldgof,
G., Siddiqui, N., Bellos, E., Wright, V. J., Coin, L. J., Newton, S. M., & Levin, M. (2018,
January 12). Mycobacterium tuberculosis exploits a molecular off switch of the immune
system for intracellular survival. Nature News. https://www.nature.com/articles/s41598-
017-18528-
y#:~:text=Abstract,molecular%20mechanisms%20are%20poorly%20understood.
51. Wang, H., Liu, D., & Zhou, X. (2023). Effect of mycolic acids on host immunity and
lipid metabolism. International Journal of Molecular Sciences, 25(1), 396.
https://doi.org/10.3390/ijms25010396
52. Windels, E. M., Michiels, J. E., Fauvart, M., Wenseleers, T., Van den Bergh, B., &
Michiels, J. (2019, May). Bacterial persistence promotes the evolution of antibiotic
resistance by increasing survival and mutation rates. The ISME journal.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474225/#:~:text=Replicating%20cells
%20develop%20resistance%20according,a%20constant%20rate%20%C2%B5persisters.
53. World Health Organization. (2023, November 6). Global tuberculosis report factsheet
2023. World Health Organization. https://www.who.int/publications/m/item/globaltuberculosis-report-factsheet-2023
47
54. World Health Organization. (n.d.-a). Tuberculosis (TB). World Health Organization.
https://www.who.int/news-room/fact-sheets/detail/tuberculosis
55. World Health Organization. (n.d.-b). Types of TB drug-resistance. World Health
Organization. https://www.who.int/teams/global-tuberculosis-programme/diagnosistreatment/treatment-of-drug-resistant-tb/types-of-tb-drug-resistance
56. Zhang, Y., Yew, W. W., & Barer, M. R. (2012, May). Targeting persisters for
tuberculosis control. Antimicrobial agents and chemotherapy.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346619/
Abstract (if available)
Abstract
Tuberculosis (TB) is an infectious disease that is cause by Mycobacterium tuberculosis (Mtb). TB remains as one of the top leading infectious killers worldwide and a major contributor of antimicrobial resistance. Effective treatment for TB is destabilized by the presence of multidrug-resistant (MDR) TB as there has been no effective treatment method available to treat MDR TB. MDR TB is Mtb strains that are resistant to two first-line TB antibiotics, which are isoniazid (INH) and rifampicin (RIF). Understanding the mechanisms underlying the emergence of MDR and exploring new treatment approaches is crucial in combating this growing public health concern. Mtb that can survive under various stress conditions is known as latent tuberculosis wherein Mtb resides in a state of persister cells. Persister cells are bacterial cells that have acquired a tolerance to various stress conditions and remain viable. From previous findings, Mtb trehalose metabolism could trigger the formation of transient drug tolerant fraction and this metabolic change can also lead to massive development of drug-resistant mutants [20]. The RIF-resistant strain has shown an increased in trehalose catalytic shift activity and exhibited cross-resistance to other antimicrobial drugs. For instances, the RIF-resistant strain exhibit resistance to INH and bedaquiline (BDQ). In a contrary, the INH-resistant strain had become more susceptible to other antimicrobial drugs due to alterations in their cell wall integrity and subsequent induction of antibiotic uptake. Due to the increase in their trehalose catalytic shift, the RIF-resistant strain serves as a main source to develop into MDR TB strain. Developing TB treatments by targeting the trehalose catalytic shift in Mtb could be a breakthrough to prevent the emergence of MDR TB.
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Targeting trehalose catalytic shift as a novel strategy to control the emergence of multidrug-resistant tuberculosis
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Molecular Microbiology and Immunology
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metabolomics,multidrug-resistant TB,mutation assay,mycobacteria,Mycobacterium smegmatis,Mycobacterium tuberculosis,OAI-PMH Harvest,persister cell,trehalose metabolism,trehalose synthase (TreS),tuberculosis (TB)
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metabolomics
multidrug-resistant TB
mutation assay
mycobacteria
Mycobacterium smegmatis
Mycobacterium tuberculosis
persister cell
trehalose metabolism
trehalose synthase (TreS)
tuberculosis (TB)