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Molecular and genetic epidemiology of Chlamydia trachomatis in the United States

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Molecular and genetic epidemiology of Chlamydia trachomatis in the United States

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MOLECULAR AND GENETIC EPIDEMIOLOGY OF CHLAMYDIA
TRACHOMATIS IN THE UNITED STATES
by
Kim Lori Millman
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(EPIDEMIOLOGY)
August 2005
Copyright 2005 Kim Lori Millman
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UMI Number: 3196859
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ii
TABLE OF CONTENTS
LIST OF TABLES iv
LIST OF FIGURES v
ABSTRACT vii
CHAPTER 1. Literature Review 1
Taxonomy 1
Clinical Relevance 4
Clinical Features of Chlamydia trachomatis Genital
Infection 7
Epidemiology of Genital Infections by Chlamydia
trachomatis 12
Chlamydial Developmental Cycle 16
Chlamydial Outer Membrane Structure 24
Molecular Genetics 31
Serotyping of Chlamydia trachomatis 35
OmpA Chlamydia trachomatis Serotyping and
Genotyping Studies 37
Virulence Factors and Pathogenesis 46
CHAPTER 2. Recombination in the ompA Gene but Not the
omcB Gene of Chlamydia Contributes to Serovar-Specific
Differences in Tissue Tropism, Immune Surveillance and
Persistence of the Organism. 50
Introduction 50
Materials and Methods 52
Results 64
Discussion 85
CHAPTER 3. Population-Based Genetic and Evolutionary
Analysis of Chlamydia trachomatis Urogenital Strain
Variation in the United States. 94
Introduction 94
Materials and Methods 95
Results 103
Discussion 127
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iii
CHAPTER 4. Population-based genetic epidemiologic
analysis of Chlamydia trachomatis urogenital infections in
the United States and association with symptoms and
clinical disease. 135
Introduction 135
Materials and Methods 137
Results 142
Discussion 149
BIBLIOGRAPHY 158
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LIST OF TABLES
Chapter 2:
Table 2-1. Properties of Chlamydial Strains Investigated
Table 2-2A. Sawyer’s Runs Test Results for ompA Intraspecies
Comparisons for C. trachomatis and C. psittaci
Table 2-2B. Sawyer's Runs Test Results for ompA Interspecies
Comparisons between C. trachomatis, C. pneumoniae and C. psittaci
Table 2-2C. Sawyer's Runs Test Results for omcB Intraspecies
Comparisons for C. trachomatis
Table 2-2D. Sawyer's Runs Test Results for omcB Interspecies
Comparisons between C. trachomatis and C. pneumoniae
Table 2-3. Break Point Analysis of Potential ompA Mosaics
Chapter 3:
Table 3-1. OmpA Primer Sequences Used for Sequence Generation
Table 3-2. Genetic Description of Unique Sequences in this
Population Organized by Serovar
Table 3-3. Distribution of Chlamydia trachomatis Serovars by
Metropolitan Area in the United States and by Gender
Chapter 4:
Table 4-1. Demographic Distribution for the Study Population from
Five Metropolitan Cities in the United States
Table 4-2. Adjusted Odds Ratios for Risk of Chlamydial-Probable
Symptoms and for Risk of Having M ucopurulent Cervicitis, Vaginal
or Urethral Discharge According to the Significant Independent
Predictors Measured in the Population-Based Study
Table 4-3. Chlamydial-Probable Symptom Status across all
Chlamydial Serotypes and Stratified by Gender
Table 4-4. Odds Ratios for Abnormal Vaginal Bleeding According to
Character States at Specific Variable ompA Positions
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V
LIST OF FIGURES
Chapter 2:
Figure 2-1A. Phylogenetic Reconstruction for the ompA Gene
Sequences using the Neighbor-Joining Method
Figure 2-1B. Phylogenetic Reconstruction for the omcB Gene
Sequences using the Neighbor-Joining Method
Figure 2-2A. Compatibility Matrix for the Chlamydia ompA Gene from
Human Hosts
Figure 2-2B. Compatibility Matrix for the Chlamydia ompA Gene from
Lower Vertebrate Mammals and Birds
Figure 2-2C. Compatibility Matrix for the omcB Gene from Human
Hosts
Figure 2-3. Linkage Between Polymorphic Codons within Successive
ompA Regions, Approximately 20 Polymorphisms in Length
Figure 2-4A. RIP Images Depicting the Similarity of Strain D/B120
Against all Others with Position along the ompA Gene
Figure 2-4B. RIP Images Depicting the Similarity of Strain G/UW-57
Against all Others with Position along the ompA Gene
Figure 2-4C. RIP Images Depicting the Similarity of Strain E/Bour
Against all Others with Position along the ompA Gene
Figure 2-4D. RIP Images Depicting the Similarity of Strain LGV-98
Against all Others with Position along the ompA Gene
Figure 2-5. Mosaic Structures for Strains E/Bour, G/UW-57, LGV-98
and D/B-120 Predicted by the Maximum Chi-Squared Test
Chapter 3:
Figure 3-1. Alignment of Ba/D Recombinant Compared to Ba and D
Prototype Sequences
Figure 3-2A. Phylogenetic Reconstructions for the Unique ompA Gene
Sequences in the B Seroclass using the Neighbor-Joining Method
Figure 3-2B. Phylogenetic Reconstructions for the Unique ompA Gene
Sequences in the Intermediate Seroclass using the Neighbor-Joining
Method
Figure 3-2C. Phylogenetic Reconstructions for the Unique ompA Gene
Sequences in the C Seroclass using the Neighbor-Joining Method
Figure 3-3. Mean Hamming Distance for Each Serotype Group Based
on Hamming Distance between all Possible Pairs of Similar Serotype
Sequences
Figure 3-4. Mean Synonymous Mutation Rate; Nonsynonymous
Mutation Rate and Synonymous to Nonsynonymous Mutation Rate
Ratio for Serotype Groups Based on the Method of Nei and Gojobori
65
66
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116
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120
122
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Figure 3-5A. Unimodal Population Structure of Serotype E
Figure 3-5B. Unimodal Population Structure with Shoulder of
Serotype D with Main and Shoulder Group Roughly 99.7% Similar
Figure 3-5C. Bimodal Population Structure of Serotype J with the
Two Main Groups Roughly 98.5% Similar
Figure 3-6. Phylogenetic Reconstructions with Starburst
Superimposed at End of Branch Corresponding to Appropriate
Serotype Group
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vii
ABSTRACT
Chlamydia trachomatis is the leading bacterial cause of sexually
transmitted diseases in the world today. Since a large proportion of
chlamydial genital infections are asymptomatic, sequelae such as pelvic
inflammatory disease (PID), ectopic pregnancy, and infertility may progress
insidiously. For this reason, significant effort has been devoted to
developing an effective vaccine. The major outer membrane protein
(MOMP), encoded by ompA, has been a major target of vaccine research as it
is one of the most highly antigenic proteins known for Chlamydia. In order to
facilitate vaccine development, this dissertation investigated the genetic and
molecular epidemiology of Chlamydia, focusing on the outer membrane
protein genes, ompA and omcB.
In Chapter 1, literature pertaining to chlamydial molecular and
cellular biology as well as molecular and genetic epidemiology is reviewed.
In Chapter 2, we analyzed ompA and omcB sequences for evidence of
intragenic recombination among the three species of Chlamydia. Multiple
analyses provided consistent evidence that there has been a history of
intragenic recombination at ompA including one instance of interspecies
recombination, but not at omcB.
In Chapter 3, we investigated the genetic variation of Chlamydia
trachomatis ompA in the United States. We generated and analyzed 507
nearly complete ompA sequences from urogenital clinical samples from five
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metropolitan areas. Among the serovariant groups, a unimodal population
structure was predominantly observed, consistent with a rapid expansion of
strains with little time for propagation of mutations. For most serovariant
groups, nonsynonymous to synonymous mutation rate ratios indicated that '
ompA is primarily under purifying selection and that it has many functional
constraints. However, the serovars with rapid expansion were under a
greater degree of diversifying selection.
In Chapter 4, we correlated the clinical phenotype for 180 men and
164 women whose clinical samples were sequenced with the serovariant
group and genotype established in Chapter 3. Based on multiple logistic
regression analyses, neither serotype nor genotype were predictive of clinical
phenotype except for an association between serovariant F and PID;
although, the number of cases was small. These results suggest there is little
if any prognostic information yielded from strain typing based on ompA for
urogenital infections.
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CHAPTER 1. Literature Review
Taxonomy
Microorganisms of the bacterial order and phylogenetic division
Chlamydiales are obligate intracellular parasites. Until recently, only four
species were recognized in the single genus, Chlamydia, belonging to the only
family, Chlamydiaceae, in the order. Currently, Chlamydiales contains
organisms of the four well-known Chlamydia species (Chlamydia trachomatis,
C. pneumoniae, C. psittaci, and C. pecorum) and recently described Chlamydia
like organisms. The latter have been isolated from clinical tissues and
environmental sources including: Simkania sp. (family Simkaniaceae),
originally coined the "Z" organism, isolated from a contaminated cell culture
(Kahane et al., 1993); Waddlia chondrophila sp. (family Waddliaceae) from an
aborted bovine fetus (Kahane et al., 1993; Rurangirwa et al., 1999); and the
two organisms, Parachlamydia acanthamoebae spp. and Neochlamydia
hartmanellae spp. (family Parachlamydiaceae), from the cytoplasms of free-
living Acanthamoebaes sp. (Amann et al., 1997; Horn et al., 2000). After the
development of a broad range PCR assay, 30 additional Chlamydia-like
organisms were identified and it is likely that others will continue to emerge
(Meijer and Ossewaarde, 1998; Meijer, Roholl and Ossewaarde, 2000). All
organisms in the order are obligate intracellular pathogens, however,
differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) profiles, growth cycles, and drug sensitivities justify placement
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of the Chlamydia-like organisms in families separate from Chlamydiaceae
(Kahane et al., 1993).
Within the genus Chlamydia, 16S rRNA sequences are at least 94%
similar, and thus, the basic cellular processes are expected to be comparable
(Tanner, Harris, and Pace, 1999). The relative degree of similarity in these
processes is currently under investigation by comparison of newly acquired
complete genome sequences for two C. trachomatis strains and three C.
pneumoniae strains. Historically, differences in biochemical characteristics
and morphology have distinguished the four species. Of the four, C.
trachomatis shares fewer characteristics with the others, reflected by the fact
that it exhibits the greatest between-species strain divergence. For example,
C. trachomatis accumulates glycogen in the inclusion, visualized by staining
with iodine, while C. pneumoniae and C. psittaci do not (Moulder, 1991; Kuo et
al., 1986; Grayston et al., 1989). In terms of morphology, C. pneumoniae is
predominantly pear-shaped while C. trachomatis and C. psittaci have mainly
spherical elementary bodies (Chi et al., 1987).
With respect to differences within the species, C. psittaci is the most
heterogeneous. It has the greatest within-species strain divergence and the
widest host range. C. psittaci infects a large number of mammalian and avian
species, as well as humans, although humans appear to be dead-end hosts
for infection. On the other hand, humans are the primary hosts for C.
trachomatis and C. pneumoniae. The exceptions are that C. trachomatis can also
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infect rodents (Nigg, 1942; Stills et al., 1991) and swine (Kaltenbock et al.,
1997) and C. pneumoniae can also infect horses and koalas (Storey et al., 1993;
Girjes, Carrick, and Lavin, 1994). C. pecorum also has a narrow host range.
To date, it has only been found to infect ruminants and koalas (Fukushi and
Hirai, 1992).
An emended description of the order Chlamydiales was accepted for
publication in the International Journal for Systematic Bacteriology in 1999
(Everett, Bush, and Andersen, 1999) and after was presented at the Fourth
Meeting of the European Society for Chlamydia Research in Helsinki, Finland,
August, 2000 (Everett, Bush, and Andersen, 2000). The system was based on
clustering patterns observed in phylogenetic reconstructions consistent
across the 16S rRNA and 23S rRNA gene sequences and five major coding
regions. The system proposes to split the single order Chlamydiales into at
least four families including Chlamydiaceae nov.; Parachlamydiaceae nov.;
Simkaniaceae nov.; and Waddliaceae nov. The family Chlamydiaceae would
hold two genera, Chlamydia and Chlamydophila nov.. The genus Chlamydia
would have three species, Chlamydia trachomatis, and Chlamydia muridarum
nov., and Chlamydia suis nov.. The novel genus Chlamydophila would have
six species: Chlamydophila pneumoniae, Chlamydophila abortus nov.,
Chlamydophila psittaci, Chlamydophila felis nov., Chlamydophila caviae nov., and
Chlamydophila pecorum nov.. The proposed taxonomy has met with sizable
opposition from researchers in the Chlamydia community. The greatest
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opposition seemed to be against the division of the single genus into two
genera and not against the division of the four species into a total of nine.
The opponents argued that based on current knowledge there are no
significant biological justifications to the new divisions and that historically,
taxonomy has not been driven solely by phylogeny. Proponents of the
system argue that species in the new system have a more homogeneous host
range and that phylogenetic differences consistent across many coding
regions may represent biologic differences that are not currently appreciated.
At this time, it is up to authors and reviewers of peer-reviewed publications
as to whether they will use the old or new taxonomy system in their
publications. For this dissertation, the old system will be used since it is the
most widely accepted.
Clinical Relevance
Chlamydiae are bacteria of major clinical importance. The two
primarily hum an pathogens C. trachomatis and C. pneumoniae represent a
substantial economic and public health burden due to their prevalence,
debilitating sequelae and potential association with multiple chronic health
conditions. C. trachomatis is the leading cause of sexually transmitted
diseases (STD) in the United States and the developed world (Centers for
Disease Control and Prevention, 1997; Dean, 1999a; Dean, 1999b). Genital
infection with C. trachomatis is common, with approximately 90 million new
cases occurring per year worldwide (Gerbase et al., 1998). Over 4 million
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cases per year occur in the United States at an annual estimated health care
cost of $2.18 billion (Centers for Disease Control and Prevention, 1997).
Among women, chlamydial infection can frequently progress to pelvic
inflammatory disease (PID), which in turn can cause tubal and extra-lumenal
scarring that results in ectopic pregnancies, tubal infertility, and chronic pelvic
pain. Since infection is often asymptomatic and insidious, diagnosis and
treatment is often delayed and ineffective in preventing these sequelae. For
this reason, adolescent young women, the population at highest risk, are
screened for Chlamydia as part of routine gynecologic care and vaccine
administration has been recommended for its prevention (Centers for
Disease Control and Prevention, 1993). C. trachomatis is also the leading
cause of preventable blindness in the developing world (Centers for Disease
Control and Prevention, 1997; Dean, 1999a; Dean, 1999b). Ocular infection
can result in trachoma, a disease that initially presents with conjunctivitis.
With repeated or persistent infection, it often progresses to cause distortion
of the lids, trichiasis, corneal fibrosis and eventually blindness. The
worldwide prevalence of trachoma is estimated to be 400 to 600 million cases
in areas where the disease is endemic, with an estimated 6 million of those
cases progressing to blindness (Thylefors et al., 1995).
C. pneumoniae is an important cause of community-acquired
pneumonia (Saikku et al., 1985) and has also been implicated in the etiology
of multiple chronic health conditions including atherosclerosis, stroke,
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Alzheimer's disease and multiple schlerosis (Braun et al., 1994; Saikku, 1999).
Infection by C. pneumoniae is ubiquitous. More than 60% of adults have had
some exposure during their lifetime (Schachter and Grayston, 1998). Even
with the high prevalence of infection, respiratory disease attributed to C.
pneumoniae occurs predominantly in school-aged children, young adults and
older individuals with chronic illness. In the young, it is most often mild in
presentation, while in the elderly infection can cause more severe disease.
In hospital settings, it is estimated that 6 to 10% of community-acquired
pneumonia is due to C. pneumoniae (Schachter, 1999).
Of the multiple chronic health conditions with which C. pneumoniae is
postulated to be associated, the greatest body of evidence supports an
association between C. pneumoniae and coronary artery disease (CAD),
although the causal evidence is not definitive. At least 50 seroepidemiologic
studies have measured antibody to whole chlamydial antigen to determine if
prior infection is associated with CAD. Many of these studies were plagued
by potential cross-reactivity of antigen to other serum antibodies, inadequate
control of confounding, small sample size, and study designs that were
mostly retrospective. Although direct detection studies found C. pneumoniae
DNA or antigen in diseased arterial tissues, the evidence that C. pneumoniae
was causally associated with disease was not compelling. Preliminary
antibiotic intervention studies in humans have been inconclusive with
benefits observed in one study and none observed in two others. Probably
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the most significant evidence that supports a causal association between C.
pneumoniae infection and CAD came from experimental animal models of
pulmonary infection. New Zealand white rabbits infected with C.
pneumoniae fed a diet with small amounts of cholesterol had larger arterial
lesions than uninfected rabbits fed the same diet. Importantly, early
treatment of infection with azithromycin prevented these lesions. Evidence
to suggest associations between C. pneumoniae and other chronic health
conditions is less substantial and while additional studies are needed to
elucidate these relationships, it seems implausible that C. pneumoniae
infection is a significant risk factor for all of the proposed conditions.
C. psittaci infection in humans is rare. It is estimated that only about
800 cases of psittacosis have been reported in the United States over the ten-
year period from 1987 to 1996 (Centers for Disease Control and Prevention,
1998). Psittacosis most often presents as atypical pneumonia or unusual
febrile disease and is associated with psittacine avian exposure. Several
outbreaks have been reported in turkey farms in the United States and duck
farms in Europe (Schachter, 1999).
Clinical Features of Chlamydia trachomatis Genital Infection
Currently, classification of C. trachomatis is based on the serological
recognition of antigenic epitopes on the major outer membrane protein
(MOMP). Serotyping has identified 18 hum an serological variants or
serovars (A-K, Ba, Da, la, L1-L3 and L2a; Dean, Suchland, and Stamm, 2000;
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Wang and Grayston, 1991). The hum an serovars have been subdivided into
two biovars based on clinical presentation: the trachoma biovar (A-K, Ba, Da,
and la) and the lymphogranuloma venereum (LGV) biovar (Ll-3, and L2a).
Within the trachoma biovar, serovars A, B, Ba, and C are the primary cause
of trachoma, a chronic ocular disease found predominantly in developing
countries of the world. Serotypes D, Da, E, F, G, H, I, la, J, Ja, and K cause
lower genital tract infections (LGTIs) and upper genital tract infections
(UGTIs; Dean, 2002). The LGV biovar, including serovars LI, L2, L2a and L3,
tend to cause more severe clinical disease, ranging from purulent
conjunctivitis to supperative inguinal lymphangitis and proctitis (Dean,
1997).
Serovars D-K of the trachoma biovar infect the superficial columnar
epithelium of the endocervix, urethra, epididymis, endometrium, oviduct
and rectum. Histopathologically, the chlamydial infection induces a
neutrophilic exudate into the mucosa associated with infiltration of the
lamina propria by activated T and B lymphocytes, histocytes, macrophages
and dendritic cells (Brunham, 1999). Discretely organized lymphoid follicles
or germinal centers are often found in upper reproductive tract infection
(Brunham, 1999).
In most individuals, a combination of systemic, mucosal humoral and
cellular immune responses including CD4 Thi -dependent cytokines,
neutralizing mucosal IgA antibodies and epithelial cell shedding contribute
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to clearing the chlamydial infection, usually over a period of a few weeks to
a few months. However, in some individuals, the chlamydial infection is not
cleared over time, but instead recurs. These recurrent infections can
represent either 1) a persistent infection from a treatment failure in the
individual; 2) a re-infection from a treatment failure in the sexual partner of the
individual when a condom was not used; or 3) a new infection from either a
new or old partner when a condom was not used. These distinctions may be
crucial as risk factors and intervention strategies may differ accordingly.
Importantly, it is recurrent infection that may be primarily responsible for the
tubal fibrosis that leads to tubal occlusion (Patton and Kuo, 1989). Thus, much
of the tissue pathology associated with the long-term sequelae of chlamydial
genital infections may be a result of recurrent, rather than initial infection.
Unexpectedly, C. trachomatis associated tissue pathology often
develops despite relatively benign symptoms and signs. In clinical studies, C.
trachomatis colonization of the tube was seen in infertile women with no
symptoms and no laparoscopic signs of active pelvic infection (Wolner-
Hanssen, Kiviat, and Holmes, 1990). Thus, an asymptomatic or seemingly
mild clinical presentation may, in fact, have considerable tissue damage, not
evident until it is exposed inadvertently or until it is manifested in late
functional or fertility impairments.
Cervicitis. The three essential findings in chlamydial cervicitis are
mucopurulent exudate of the endocervix, friability of the endocervical
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mucosa and laboratory evidence of Chlamydia. Since multiple STDs are
common and cervicitis caused by Chlamydia is often clinically
indistinguishable from that caused by other organisms, it is often difficult to
determine the causative agent in cervicitis. Of those cases that can be
ascribed to Chlamydia, more than 70% are asymptomatic (Schachter et al.,
1983). Also, in more than one-third of chlamydial cervicitis, cervical
infection has been shown to ascend into the endometrium (Jones et al., 1986).
Salpingitis. Chlamydial PID is difficult to accurately diagnose
without invasive procedures. Clinical suspicion is warranted when two of
the three signs, adnexal, fundal and cervical motion tenderness, are elicited
on physical examination with laboratory evidence of Chlamydia. Other major
signs and symptoms include mid-cycle bleeding, fever, and lower abdominal
pain. It is widely believed that "silent salpingitis", tubal damage without any
signs and symptoms, may be more common than clinically apparent PID.
These cases can only be diagnosed through invasive procedures and without
them they are deemed clinically uncomplicated cases. It is estimated that 12
to 13% of women with one attack of chlamydial PID will become infertile;
while 75% of women with more than two episodes will become infertile
(Westrom, 1975).
Female urethral syndrome. A combination of frequency, dysuria and
sterile pyuria constitute this syndrome that disproportionately affects
adolescent or college-aged young women. In this younger age group as
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many as 62% of women are infected with C. trachomatis (Stamm et al., 1980).
In older women, the chlamydial contribution is much less.
Male urethritis. Nongonococcal urethritis (NGU) is characterized by
dysuria with or without discharge in the absence of laboratory evidence of N.
gonorrhoeae (GC) infection. In 30 to 50% of these cases, Chlamydia is the
etiologic agent. In 11 to 30% of men with gonococcal urethritis, a chlamydial
infection is also present (Oriel and Ridgway, 1982).
Epididymitis. Any of the signs and symptoms of NGU may be
present. Painful enlargement of the epididymis and often fever differentiates
epididymitis from NGU. In young sexually active men, chlamydiae may
cause up to 50% of cases of epididymitis (Schachter, 1999).
Arthritis (Reiter’s syndrome). The full clinical tetrad of Reiter's
syndrome is comprised of urethritis, conjunctivitis (or uveitis),
mucocutaneous lesions and arthritis. The arthritic component is most often
asymmetric, primarily affects the large weight-bearing joints including the
knee, ankle and the sacroiliac joints and often occurs one to four weeks from
the onset of urethritis. Most often, only arthritis follows urethritis. At least
one third of cases of Reiter's syndrome may be attributed to C. trachomatis
infection however, this estimate may be too conservative. Other estimates
based on serological evidence suggest as many as one half to one third of
these cases are due to C. trachomatis. Reiter's syndrome is an auto-immune
condition in which two thirds of those with the syndrome have the
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histocompatibility antigen HLA-B27. It has been postulated that C.
trachomatis antigens act as a trigger in susceptible individuals.
Epidemiology of Genital Infections by Chlamydia trachomatis
Prevalence rates for the group at highest risk for urogenital
chlamydial infection, sexually active adolescent women, exceed 10% in the
United States (Centers for Disease Control and Prevention, 1993). Among
women, the greatest prevalence rates were seen at STD clinics (15-19%; Kent,
Harrison, and. Berman, 1988, Magder et al., 1988); mid-range rates were seen
at family planning clinics (6-13%; Weinstock, Dean, and Bolan, 1995); while
the lowest prevalence rates were seen at university (5-10%; Weinstock, Dean,
and Bolan, 1995) and prenatal clinics (4-8%; Weinstock, Dean, and Bolan,
1995). Since women are more likely to be screened, prevalence rates are
estimated to be seven times higher for women than men (Centers for Disease
Control and Prevention, 1993).
Among women, young age (<25 years) is by far the strongest and
most consistent of all risk factors (Weinstock, Dean, and Bolan, 1995).
Increased risk of infection and increased risk for PID in young women may
in part be attributed to the fact that they have a higher likelihood of cervical
ectopy than do older women. Cervical ectopy results in a larger number of
cells exposed to the organism as well as increased shedding of the organism
(Dean, 1997; Barnes et al., 1990) and it is reasonable that these conditions
may lead to increased transmission. Among older women, C. trachomatis
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genital infection occurs more frequently in nonwhite, nulliparous, unmarried
women of lower socioeconomic status (Weinstock, Dean, and Bolan, 1995).
Additional risk factors include concurrent GC infection, multiple or new sex
partners, early first sexual experience and intercourse with a male with
chlamydial urethritis (Weinstock, Dean, and Bolan, 1995). Barrier
contraception including the cervical cap, diaphragm, and condoms protect
against chlamydial urogenital infection, while it is inconclusive whether oral
contraceptive pills increase risk (Weinstock, Dean, and Bolan, 1995). While
limited data is available for men, those who are seen at STD clinics have the
highest prevalence rates (15-20%; Weinstock, Dean, and Bolan, 1995)
followed by those seen at adolescent clinics (13%; Weinstock, Dean, and
Bolan, 1995). Similar to that reported for women, young age, non-white
race/ethnicity and heterosexuality are risk factors for chlamydial urogenital
infection in men (Karam et al., 1986).
Prior infection and repeat infection are strongly associated w ith an
immunopathogenic response that characterizes progression of disease as
well as late sequelae including ectopic pregnancy (Westrom, Bengtsson, and
Mardh, 1981; Morrison, Manning, and Caldwell, 1992). Data suggest that
immunologic factors may play an important role in the development of
complications associated with PID. There is a very strong association
between high levels of antibody against Chlamydia MOMP and HSP-60 and
infertility and ectopic pregnancy (Wagar et al., 1990; Cates and Wasserheit,
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14
1991; Toye et al., 1993). Moreover, the protein encoded by hsp-60, GroEL
produces a hyper-inflammatory response in animal models (Morrison et al.,
1989; Patton, Sweeney, and Kuo, 1994).
Recurrent infection is common, particularly for young women, with
estimated rates for adolescent women within 2-3 years of their initial
infection between 19-30% (Fortenberry and Evans, 1989; Blythe et al., 1992;
Hillis et al., 1994). In 1992, Blythe et al. reported that more than half of
recurrent infections occurred within 9 months of the initial infection, and about
30% within six months. Among adolescents with recurrent infection, those
with a new partner had a longer time to recurrence (median = 19.6 months)
than those without a new partner (median = 12.9 months) supporting the fact
that risk factors for "early" and "late" recurrences may differ (Blythe et al.,
1992).
Early recurrence may be a result of continued sexual experiences with
an untreated sex partner. In fact, over 96% of sexually active women report
only one sex partner in a three-month period (Seidman, Mosher, and Aral,
1992). Moreover, regular condom usage declines in what is perceived to be a
monogamous relationship (7% of women with one partner compared to 34%
of women with multiple partners; Marin, Gomez, and Hearst, 1993). Other
risk factors for severity of disease, late sequelae, re-infection and persistent
infection have not been clearly identified.
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15
It is unclear whether there is differential transmission of Chlamydia for
different genders due to difficulties in enrolling partners, differential
sensitivity of culture and other diagnostic tests for detecting chlamydiae, and
selection bias. In several studies, women (45-65%) appeared to be infected
more often than men (25-45%) by their Chlamydia positive opposite-sex
partners (Ramstedt et al., 1991; Lycke et al., 1980; Viscidi et al., 1993).
However, differences seen may be biased by difficulties in diagnosing male
infection since sampling the urethra is an invasive procedure and is less
often attempted in light of an asymptomatic course. There is also some
evidence to suggest that tissue culture methods may be less sensitive in
diagnosing Chlamydia in men than in women. In a 1994 study examining
transmission rates in a heterosexual population, PCR detected infection more
often than tissue culture and the authors found that by PCR there were equal
transmission rates for men and women (Quinn et al., 1994). Co-infection
with GC is also a risk factor for urogenital chlamydial infection. A higher
titer of infectious chlamydial organisms is associated with concurrent
GC/ chlamydial infection and recurrence rates are higher with concurrent
infection than C.trachomatis infection alone (Batteiger et al., 1989a). Since
C.trachomatis is considered to be less easily transmitted than GC, co-infection
may increase transmissibility.
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16
Chlamydial Developmental Cycle
Chlamydiae are bacteria restricted to an intracellular life cycle. Stage-
specific events primarily involve two cell types, a predominantly extra
cellular infectious form, the elementary body (EB), and an intracellular
metabolically and reproductively competent form, the reticulate body (RB).
The life cycle can be simplified as the attachment and entry of the EB to the
host cell, the structure and assembly of the inclusion membrane, the
conversion of EB to RB, the logarithmic division of RB, the reorganization of
RB back to EB, and host cell exit.
The primary purpose of the chlamydial EB is to withstand the
chemical and physical pressures of an extra-cellular environment and the
subsequent infection of a host cell. Well-adapted for this purpose, the EB has
a rigid, osmotically stable membrane with reduced surface area and is
metabolically inert. Metabolic shutdown is accomplished by complex gene
regulation and condensation of chromatin into a hyper-pyknotic nucleoid
structure, unique to chlamydiae.
The nucleoid structure contains two histone-like proteins, Hcl and
Hc2, with significant homology to eukaryotic H l-type histones (Hackstadt,
Baehr, and Yuan, 1991; Tao, Kaul, and Wenman, 1991). Both Hcl and Hc2
bind to DNA in vitro (Perara, Ganem, and Engel, 1992; Barry, Brickman, and
Hackstadt, 1993) and when expressed in E. coli, induce DNA conformational
changes (Barry, Hayes, and Hackstadt, 1992; Barry, Brickman, and
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17
Hackstadt, 1993). Based on quantitative estimates that histones make up 4 to
7% of the protein constitution of EBs, histone proteins could be spaced as
closely as 100 bp apart along the length of the genome (Brickman, Barry, and
Hackstadt, 1993). H cl has a higher affinity for supercoiled DNA in vitro
while Hc2 has equal affinity for linear as well as supercoiled DNA (Petersen,
Birkelund, and Christiansen, 1996). It is thus been proposed that Hcl may
hinder RNA synthesis by condensing DNA while Hc2 may regulate stage-
specific gene expression (Peterson, Birkelund, and Christiansen, 1996).
Additionally, Hcl and Hc2 in concert may decrease superhelicity of the DNA
and thereby regulate gene expression at promoters constrained by topology.
EBs are rapidly internalized by eukaryotic cells, however the precise
mechanism remains uncertain. It is clear that electrostatic interactions are
important in attachment, at least initially. The surface of a native EB is
relatively hydrophobic with a net negative charge at neutral pH. Treatment
with divalent cations (Ca2 + , Mg2 + , or Mn2 + ) or Na+ enhance binding of C.
psittaci and C. trachomatis L2 strains to host cells, presumably by neutralizing
surface charge (Hatch, Vance, and Al-Hossainy, 1981; Sneddon and
Wenman, 1985).
Attachment of Chlamydia to its host also involves a host cell receptor-
bacterial ligand interaction. Based on early evidence that the kinetics of
attachment does not reach saturation and is sensitive to proteolysis, the host
cell receptor is presumed to be a ubiquitous surface molecule with protein or
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18
glycoprotein moieties (Friis, 1972; Byrne, 1976; Byrne, 1978; Byrne and
Moulder, 1978). It has been postulated that a Chlamydia-specific
glycosaminoglycan (GAG) may aid in the stabilization of the receptor-ligand
interaction by forming a noncovalent link between an unidentified Chlamydia
ligand and the host cell receptor in a novel tri-molecular mechanism. This
mechanism, first proposed by Zhang and Stephens (1992), is consistent with
the complex inhibition patterns by heparin sulfate-like proteoglycans of
different sulfation levels. However this inhibition is not consistently
observed for all chlamydial serovars. For example, infection by LGV strains
is dramatically reduced by heparin sulfate (Kuo and Grayston, 1976; Zhang
and Stephens, 1992) while infection by the trachoma biovars is not inhibited
to the same degree (Kuo and Grayston, 1976; Chen and Stephens, 1997; Davis
and Wyrick, 1997). This suggests that the GAG-dependent mechanism may
stabilize some serovars and not others and that additional GAG-independent
mechanisms may be required for irreversible binding and internalization
(Hackstadt, 1999). Due to its obligate intracellular life cycle, it is conceivable
that there exist many mechanisms for chlamydial host cell attachment.
The major outer membrane protein and OmcB were both proposed as
possible GAG binding proteins (Stephens, 1994; Ting et al., 1995). Several
lines of evidence support a role for MOMP as an adhesin. It has been known
for many years that monoclonal antibodies against hypervariable regions in
MOMP, specifically variable segments (VS) I, II and IV, neutralize
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19
chlamydial infection by inhibiting attachment (Moulder, 1991). These
regions exhibit a net negative surface charge and in addition VS IV has a
conserved hydrophobic pocket. It has been suggested that these domains
mediate attachment through electrostatic and hydrophobic interactions with
the host cell (Su et al., 1990b). Furthermore, there are differences in trypsin
inhibition for different serovars. Trypsin treatment does not reduce
attachment for serovar L2, however it dramatically reduces attachment for
serovar B. It has been suggested that this difference may be due to the
presence of trypsin-sensitive lysine residues in VS II and IV of serovar B and
the absence of these residues for serovar L2. Thus, VS II and IV of MOMP
appear to be critical for attachment (Hackstadt, 1999).
Studies utilizing maltose-binding protein (MBP) -MOMP fusion
proteins have provided evidence that MOMP may be the GAG receptor on
the chlamydial surface. First, MBP-MOMP fusion proteins specifically bind
hum an epithelial cells and are internalized at 4°C. Also, binding to hum an
epithelial cells was inhibited by heparin or heparin sulfate and binding to
HeLa cells was inhibited by heparitinase treatment. Lastly, MBP-MOMP
inhibited binding of intact EBs.
The evidence that supports OmcB's role as a cellular adhesin is less
convincing. OmcB is a cysteine-rich envelope protein found in EBs, but not
in dividing RBs. Early studies evaluating binding of sarkosyl-insoluble EB
cell wall components to HeLa cells revealed that OmcB and MOMP were the
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20
proteins bound to monolayers in greatest quantity (Ting et al., 1995).
Further, inhibition of attachment by proteolysis and heat treatment
correlated well with inhibition of OmcB binding. However, inhibition of
attachment by proteolysis would suggest a surface exposed location for
OmcB and the cellular location of OmcB is controversial. In fact, the most
recent model for the elementary body outer membrane proposes that OmcB
is periplasmic based on detergent partitioning and hydrophobic affinity
labeling (Everrett et al., 1991).
Entry into the host cell is thought to occur by a zipper-type
mechanism that involves direct contact between bacterial ligands and host
receptors. This results in the activation of signal transduction processes and
subsequent rearrangement of the host cytoskeleton (Ward and Murray,
1984). Recent new evidence suggests that the newly identified chlamydial
type III secretion system (see Virulence and Pathogenesis section) may
deliver virulence proteins. In other gram-negative bacteria, these proteins
have the potential to disrupt signal transduction pathways and thereby affect
cytoskeleton rearrangement and other cellular processes. It is thus possible
that the chlamydial type III secretion system may play a role in host cell
entry and undoubtedly this will prove to be an intriguing new avenue of
research in the coming years. Regardless of the mechanism by which signal
transduction is induced, as early as 15 minutes after chlamydial infection,
multiple host proteins have been tyrosine phosphorylated (Birkelund,
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21
Johnsen, and Christiansen, 1994; Fawaz et al., 1997). One of the
phosphoproteins may be associated with the cytoskeleton and an actin-
associated protein, cortactin, has been colocalized with the tryrosine-
phosphorylated proteins (Fawaz et al., 1997).
Depending upon culture and inoculation conditions, chlamydiae may
induce microfilament-dependent uptake into non-clathrin coated vesicles
(phagocytic) (Ward and Murray, 1984; Prain and Pearce, 1989; Reynolds and
Pearce, 1990) and receptor-mediated endocytosis into clathrin-coated pits
(pinocytotic) (Flodinka and Wyrick, 1986; Hodinka et al., 1988). Based on the
results of several studies, a greater frequency of pinocytosis was observed
when epithelial or FleLa cells were grown on collagen-coated fibers as
opposed to when cultured on plastic (Wyrick et al., 1989). The same effect
was also observed when static inoculation was employed over centrifugation
(Prain and Pearce, 1989). The cumulative evidence suggests that chlamydiae
may employ both methods for entry into the host cell.
After internalization, chlamydiae inhabit endosomes that do not fuse
with lysosomes. This is based on the absence of several lysosomal markers
including acid phosphatase, transferrin receptor, mannose-6-phosphate
receptor, lysosomal glycoproteins 1 and 2, cathepsin D, H+ ATPase and the
inability to fuse with secondary lysosomes (Friis, 1972; Lawn, Blyth, and
Taverne, 1973; Wyrick and Brownridge, 1978; Heinzen et al., 1996; Sddmore,
Fischer, and Hackstadt, 1996; Taraska et al., 1996; van Ooij, Apodaca, and
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22
Engel, 1997). The immediate-early gene product, CT147, has the potential to
modify the bacterial phagosome in order to escape fusion with lysosomes
and is most likely responsible for the organism's ability to avoid internal
degradation (Belland et al., 2003). A group of Chlamydia-specific proteins
that become inserted into the inclusion membrane, termed inclusion
membrane proteins (Inc), have been described for both C. psittaci and for C.
trachomatis (Rockey and Rosquist, 1994; Rockey, Heinzen, and Hackstadt,
1995; Bannatine et al., 1998). Their function at this time is not known;
however, they may contribute to controlling the intracellular movement of
the vesicle (Hackstadt, 1999).
Proteolytic degradation of the histone proteins is thought to
contribute to the breaking down of the nucleoid structure and the
concomitant beginning of parasite metabolism (Kaul et al., 1997). Within two
hours post internalization, chlamydiae initiate protein synthesis. Early gene
products support multiplication and direct the vesicle to the peri-Golgi
region (Plaunt and Hatch, 1988; Lundemose et al., 1990; Scidmore, Fischer,
and Hackstadt, 1996). This redistribution appears to be dependent on
microtubules and the microtubule motor protein dynein, since EBs aggregate
at the microtubule organizing center with dynein colocalized with
phosphoproteins (Clausen et al., 1997). However, the dependency on
microtubules appears to be species specific. Microtubules were involved for
C. trachomatis serovars E and L2 but not for C. pneumoniae (Clausen et al.,
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23
1997). There are other species-specific characteristics of intracellular
inclusion formation. For example, for C. trachomatis, and not for C. psittaci,
multiple inclusions fuse into a large vesicle (Ridderhof and Barnes, 1989).
This even occurs when the cell is infected with multiple serovars.
After redistribution to the peri-Golgi region, the chlamydial inclusion
eventually fuses with an exocytic vesicle that delivers sphingolipids from the
cis or medial Golgi to the plasma membrane (Hackstadt, Scidmore, and
Rockey, 1995; Hackstadt et al., 1997). The transformation of the vesicle from
nonfusogenic with lysosomes to fusogenic with exocytic vesicles is
dependent upon Chlamydia gene products. After this transformation, it is
considered a mature inclusion (Scidmore, Fischer, and Hackstadt, 1996). A
substantial proportion of the sphingomyelin that is transported within the
vesicle is retained and incorporated into the inclusion membrane (Hackstadt,
Scidmore, and Rockey, 1995). Late-gene products in the chlamydial
development cycle play a role in the cessation of replication; the formation of
the outer membrane complex; and the attachment and invasion of new host
cells (Belland et al., 2003). Many of the immediate-early and late-gene
products show an evolutionary relatedness to eukaryotic lineages (Belland et
al., 2003).
Host cell functions including DNA and protein synthesis are
minimally disturbed by chlamydial infection (Schechter, 1966; Alexander,
1968; Bose and Leibhaber, 1979), whereas glycolysis and respiration are
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24
stimulated (Moulder, 1970; Ojcius et al., 1998). Metabolites that are derived
from the host include amino acids (Hatch, 1975a), nucleotides (Hatch, 1975b;
McClarty and Tipples, 1991; Tipples and McClarty, 1993; McClarty, 1994)
and lipids (Hackstadt, Scidmore, and Rockey, 1995; Wylie, Hatch, and
McClarty, 1999). Mechanisms of nutrient entry from the cytoplasm into the
vesicle are unknown. Proteins that may play a role in nutrient exchange
include MOMP (Bavoil, Ohlin, and Schachter, 1984), an ATP translocase
(Hatch, Al-Hossainy, and Silverman, 1982) and putative specific transporters
identified in the complete genome sequence (Stephens et al., 1998).
Additionally, RBs maintain a tight association with the inner surface of the
inclusion membrane and it has been proposed that this association may
mediate nutrient exchange (Moulder, 1991).
The developmental cycle for chlamydiae is complete as early as 36
hours post infection. Release from the cell is by lysis for the rapidly growing
C. psittaci and C. trachomatis LGV strains and fusion with the plasma
membrane for certain serovars of C. trachomatis (Todd and Caldwell, 1985).
Chlamydial Outer Membrane Structure
The characterization of the chlamydial outer membrane structure, as
well as the organism's metabolism and developmental biology, have been
hindered by the lack of a cell-free growth system or a gene transfer system.
Thus, host cell contamination has been a ubiquitous problem with
complications that arise from the asynchrony of the developmental cycle.
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Due to these difficulties, purified preparations of chlamydial inclusions can
contain a mixture of RBs, EBs and intermediate forms, as well as host
components.
Early work on the structure of the outer membrane included
investigating whether chlamydiae possess peptidoglycan in their cell walls.
Chlamydiae are penicillin and D-cycloserine sensitive (Weiss, 1950; Moulder,
Novosel, Officer, 1963) and C. trachomatis serovar D contains a full
complement of genes that encode enzymes required for peptidoglycan
synthesis and assembly (Stephens et al., 1998). Unexpectedly, in a number of
studies over the past decades, N-acetylmuramic acid, a sugar found only in
peptidoglycan, has only been found in chlamydiae in trace amounts (Jenkin,
1960; Perkins and Allison, 1963; Barbour et al., 1982; Garrett, Harrison, and
Manire, 1974). Thus, the function and relative importance of peptidoglycan
in cell wall formation remain unclear.
Proteins associated with the outer membrane complex of EBs were
initially determined by insolubility in the anionic detergent, sodium lauryl
sarcosinate (Sarkosyl). The three proteins that were found in highest
quantity were MOMP and two cysteine-rich proteins, outer membrane
complex A (OmcA) and outer membrane complex B (OmcB), in the ratio of 5
MOMP: 2 OmcA: 1 OmcB (Everett and Hatch, 1991).
MOMP is the most abundant protein in both EBs and RBs and is
constitutively expressed (Stephens et al., 1986). In RBs, MOMP is in its
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26
reduced form, and in EBs it is extensively cross-linked and disulfide bonded
(Newhall and Jones, 1983; Hatch, Allan, and Pearce, 1984; Hackstadt and
Caldwell, 1985; Hatch, Miceli, and Sublett, 1986; Newhall, 1987; Newhall,
1988). In most serovars of Chlamydia, MOMP is surface exposed. The gene
that encodes MOMP, ompA, exhibits extensive DNA sequence variation
mainly confined to four variable segments (VS I to IV) (Yuan et al., 1989) and
these variable segments tend to be surface exposed (Hatch, 1999). They also
contain subspecies- and serovar-specific antigenic determinants and within
MOMP are important T-cell epitopes (Allen, Locksley and Stephens, 1991;
Ishizaki et al., 1992). In fact, in all chlamydial strains examined except for C.
pneumoniae, MOMP is an immunodominant protein (Campbell, Kuo, and
Grayston, 1990; Christiansen, Ostergaard, and Birkelund, 1997).
An early function attributed to MOMP was that of a porin. More
specifically, Wyllie et al. (1998) determined that a trimer of MOMP
functioned as an ATP-translocase. Earlier studies demonstrated that only
reduced non cross-linked MOMP functioned as a porin (Bavoil, Ohlin, and
Schachter, 1984). Reduction of MOMP is dependent upon chlamydial gene
products and occurs directly after internalization of the EB (Hatch, Miceli,
and Sublett, 1986). In contrast, cross-linking of MOMP is a late stage-specific
event that occurs during the conversion of a RB back to an EB. Thus, the fact
that only reduced MOMP functioned as a porin suggested that cross-linking
of MOMP with other membrane proteins contributed to the impermeability
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27
of an EB. However, more recent studies have refuted these original findings
(Hatch, Miceli, and Sublett, 1986; Wyllie et al., 1998).
Additionally, there is substantial evidence to support MOMP's
putative role as a cellular adhesin (see Chlamydial Developmental Cycle).
Given the immunogenieity of MOMP and its potential role in vital functions,
MOMP has been the leading candidate for a subunit vaccine. However,
despite tremendous effort and numerous experimental designs, protection
against MOMP has been short-lived (Tan et al., 1990; Batteiger et al., 1993;
Pal et al., 1997a; Pal et al., 1997b; Zhang et al., 1997).
In contrast to MOMP, OmcA and OmcB are late-cycle proteins found
only in EBs. OmcA is a lipoprotein (Everett, Desiderio, and Hatch, 1994) and
OmcB is post-transcriptionally processed to form a doublet, except for C.
trachomatis serovar B, where only one mature protein has been detected
(Everrett and Hatch, 1991). Studies with a lipophilic outer membrane probe
suggested OmcA is surface exposed while OmcB is not (Everett and Hatch,
1995). Everrett and Hatch (1995) proposed that Sarkosyl insolubility may be
explained by extensive cross-linking of OmcB and not by an outer membrane
location. However, there is some evidence that a portion of one of the OmcB
doublets is surface exposed. Ting et al. (1995) found that trypsin treatment of
EBs degraded a portion of the large OmcB doublet. Based on these findings
and other experimental evidence, the most recent model for the EB outer
membrane structure includes a supramolecular lattice of extensively cross
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28
linked OmcB proteins in the periplasm interacting with the amino acid
portion of OmcA. The lipophilic end of OmcA is inserted in the inner leaflet
of the outer membrane and a trimer of MOMP forms a surface exposed
channel (Everrett and Hatch, 1995). Consistent with the findings of Ting et
al. (1995), the larger protein of the doublet may extend to the surface or it
may be associated with an outer membrane channel (Mygind, Christiansen,
and Birkelund, 1998).
A family of 90-190 kDa proteins originally detected in insoluble
Sarkosyl fractions and further identified in the genome sequences and
immunogold studies include the putative outer membrane proteins (Pmp).
In the C. trachomatis genome, loci encode for nine Pmps (PmpA to PmpI;
Stephens et al., 1998), a sizablelO% or so of the genome (Grimwood and
Stephens, 1999). In the C. pneumoniae genome, loci encode 21 Pmps
(Grimwood and Stephens, 1999; Stephens et al., 1998) and in the C. psittaci
genome, loci encode at least six Pmps (Omp90A, Omp90B, Omp91A,
Omp91B; Longbottom et al., 1996; Giannikopoulou et al., 1997; Longbottom
et al., 1998). Amino acid sequence variation between genes in the Pmp
family for a single strain is at most 50% for C. trachomatis while it ranges 25-
52% for C. pneumoniae (Grimwood and Stephens, 1999). Inter-strain
nucleotide comparison between four strains of C. pneumoniae (CWL029,
CWL02, AR39, and J138) reveal deletions, frameshift mutations, and
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29
intragenic duplications, however, no quantitative assessment of inter-strain
variation has been done.
Predicted structures of the C. psittaci and C. trachomatis genes within the
various Pmp loci have characteristics of outer membrane proteins. These
characteristics are conserved and include motifs [GGA(I, L,V) and FXXN] at
the N-terminus; a phenylalanine C-terminus residue; and ( 3 sheet motifs
(Longbottom et al., 1998; Stephens et al., 1998). Immunogold studies reveal
that at least 3 of 6 (50%) C. psittaci Pmps (Tanzer, Longbottom, and Hatch,
2001) and 10 of 21 (48 %) C. pneumoniae Pmps (Grimwood, Olinger, and
Stephens, 2001; Vandahl et al., 2001) are surface exposed. Three late genes
that encode C. trachomatis serovar L2 Pmps were confirmed as outer
membrane proteins (Tanzer, Longbottom, and Hatch, 2001; Mygind et al.,
2000).
Like one of the OmcB doublets, evidence suggests that a portion of
Hsp70, a cytoplasmic chaperone protein, is associated with the inner leaflet
of the outer membrane and may be surface exposed under reducing
conditions. A portion of Hsp70 was found in outer membrane complexes
and was recognized by a monoclonal antibody following exposure to the
reducing agent, dithiothreitol (Raulston et al., 1993; 1998). Based on these
studies, Raulston et al. (1998) proposed that after reduction of the cross-
linked envelope proteins, Hsp70 or a ligand presented by Hsp70, adheres to
a host receptor. However, evidence suggests that Hsp70 may be surface
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30
exposed for C. trachomatis serovar D (Danilition et al., 1990) but not for C.
trachomatis serovar L2 (Birkelund, Lundemose, and Christiansen, 1989) and
these differences may reflect the degree to which envelope proteins are cross-
linked in these serovars (Hatch, 1999).
As discussed earlier, chlamydial surface projections on both RBs and
EBs identified through electron microscopy may represent a type III
secretion apparatus (Matsumoto, 1982a). These structures are regularly
spaced over the surface of the envelope and are dome-shaped with spike-like
projections reminiscent of bacteriophage tails. Interestingly, in RBs the
projections appear to extend through the membrane (Matsumoto, 1982b).
The function of these projections is unclear; however, they may contribute to
virulence and entry of the parasite (see Chlamydial Developmental Cycle).
Lipopolysaccharide (LPS) resembling the rough form of Salmonella minnesota
Re is present in the outer membrane for both RBs and EBs (Nurminen et al.,
1983; Brade et al., 1987). Key differences include the presence of a 1-8 linkage
and a genus-specific epitope on the trisaccaride 3-deoxy-D-mannose-
octulosonic acid (KDO) and low endotoxicity (Belunis et al., 1992; Brade,
Brade, and Nano, 1987; Ingalls et al., 1995). Surface exposure and epitope
configuration appears to depend on the developmental cycle (Kuo and Chi,
1987; Collett et al., 1989; Birkelund, Lundemose, and Christiansen, 1988).
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31
Molecular Genetics
The chlamydial genome consists of a double-stranded circular
chromosome ranging from 1.1-1.2 Mbp and a double-stranded circular
plasmid of 723 bp. At the time of this writing, full genome sequences for C.
trachomatis serovars D and L2 and MoPn biovar and C. pneumoniae strains
J138, CWL029 and AR39 have been determined (Stephens et al., 1998; Read et
al., 2000) whereby our understanding of chlamydial biology has increased
dramatically (Kalman et al., 1999; Read et al., 2000; Shirai et al., 2000).
Chlamydiae do not have the ability to synthesize nucleotides either de
novo or by the salvage pathway (Hatch, 1975b; McClarty, 1994; McClarty
and Tipples, 1991). As a result, chlamydiae are auxotrophic for three of the
four ribonucleotides (ATP, GTP and UTP) (Tipples and McClarty, 1993).
CTP can be synthesized de novo utilizing a CTP synthetase homolog and can
also be acquired from the host cell (Tipples and McClarty, 1995; Wylie, Berry,
and McClarty, 1996; Wylie et al., 1996). The chlamydial adt2 gene may
encode an NTP-translocase used for transporting NTPs from the host cell
into the inclusion (McClarty, 1999). In contrast to ribonucleotides,
deoxyribonucleotides are not transported from the host cell, but rather, are
synthesized via reduction of the corresponding ribonucleotides (Reichard,
1993; 1997). The exception is for dTTP wherein the reduction of UTP and
thymidylate synthase is utilized in combination (Montfort and Weichsel,
1997).
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In terms of amino acids, chlamydiae compete with the host cell for the
available pool although they also have the ability to synthesize a few on their
own (Hatch, 1988; Moulder, 1991). Amino acid transporters with broad
specificity and the oligopeptide ABC transport system have been identified,
as well as genes involved in amino acid biosynthesis (Stephens et al., 1998).
Chlamydiae synthesize DNA, RNA and protein using a combination
of host and parasite derived substrates, cofactors and host ATP (Hatch,
1988). Peak levels of synthesis occur during periods of logarithmic growth
(McClarty, 1994). These processes are not dependent on host replication
enzymes since they are inhibited by prokaryotic inhibitors and not by
eukaryotic inhibitors. The genome contains homologs of multiple subunits
of DNA polymerase III, a DNA polymerase I, and a type I and two type II
topoisomerases (Stephens et al., 1998). Also, homologs for three subunits of
the core RNA polymerase, a6 6 and two putative alternative sigma factors (a2 8
and a 5 4 ) were identified. Several homologs of genes encoding enzymes
involved in transcription elongation and termination and in RNA processing
are encoded in the genome. Two copies of a 16S-23S-5S rRNA operon are
present, as well as all required ribosomal protein genes. Thirty-seven tRNA
genes and eighteen tRNA synthetase genes are present. Of note, is the
absence of the glutaminyl- and the aspariginyl- tRNA synthetase genes,
however, this is not uncommon among other bacteria with minimal
genomes.
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33
Chlamydiae exhibit considerable capacity for DNA repair. Enzymes
required for uvr excision repair and m utL/ mutS mismatch repair as well as
several enzymes in the RecBCD and RecF recombination pathways have
been identified in the genome sequences (Stephens et al., 1998).
To date, several differences between chlamydial and E. coli gene
regulation are apparent. Chlamydial transcription machinery recognizes a
widely divergent population of transcription initiation sequences that at best
roughly resemble the canonical E. coli -35 and -10 hexamers of TTGACA and
TATAAT and at worst differ from them significantly (Mathews and
Sriprakash, 1994). It was originally thought that this may be explained by
the utilization of multiple alternate sigma factors, however, only two
alternate sigma factors, a 2 8 and cr5 4 , have been identified in the genome
(Stephens et al., 1998). Another difference is that the chlamydial
transcription machinery appears to use a homolog of a6 6 as its major
transcription factor. This homolog has only 45% similarity to a 7 0 , the major
sigma factor of E. coli (Koehler et al., 1990; Engel and Ganem, 1990; Douglas,
Saxena, and Hatch, 1994). Also chlamydial genes often contain AT-rich
sequences in a -35 to -10 spacer region just downstream of the -35 promoter
element. In the ompA gene, a poly-A tract in this region is required for
optimal transcription by the chlamydial RNA polymerase preparation but
not by the £. Coli polymerase (Tan et al., 1998). This evidence suggests this
AT-spacer region may be a promoter element unique to chlamydiae (Hatch,
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34
1999). Tan et al. (1998) suggested this region may enhance transcription by
localized strand melting, bending of DNA, or recognition by a transcription
activator.
In E. coli, transcription of the stress-induced genes groE and dnaK
utilize the alternate transcription factor a 3 2 . Chlamydiae appear to lack a
specific stress-related sigma factor, however, located just upstream of the
groE and dnaK operons is an inverted repeat very similar in sequence to
CIRCE (controlling inverted repeat for chaperone expression). In other
bacterial species without a stress-related sigma factor, transcription of these
operons is mediated through CIRCE and it appears likely this is the case for
chlamydiae also (Schmiel and Wyrick, 1994; Tan, Wong, and Engel, 1996).
The fact that several chlamydial genes generate multiple transcripts has
intrigued chlamydial researchers for some time. Two transcripts that encode
the entire MOMP protein are generated from ompA with different 5' ends
(Fahr et al., 1995; Stephens et al., 1998) and two transcripts are generated
from the omcAB operon with the same 5' ends (Lambden et al., 1990; Fahr et
al., 1995; Watson et al., 1995). The plasmid counter transcript (PCT) also
generates two transcripts with the same 5' ends (Fahr, Sriprakash, and Hatch,
1992; Ricci et al., 1993; Ricci, Ratti, and Scarlato, 1995). Each of the transcripts
from these operons with the exception of ompA is apparently generated by
post-transcriptional processing or differential termination. It is thus likely
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35
that these mechanisms are important in the gene regulation of chlamydiae
(Hatch, 1999).
Serotyping of Chlamydia trachomatis
Originally, microimmunofluorescence (MIF) with polyvalent antisera
was used to identify the 15 serovars of C. trachomatis (A-K, Ba, L1-L3; Wang,
Grayston, and Gale, 1973). This early in vitro MIF test used antisera derived
from mice immunized with the isolate under investigation (unknown
antisera) and from mice immunized with the prototype strains (prototype
antisera). Reaction of the immune sera to elementary bodies used as
antigens was determined by immunofluorescence. Immune profiles were
determined for serial dilutions of the unknown antisera against prototype
antigens and for serial dilutions of the prototype antisera against the
unknown antigen (two-way test). A one-way test was adopted that
restricted the procedure to determining the reaction profiles of serial
dilutions of unknown antisera against prototype antigens. Based on the
cross-reactivity of antibodies to the serovars in the MIF test, two major
groupings were established: the B complex including B, Ba, E, D, LI, L2, G
and F and the C complex including C, J, A, H, I, K and L3. However, G /F
serovars were less closely related to serovars in the B complex and K/L3
were related to both the B and the C complex groups (Grayston and Wang,
1975; Wang and Grayston, 1982).
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36
Serotyping was improved by the development of monoclonal
antibodies (mAb) with type and subspecies specificity that more precisely
differentiated strains with less time. Twenty-two monoclonal antibodies
with different specificities were used. Monoclonal antibody LE-4 reacted to
most of the C complex serovar strains while KD-3 and BB-11 reacted to most
of the B complex strains. The other nineteen mAbs reacted to one serovar or
to two closely related serovars. This led to a two-step process of
differentiation that included first determining the complex by the use of LE-
4, KD-3 and BB-11 mAbs and once the complex was known utilize the mAbs
of that complex to determine the serovar (Wang et al., 1985). The
implementation of this system led to the identification of three additional
serovars Da, la, and L2a (Wang and Grayston, 1991).
The strength of serotyping is that immunologic markers presumably
reflect attributes related to pathogenesis and immunity and a large number
of isolates can be typed quickly. However, relatively large quantities of
cultured chlamydiae are required, some specimens can not be propagated
due to their growth characteristics and it is expensive. Probably the most
compelling argument against immunotyping is that only those nucleotide
changes that result in a change in the antigenicity of the protein are
differentiated and monoclonal antibodies only detect the currently known
serological antigens in MOMP (Dean et al., 1992). Unlike immunotyping,
ompA genotyping detects all nucleotide substitutions including those that are
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37
synonymous and those that are nonsynonymous which do not result in a
change in antigenicity. Thus, ompA genotyping can more precisely
differentiate strain variation in chlamydiae compared to serotyping.
OmpA Chlamydia trachomatis Serotyping and Genotyping
Studies
Because of its sequence diversity and immunogenicity, MOMP is one
of the most widely used targets for identification of chlamydiae. Thus,
numerous ompA serotyping and genotyping studies in the past decade have
expanded our previous appreciation for strain variation, and in many cases
have identified substitutions that correlate with serotyping. However few
have identified serovars associated with severity of disease, tissue tropism or
clinical presentation and none have identified specific genotypes or sequence
patterns associated with disease markers.
Serotyping studies. Many studies have assessed the serovar
distribution of chlamydial urogenital infections throughout the world.
Populations that have been investigated include metropolitan areas of the
United States, Finland, Tahiti, France, and the Netherlands. Serovar has
primarily been based on monoclonal antibody recognition and restriction
fragment length polymorphism (RFLP) analysis of PCR products. While
most have focused on heterosexual populations, several have examined
rectal infections within homosexual male populations. Examination of
cervical and urethral infections within heterosexual populations has revealed
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38
that serovar E, F and D were consistently the most prevalent serovars
(Wagenvoort, Suchland, and Stamm, 1988; Saikku and Wang, 1987; Chungue
et al., 1994; Moncan, Eb, and Orfila, 1990; Gaydos et al., 1992). In 1987,
Barnes, Rompalo, and Stamm revealed that there was a difference in the
serovar distributions between rectal and cervical chlamydial infections.
They found that D was the predominant serovar among homosexual patients
with rectal chlamydial infections while E was the predominant serovar
among women with cervical chlamydial infections. In 2002, Geisler et al.
confirmed the high prevalence of serovar D among homosexual men in
Seattle. In this population, they found 47.9% of rectal chlamydial infections
were serovar G and 29.6% were serovar D. In 2000, Dean et al. found a
preponderance of C class serovar infections (44.6%) among women with at
least three chlamydial infections of the same serovar over a 2 year period
compared to the overall Seattle population (28%; odds ratio (OR) = 2.4 [Cl:
1.7-3.5]; p < 0.0001).
Mixed infections in population based serotyping studies, whereby
more than one serovar is present in a clinical specimen, occur in an estimated
2% of cases (Barnes et al., 1985; Jurstrand et al., 2001; Bandea et al., 2001).
However, in a study that evaluated sex partners with Chlamydia and GC, Lin
et al. (1998) revealed that mixed infections were found roughly half of the
time regardless of whether the specimen was derived from the male or
female partner. In another study evaluating strains from heterosexual
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39
partners, Lan et al. (1995) found that identical serovars were found between
partners 89% of the time (31 of 35 pairs were concordant).
Several studies have investigated the relationship between
demographics and serovar distribution and one examined longitudinal
trends in serovar. For a heterosexual population seen at STD clinics in
Seattle, black race increased risk of infection with serovar la (p<0.02) and
black race decreased risk of infection with serovar D. However, the
association with D was solely at the urethra (p=0.001; Workowski et al.,
1992). For a similar Seattle population that included homosexual and
bisexual patients, serovar class C infections were associated with older age
(p<0.001; Suchland et al., 2003). The latter study also assessed longitudinal
trends in serovar distribution. The authors determined that over a nine year
time period in Seattle, the proportion of serovars F and G increased (p =
0.007, p = 0.009); while the proportion of serovars I and K (p < 0.001, p =
0.008) decreased (Suchland et al., 2003). In the Netherlands, heterosexual
women seen at a STD clinic younger than 18 years at age of first intercourse
were more likely to be infected with serovar class C (p=0.05; van de Laar et
al., 1996).
There have been several studies that have examined the relationship
between serovar and clinical signs and symptoms of chlamydial urogenital
disease in different heterosexual populations, although they have produced
widely varying conclusions. Batteiger et al. (1989b) found no strong
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40
association between serovar based on MAbs and clinical signs of
inflammation including the number of polymorphonuclear leukocytes and a
clinician's assessment of the presence or absence of cervicitis. In contrast,
Workowski et al. (1994) found serovar F was associated with fewer signs of
severe disease including easy bleeding, cervical ectopy and mucopus than
other serovars. Studies based on RFLP have reported other spurious
associations including: Ga in men with dysuria; la in men and women with
asymptomatic infection; K in women with discharge; H and J among men
with discharge and dysuria; F and G among women with lower abdominal
pain; F and G in men without discharge (Morre et al., 2000; van Duynhoven
et al., 1998; van de Laar et al., 1996). Discrepencies between the studies could
be attributed to differences in the methods to determine serovar, outcomes
measured, populations studied and inadequate adjustment for confounding
and multiple comparison testing. In the sole study examining these
relationships while adjusting for confounders, Geisler et al. (2003) found that
after adjusting for age and race, women infected with serovar F were more
likely to report abdominal pain and/or dyspareunia than those infected with
serovars D, E, la, and J (p = 0.048). No other associations between clinical
manifestations and serovar were found for the Seattle population. The
authors concluded that clinical signs and symptoms were not strongly
influenced by the infecting serovar.
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41
For homosexual populations, Boisvert et al. (1999) determined that
among men with rectal infections in Seattle, those infected with Intermediate
class serovars were 10.5 times more likely to have mucopus (95% Cl: 1.2-95.5)
and 4.2 times more likely to have proctitis than those infected with class B
serovars (95% Cl: 1.1-16.7). Similarly, men infected with serovar class B were
2.5 times more likely to report symptoms (95% Cl: 0.1-0.8) and 3.3 times
more likely to have erythema, bleeding or mucopus (95% Cl: 0.1-0.8) than
those infected with serovar class C (Boisvert et al., 1999).
Genotyping studies. Most early ompA genotyping studies focused on
sequence variation in the four variable segments, designated VS1-4. In 1989,
Yuan et al. determined the nucleotide and deduced amino acid sequences of
the four VSs for the 15 original C. trachomatis serovars used by Wang et al.
(1985) to develop the monoclonal antibody serotyping system. This
sequencing effort was followed by the elucidation of the nucleotide and
deduced amino acid sequences of VS1-4 for the newly identified serovars Da;
la; and L2a (Dean, Patton, and Stephens, 1991; Lampe, Suchland, and Stamm,
1993). Genotyping studies which estimated serovar from the ompA
nucleotide sequence data evaluating populations on the American and
European continents have found similar serovar distributions than
serotyping studies. However in Nairobi, there was a preponderance of the
LGV biovars and among asymptomatic pregnant women from Thailand,
serovar F instead of E was most prevalent (Poole and Lamont, 1992; Yang,
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42
Maclean, and Brunham, 1993; Branham et al., 1994; Morre et al., 1998;
Bandea et al., 2001). In 1985, a source outbreak of symptomatic proctitis
among homosexual men in Seattle from 1982-1983 was identified by VS
ompA sequence comparison (Bauwens et al., 1995). Identical variable
segments encoding the LI serovar MOMP were found for all five patients
with proctitis during this time interval. In 1992, Poole and Lamont were able
to distinguish between different chlamydial serovars solely by VS4 sequence
comparison and reported that serovar D was found significantly more often
in men than women; while G was found significantly more often in women
than men.
Multiple genotyping studies found sequences that were designated as
putative recombinants based on sequence comparison. In an early
population-based study, Yang, Maclean, and Brunham (1993) described VS1,
2, and 4 ompA polymorphisms for urogenitial infections in Canada. The
ompA gene sequences had multiple substitutions, insertions and deletions
and 4 of 49 (8%) had mosaic structures composed of either C /J or I/H by
sequence comparison. Further evidence of recombinant genotypes was
found by Lampe, Suchland, and Stamm, who in 1993, suggested that la was a
composite of I/H , and by Hayes et al., who in 1994, identified three strains
(LGV- 98/ 224 and LGV-115) from South Africa that appeared to be
composed of LI and L2. In two other population-based studies assessing VS
ompA polymophisms for a commercial sex worker core group in Nairobi, 4
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43
(6%) of 60 strains and 7 (4%) of 172 strains were thought to be putative
recombinants composed of L1/L2, L2/L1, L3/H, and I/H (Brunham et al.,
1994; Brunham et al., 1996). In the Brunham et al. (1996) study, signifcant
allelic ompA differences were noted over the 2.5 years of observation and
were ascribed to immune selection pressure. Breakpoint analyses were not
available for the Yang, Maclean, and Brunham (1993) or the Brunham et al.
(1994; 1996) studies as only the VSs were sequenced.
Only a few ompA genotyping studies have analyzed a large
contiguous segment of the ompA gene for quantitative and phylogenetic
reconstructions or have analyzed additional constant regions (Dean et al.,
1995; Bandea et al., 2001; Jurstrand et al., 2001; Stothard, Boguslawski and
Jones, 1998; Dean and Millman, 1997; Fitch, Peterson, and de la Maza, 1993;
Frost et al., 1995). Phylogenetic analyses of the complete or nearly complete
ompA gene have been performed for various populations including the
metropolitan United States and Sweden (Jurstrand et al., 2001; Stothard,
Boguslawski, and Jones, 1998; Dean and Millman, 1997). In 1995, Frost et al.
determined ompA nucleotide signatures in the constant region upstream of
VS1 that differentiated urogenital serovar B sequences from trachoma
serovar B sequences. Interestingly, these same changes were also found in
several of the serovar D urogenital sequences. In 1997, Dean and Millman
evaluated ompA polymorphisms for serovar E within a San Francisco
heterosexual population. Using pattern recognition analysis over the
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44
complete gene, the authors determined a 57-deduced amino acid signature
pattern that distinguished serovar E from non-serovar E strains.
The first computational evidence for recombination in Chlamydia was
provided by Fitch, Peterson, and de la Maza (1993) based on the analysis of
24 complete ompA and 10 omcB sequences. They found that phylogenetic
reconstructions were not congruent for the C. trachomatis strains and that the
genetic distance between LI and B was 10 times greater for MOMP than for
OmcB, despite the fact that the genetic distance between species was only
25% greater for MOMP than for OmcB. They suggested that the most
plausible explanation was genetic exchange among strains with a bias in the
direction of recombination.
Limited studies have correlated ompA polymorphisms with clinical
and demographic data. Similar to serotyping studies, those available have
had conflicting results. In 1995, Dean et al. reported that serovar F was
associated with severe histopathology and serovar E was associated with
milder disease. Further, in all cases where the infecting serovar was F and
severe disease was present, the infecting strain had nucleotide and deduced
amino acid changes compared to the serovar F protoptype sequence. In
contrast, when comparing the VSs from the endometriums of women with
confirmed PID to the VSs from the cervices of women with presumed first
time infection, Lampe, Wong, and Stamm (1995) found no greater
preponderance of variant genotypes within the PID group.
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45
Genotyping was shown to be superior to RFLP for examining
recurrent chlamydial infections (Pedersen et al., 2000). Using genotyping,
after 24 weeks of follow-up, 6 of 31 (19%) cases had recurrence and of these,
five of six (83%) had identical genotypes and one of six (17%) had a different
genotype. However, the percentage of identical genotypes in the genotyping
study was similar to that reported by Lan et al. (1995) which used RFLP to
determine the genotypes. In 2000, Dean, Suchland, and Stamm examined
recurrent infection genotypes in the Seattle area from women with at least
three culture positive C. trachomatis infections of the same serovar over a 2-
year period. Only minor sequence variations were observed in the recurrent
infections with respect to the initial infecting strain and these recurrent
infections occurred over a period of 5 to 10 years. After treatment, many
intervening culture-negative specimens were positive by LCR. This
cumulative evidence suggests that it is unlikely that these recurrences
represented re-infection from a single untreated partner, that they were
instead persistent infections. Moreover, the investigators determined that it
was unlikely that the recurrent infections were due to antimicrobial
resistance given that only one of the seven isolates tested had increased MIC
compared to controls.
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46
Virulence Factors and Pathogenesis
For some time it has been appreciated that high titers of GroEL are
found in persistent infections induced by interferon-gamma (Beatty,
Morrison, and Byrne, 1995); that antibody titers against GroEL are associated
with pelvic inflammatory disease (Wagar et al., 1990) and that GroEL
produces a hyper-inflammatory response in animal models (Morrison et al.,
1989; Patton, Sweeney, and Kuo, 1994). Recent genome work revealed that
the six chlamydial genomes available for study universally had three groEL-
like genes that were substantially conserved (groELl, groEL2, and groEL3;
Karunakaran et al., 2003). Constitutive expression throughout the
development cycle with g ro E L l» groEL2 and groEL3, and increased with
heat and shock characterized their gene transcription.
As early as 1982, regularly spaced dome-shaped envelope projections
that appeared to extend through the membrane of chlamydial reticulate
bodies had been recognized (Matsumoto, 1982; Matsumoto, 1988). These
projections, representing the type III secretion system of Chlamydia, are
morphologically similar to those of Salmonella typhimurium (Hsia et al., 1997;
Bavoil and Hsia, 1998) and the loci that encodes these proteins has been
linked to a gene that encodes a putative serine/threonine protein kinase
(Stephens et al., 1998). In other bacteria, type III secretion systems secrete
proteins that function as inclusion proteins or disrupt signal transduction in
the cell whereby giving the organism the ability to direct vesicular
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47
trafficking, degrade transcription factors, and ultimately to prevent
apoptosis (Stephens, 1998). It is reasonable that differences in the expression
and implementation of this system contribute to differences in strain
virulence for Chlamydia as well as other bacteria.
In May 2000, it was proposed by Shaw et al. that since interferon-
gamma induced degradation of tryptophan inhibits multiplication of
C.trackomatis in epithelial cells, differences in tryptophan levels could explain
variations in serovar pathogenesis. They found that serovars D and L2
encode a 30 kDa tryptophan synthase protein while serovars A and C encode
a truncated 7.7 kDa protein. They proposed that truncation impairs TrpA,
thus reducing levels of tryptophan in the cell and ultimately resulting in
inhibition of bacterial multiplication for serovars A and C. Recently,
Fehlner-Gardiner et al. (2002) showed that the truncation seen for the ocular
serovars resulted in an inability for those serovars to utilize indole glycerol 3-
phosphate as a substrate. In contrast, the full-length protein encoded by the
genital serovars retained the ability to utilize exogenous indole for the
biosynthesis of tryptophan. They proposed that this property might explain
serovar-specific tissue tropism.
Recently, two proteins have been implicated in chlamydial virulence
through genomic sequence analysis. PorB is a surface exposed, outer
membrane porin with weak similarity to MOMP (Kawa and Stephens, 2002).
Unlike MOMP, there is little sequence variation within-species, however,
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48
there is substantial sequence variation between- C. trachomatis and C.
pneumoniae species (Kubo and Stephens, 2000). In addition to functioning as
a porin, PorB is capable of eliciting neutralizing Abs with four major clusters
at Phe34-Leu59, A spll2 -Glul45, Glyl79~Ala225, and Val261-Asn305 (Kawa
and Stephens, 2002).
In addition to PorB, genomic sequence comparison has also revealed
that Chlamydia encodes proteins with substantial homology to the large
clostridial cytotoxins. These cytotoxins produce morphological and
cytoskeletal changes in epithelial cells indistinguishable from those caused
by clostridial toxin B. The authors propose that the elementary body (EB)
proteins are produced and delivered to host cells very early during infection
and that they represent a virulence factor for Chlamydia (Belland et al., 2001).
Finally, Chlamydia produces a protease- or proteasome-like activity
factor (CPAF) that is capable of splitting host cell transcription factors
involved in MHC class I and II antigen presentation (Shaw et al., 2002).
Interestingly, cleavage appears to be fully functional during acute infection
while it is fully or somewhat inhibited under IFN-induced persistence or iron
deprivation conditions (Heuer et al., 2003). CPAF appears to be unique to
Chlamydia in that it has no significant homology to other known proteins
(Zhong et al., 2001). Further elucidation of the function of these proteins is
an exciting research avenue. Differences in expression and function of these
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49
proteins on a population level will aid researchers in their goal of elucidating
the molecular mechanisms behind pathogenic strain differences.
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CHAPTER 2. Recombination in the ompA Gene but
Not the omcB Gene of Chlamydia Contributes to
Serovar-Specific Differences in Tissue Tropism,
Immune Surveillance and Persistence of the Organism
Introduction
The inability to serotype several C. trachomatis strains in the last
decade has prom pted investigators to examine the sequence variation of
ompA. Some variant strains were observed to have a mosaic structure based
on the presence of nucleotide runs with different ancestries. For example, la
was composed of I/H (Lampe, Suchland, and Stamm, 1993); LGV stains
(LGV- 98, LGV-224, and LGV-115) were composed of LI and L2 (Hayes et
al., 1994); and 4-8% of sexually transmitted (STD) stains were mosaics of C /J
and I/H (Yang, Maclean, and Brunham, 1993), or L1/L2, L2/L1, L3/H, and
I/H (Brunham et al., 1994). However in other populations, there were fewer
or no recombinant sequences observed. There were no recombinants
among 68 STD ompA strains in San Francisco (Dean et al., 1995), among 188
trachoma strains in Gambia (Hayes et al., 1995), or among 27 trachoma
strains in Tunisia (Dean et al., 1992). The first computational evidence for
recombination in Chlamydia was provided by Fitch, Peterson, and de la Maza
(1993) based on the analysis of 24 ompA and 10 omcB sequences. They found
that phylogenetic reconstructions were not congruent for the C. trachomatis
strains and that the genetic distance between LI and B was 10 times greater
for MOMP than for OmcB, despite the fact that the average genetic distance
between species was only 25% greater for MOMP than for OmcB. They
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51
suggested that the most plausible explanation was genetic exchange among
strains with a bias in the direction of recombination. Jordan et al. (2000)
identified one apparent gene conversion event between two genes that
encode putative outer membrane proteins of C. pneumoniae strain AR39 by
comparing complete genome sequences.
While the observational data and computational analyses indicated
that recombination had occurred, statistical methods able to detect patterns
of apparent recombination among and between chlamydial species in the
absence of obvious mosaic structures were not applied to these data.
Furthermore, neither the significance of the mosaics nor an accurate
identification of breakpoints was assessed. Our approach to providing a
comprehensive statistical analysis of recombination at these two loci was
two-fold. Methods designed to detect unique or rare, as well as those
designed to detect repeated, recombination were applied to forty ompA and
nineteen omcB sequences from C. trachomatis, C. psittaci and C. pneumoniae.
We also evaluated whether any significant intraspecies and/or interspecies
gene conversion events had occurred and if there were any barriers to these
events. We also analyzed potential mosaics identified by observation to
determine their significance and breakpoints. In agreement with Fitch,
Peterson, and de la Maza (1993), our results revealed an incongruence in the
branching orders of phylogenetic trees for C. trachomatis, including one
cluster of strains not previously identified. Our data show for the first time
statistical evidence to support ompA intragenic recombination for C.
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52
trachomatis strains, including a determination of the relative degree of
recombination for different regions of ompA and for different classes. Our
analyses also reveal ompA intragenic recombination between an equine C.
pneumoniae strain and a rodent C. trachomatis strain. We interpret these
results in light of their implications for immune evasion, tissue tropism,
persistence, and vaccine design.
Materials and Methods
DNA Sequences. Sequence analysis was performed for forty full-
length ompA sequences and for nineteen nearly full-length omcB sequences
from C. trachomatis, C. pneumoniae, and C. psittaci strains. All of the full-
length ompA sequences and twelve full-length omcB sequences were from
GenBank. Seven omcB sequences were determined in this study by direct
sequencing of the following archival non-amplified remnant chlamydial
strains including A/Har-13, D/UW-3, G/UW-57, H/UW-4,1/UW-12, J/UW -
36 and K/UW-31 obtained from the original authors. The properties of the
chlamydial strains from GenBank and from those sequenced in this study are
shown below in Table 2-1. With the exception of strains E-DK20 and D/UW -
3, all omcB sequences were derived from the same strains as for ompA.
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IT,
Table 2-1. Properties of Chlamydial Strains Investigated
Species:
Serovar /
Strain1 3
Yr
isolated
Location Anatomic
Sitea/
Species
Disease Reference
ompA omcB
C.
trachomatis:
A / Har-13 1958
Egypt
Cc./Hu Trachoma Baehr et al., 1988 This study
A/Sa-1 1957 Saudi
Arabia
Co/Hu Trachoma Hayes and Clarke, 1990
B/Jali20 1985 Gambia Co/ Hu Trachoma Hayes et al., 1990 Watson et al., 1989
B/TW -5 1959 Taiwan Co/Hu Trachoma Dean and Millman, 1997
B a/A pache-2 1960 Arizona Co/Hu Trachoma Stothard, Boguslawski,
and jones, 1998
C/TW -3 1959 Taiwan Co/Hu Trachoma Dean and Millman, 1997 de la Maza et al., 1991
D/B-120 1983?
■ > ?
STD Sayada, Denamur, and
Elion, 1992
D /IC -C al-8 1991?
7 7
STD Sayada, Denamur, and
Elion, 1992
D f UW-3 1965 W ashington
7
Cx?/Hu Cervicitis? This study
Da/TW -448 1985? Taiwan Co/Hu Trachoma Savada, Denamur, and
Elion, 1992
E/Bour 1959 California Co/ Hu Trachoma Peterson, Markoff, and
de la Maza, 1990
de la Maza et al., 1991
E/DK-20 1967? Denmark Co/Hu Conjunctivitis Coles, Allan, and Pearce,
1990
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tr,
Table 2-1. Continued
Spedes:
Serovar /
Strainb
Yr
isolated
Location Anatomic
Site3/
Species
Disease Reference
ompA omcB
C.
trachomatis:
F/IC-Cal-3 I960? California Co/ Hu Conjunctivitis Zhang, Morrison, and
Caldwell, 1990
None
G/UW -57 1971 W ashington Cx/Hu C ervicitis Stothard, Boguslawski,
and Jones, 1998
This study
H /U W -4 1965 W ashington Cx/Hu C ervicitis Dean and Millman, 1997 This study
l/UW -12 1966 W ashington Ur/Hu U rethritis Stothard, Boguslawski,
and Jones, 1998
This study
la / IU-4168 1987 Indiana Ur/Hu
1
Stothard, Boguslawski,
and Jones, 1998
l/UW -36 1971 W ashington Cx/Hu Cervicitis Dean and Millman, 1997 This study
ta/ IU-37538 1985 Indiana Cx/Hu
?
Stothard, Boguslawski,
and Jones, 1998
K/UW -31 1973? W ashington Cx/Hu Cervicitis Stothard, Boguslawski,
and Jones, 1998
This study
L I/440 1968 California Ln/Hu LGV Pickett, Ward, and
Clarke, 1987
Clarke, Ward, and Lambden
1988
L2/434 1968 California Ln/Hu LGV Stephens et al., 1986 Allen and Stephens 1989
L2a/ UVV-396 1985? US/Europe Ln/Hu LGV? Dean and Millman, 1997
13/404 1967 California Ln/Hu LGV Fielder, Peterson, and
de la Maza, 1991b
de la Maza et al., 1991
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ir,
Table 2-1. Continued
Species:
Serovar /
Strain*3
Yr
isolated
Location Anatomic
Site3/
Species
Disease Reference
ompA omcB
C.
trachomatis:
LGV-98 1994? South
Africa
Ln?/Hu LGV Hayes et al., 1994
LGV-224 1994? South
Africa
Ln?/Hu LGV Hayes et al., 1994
LGV-115 1994? South
Africa
Ln?/Hu LGV Hayes, et al., 1994
MoPn / Niggll 1939 Minnesota Lu/ Mo Asymptomatic Fielder, Peterson, and
de la Maza, 1991a
Read et al., 2000
Hamster/
SFPD
1991
?
Ileum ?/H a Ileitis Zhang et al., 1993
c.
pneumoniae:
/TWAR-IOL-
207
1967 Iran Co/Hu Trachoma Carter et al., 1991 Watson et al., 1990
/N16 1990? England? NP/horse Nasal discharge Storey et al., 1993
/TWAR-AR-39 1983 W ashington Throat/ Hu Pharyngitis Melgosa, Kuo, and
Campbell, 1993
/K oala 1993? Australia Co / koala Conjunctivitis Girjes, Carrick, and
Lavin, 1994
/liV-BL 1996 Tennessee CNS/H u MS Sriram, Mitchell, and
Stratton, 1998
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o
Table 2-1. Continued
Species:
Serovar /
Strainb
Yr
isolated
Location Anatomic
Site3/
Species
Disease Reference
ompA omcB
C. psittaci:
/ EAE-A22-M 1949 Scotland Lamb Abortion Pickett, Everson, and
Clarke, 1988
Watson, Lambden, and
Clarke,
1990
/ EAE-S26-3 1984? Scotland Lamb Abortion Herring et al., 1989
/6BC 1941 California ? / Parakeet
?
Everett et al., 1991 Everett and Hatch, 1991
AvianC /1 1994? ? ? / Avian
7
None
A vianC /2 1994?
?
? /Avian
7
None
/N352 1982? England Cloaca/
Duck
Asymptomatic? Storey et al., 1993
/ Fe-pring 1984 England? Co/Cat Conjunctivitis Storey et al., 1993
aCo=conjunctiva; Hu=human; Cx=cervix; Ur=urethra; Ln=lymph node; Lu=lung; RT=respiratory tract, CNS=central
nervous system; MS=Multiple Schlerosis; STD=sexually transmitted disease. b LGV=lymphogranuloma venereum;
Mo=mouse; GP=guinea pig; and Ha=hamster.
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57
Chlamydial samples from the archival strains were processed in the
following manner. To extract the DNA, approximately 0.5mL of sample was
centrifuged at 14,000 g for 30 minutes. The pellet was washed once in 200 pi
of IX phosphate buffered saline and centrifuged at 14,000 g for 5 minutes.
The pellet was re-suspended in 100 pi of 10 mM Tris Cl (pH 8.1), 1 mM
EDTA and 20 mM dithiothreitol and then boiled for 10 minutes before
amplification. Purified DNA was stored at -80°C until use.
OmcB PCR primers were synthesized on a DNA synthesizer (380A;
Applied Biosystems, Foster City, CA) by the solid-phase triester method. To
construct the entire gene, three fragments with an approximately one
hundred bp overlap between were generated and joined using the following
primer pairs: omcBFl: 5'-AAAGTTAGTTAAT AACAATT-3' (nucleotides (nt)
-71 to -52) and omcBBl: S'-CGGATCTCTGGACAAG CGCAT-3' (nt 632 to
612); omcBF2:5'-TCCTACT-GCTGATGGTAAG-3' (nt492 to 510) and
omcBB2:5'-GCTCCTGC AGCTT C A AG A ACT-3' (nt 1133 to 1113); and
omcBF3:5'-TGTAGAATA-TGTGATCTCC-3' (nt 1026 to 1044) and omcBB3:
5'-AAAGCCGCCCAG GAATCCCT-3' (nt 1754 to 1733). Using these
primers, several fragments of A/Har-13 could not be amplified. The
following modified primers were designed and used for this strain:
omcBAFl: 5'-AATGTTGAGGGTAAAAGTT-3' (nt -65 to -47) and omcBABl:
5/ACCTTCTT-TAAGAGGTTTTACC3, (nt 591 to 570); and omcBAF3:
5'ACGAGCCTT GCGTACAAGT3' (nt 971 to 988) and omcBAB3:
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58
5'AAACTCTACAGAT-TCCTTA3' (nt 1566 to 1548). The upstream primers
induding omcBFl, omcBF2, omcBF3, omcBAFl and omcBAF3 contained the
-21 M13 universal sequence (TGTAAAACGACGGCC-AGTGCC) 5’ to the
sequence shown above.
For the PCR reaction, one microliter of extracted sample was used in a
100 pi reaction volume which contained 50 mM KC1,10 mM Tris Cl (pH 8.1),
1.5 mM MgCl2 , 100 pM (each) of dATP, dCTP, dGTP, and dTTP, 2.5 U of
ampli-Taq DNA polymerase (Perkin Elmer, Foster City, CA) and 150 ng of
each primer. The sample was subjected to the following thermocycler
temperature profile: 95°C for 2.5 minutes followed by 35 cydes of 94°C for
45 seconds, 55°C for one minute, and 72°C for two minutes with a final
extension of 10 minutes at 72°C after the last cycle. Each amplification
product (10 pi) was run on a 2% agarose gel stained with Ethidium bromide
to confirm the size.
All double stranded DNA products of the correct molecular weight
were purified using GeneClean according to the manufacturer's instructions
(Bio 101, La Jolla, CA). Approximately 60 ng of purified template was
annealed to the M l 3 universal dye primers (Applied Biosystems) and
amplified by PCR using a standard or modified protocol prior to sequencing
with taq polymerase (Perkin-Elmer Cetus) using dye primers or dye
terminators and gel or capillary electrophoresis (373A or 370 automated
sequencing system; Applied Biosystems, Foster City, CA). Each sequence
was read by automation and semiautomation in a blinded fashion. Samples
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59
with ambiguous nucleotides were re-sequenced using newly purified DNA
from the respective remnant sample. The sequences of the omcB genes
determined in this study have been cataloged in GenBank under the
accession numbers AF304326-AF304332.
Phylogenetic and Statistical Analyses. Sequences were aligned
manually using the multiple alignment sequence editor (MASE; version 3.1,
Dana-Farber Cancer Institute, Harvard School of Public Health, [ftp.ebi.ac.
u k /p u b /so ftw are/u n ix /]). The omcB sequences were truncated to the
length of the shortest sequence available (nt 1-1566 of the complete 1676 nt
omcB gene).
Neighbor joining tree topologies (Saitou and Nei, 1987) were
generated by the Molecular Evolutionary Genetics Analysis (MEGA; version
1.01, Institute of Molecular Evolutionary Genetics, Pennsylvania State
University, [http: / / www.megasoftware.net]) program based on distance
estimates using a Kimura (1980) two-parameter model for substitution
events. Bootstrap confidence levels (BCL) were determined by randomly re
sampling the sequence data 1000 times.
Sawyer's runs test was used to test whether significant intraspecies or
interspecies recombination had occurred among the sequences analyzed at
the ompA or the omcB locus of Chlamydia. An extension of the nonparametric
method of Sawyer (1989) that required no phylogenetic inference was
performed using GENECONV (version 1.70; Department of Mathematics,
Washington University St. Louis, [http: / / www.math.wustl.edu/~sawyer1 ).
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60
The basic procedure in the method was the following. Each silent
polymorphic codon in the gene was identified. For each sequence pair, the
gene was partitioned into fragments. The first fragment was from the
beginning of the sequence to the first silent polymorphic codon that differed
between the two; the last fragment from the last difference to the end of the
sequence; and fragments in between were bounded by consecutive
differences between the two. The fragment length for a given pair was the
number of silent polymorphic codons that differed among the other
sequences in the dataset but were not polymorphic with respect to the given
pair. The global fragment score was a linear function of the sum of squares
of the fragment lengths over all fragments over all pairs of sequences. We
tested the null hypothesis that no significant gene conversion events
occurred among the sequences analyzed. The p-value was determined
empirically.
Sequence data were subdivided in order to make valid intraspecies
and interspecies comparisons. We chose to test these hypotheses for strains
that infected humans separately from those that infected lower vertebrate
mammals and birds. This was a conservative approach since dramatic
differences in coalescence times of the hosts may have influenced the results
in ways that were unpredictable. For ompA, we subdivided the sequence
data into five groups: hum an C. trachomatis (all C. trachomatis except
M oPn/Niggll and SFPD), lower vertebrate mammal C. trachomatis
(MoPn/Niggll and SFPD), hum an C. pneumoniae (all C. pneumoniae except
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61
N16 and Koala), lower vertebrate mammal C. pneumoniae (N16 and Koala)
and C. psittaci. For omcB, the data were similarly subdivided into four
groups: hum an C. trachomatis (all except M oPn/Niggll), lower vertebrate
mammal C. trachomatis (MoPn/Niggll), hum an C. pneumoniae (TWAR-IOL-
207), and C. psittaci (EAE-A22-M and 6BC).
Intraspecies hypothesis testing involved inspection of the global
Sawyer's run test score for the appropriate dataset. Intraspecies testing was
possible for hum an C. trachomatis and lower vertebrate mammal C. psittaci
for ompA, and for hum an C. trachomatis for omcB. Testing was not possible
for the other groups because there were either too few sequences or too few
polymorphisms to evaluate. Interspecies hypothesis testing was
accomplished by combining appropriate data. Evidence was only considered
significant if the global score for the combined dataset was significant and if
there were significant fragments detected between the two species. For the
same reasons noted above, interspecies hypothesis testing for omcB was not
possible for lower vertebrate mammal C. trachomatis and C. psittaci. To test
hypotheses about barriers to recombination, we compared the global scores
of datasets before and after the inclusion of strains from a different species.
If the global score decreased from significant to insignificant, a barrier to
recombination was considered suggestive for the strains involved.
These hypotheses were further evaluated using compatibility
matrices. Compatibility matrices and neighborhood similarity scores were
calculated using the program RETICULATE (Human Genetics Group, John
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62
Curtin School of Medical Research, Australian National University,
[h ttp ://jcsm r.anu.edu.au/dm m /hum gen/ingrid/reticulate.htm ]) following
the methods of Jakobsen and Easteal (1996). In this method, matrices
representing the compatibility of all possible pairs of informative sites with a
single maximum parsimony tree were calculated. The neighbor similarity
score indicated the degree to which sites were compatible and its significance
was determined empirically. The null hypothesis tested was that sites were
randomly distributed with regard to type (incompatible / compatible),
where clustering of sites suggested recombination. In this analysis, only
parsimoniously informative binary sites (sites with only two different
nucleotides present more than once) were included. For these analyses, the
ompA and omcB data were subdivided into strains that infect hum an hosts,
and those that infect lower vertebrate mammals and birds for the same
reasons as stated above.
The Index of Association between codons following the methods of
Feil et al. (1996) was used to test the null hypothesis that ompA polymorphic
codons were in complete linkage equilibrium. Briefly, IA was computed by
comparing the observed between strain variance (V0 ) with the variance that
would be expected if polymorphic codons were randomly assorted (VE ).
Any observed variance that exceeded that expected by chance represented
linkage between codons. If the polymorphic codons were randomly
assorted and the data were in linkage equilibrium, IA was expected to be
zero. The significance of IA was determined empirically. Since Vw and in
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63
turn IA increased with the number of polymorphic codons (r), it was
important to compare values only if r was the same, and in other cases, only
to distinguish those IA that were significantly different from zero from those
that were not.
The Recombination Identification Program (RIP; version 1, HIV
Database Group, Los Alamos National Laboratories, [http: / /hiv-web.lanl.
gov/]) was used as a graphical tool to predict a strain's mosaic structure
(Siepel et al., 1995). For each of these strains, the Maximum Chi-Squared
program (version 1.0, Molecular Microbiology Group, University of Sussex,
[http:/ / www.biols.susx.ac.uk/ Biochem/Molbiol/maximum-chi-
squared.html]) following the methods of Maynard Smith (1992) was used to
refine and assess the significance of the structure. In the test, polymorphic
sites were defined as those sites that varied between the potential
recombinant and its possible parental strains. For each of the parental
strains, the number of differences between the parental strain and the
putative recombinant divided by the total number of differences in the data
was calculated before and after a proposed cut. A cut was optimal when the
difference in these proportions was maximized. The significance of the
division was tested by empirically determining a p-value by randomization.
An iterative procedure was used to evaluate strains with multiple divisions
as described in the method (Maynard Smith, 1992).
For all statistical analyses, sites that contained gaps were ignored and
analyses were considered significant at a p-value of s 0.05 except for the
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64
maximum chi squared analysis, which was considered significant at p < ; 0.001
in order to be conservative.
Results
Phylogenetic analysis of ompA and omcB. In the ompA alignment,
the locations of the VSs as defined by Yuan et al. (1989) were as follows: VS1
from 262-333; VS2 from 499-576; VS3 from 766-813 and VS4 from 964-1068.
At the nucleotide level, there were 691 polymorphic sites within the 1215
base pairs (bp) of the complete ompA gene, and 609 polymorphic sites within
the 1566 bp of the partial omcB gene analyzed. There were 142 polymorphic
sites among the B class strains, 100 polymorphic sites among the C class
strains, and 36 polymorphic sites within the Indeterminant class strains.
Phylogenetic reconstructions suggest potential differences in the
evolutionary histories of ompA and omcB as seen in Figure 2-1A-B. Major
branching orders of the neighbor- joining ompA trees agreed with those
already reported: the three species were represented by three distinct clades
and the hum an C. trachomatis strains clustered into B, C, and Indeterminant
classes as described above. However, for omcB, the C. trachomatis strains did
not form three classes. The LI, L2 and L3 serovars of the LGV biovar
formed a distinct group instead of being split into the B and C classes,
consistent with the findings of Fitch, Peterson, and de la Maza (1993). In
addition, the inclusion of the seven new omcB strains sequenced in this study
revealed that F did not cluster with G in the Indeterminant class but instead
grouped with E. These incongruent patterns suggested that recombination
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65
had been responsible for driving the divergence of several of the C.
trachomatis strains including E,F,G,L1 and L3.
Figure 2-1A. Phylogenetic Reconstruction for the ompA Gene Sequences
using the Neighbor-Joining Method
m
IG D
1 0 0
45
m
-BBour
-L2/434
-B/Jafi20
ICO
42
55
34
57
ICO
1 00
3 0 0
- i m m
-Ll/44©
-F/TC-Cat-3
QUW-5?
C/TW-3
HOT-4'
-K/UW-31
-WW-12
-JW U W -36 ■
-0/404
-MIar-13
■ MpPn/Niggll
TWAR-1OL-207
-6BC
•EAB-A22-M
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66
Figure 2-1B. Phylogenetic Reconstruction for the omcB Gene Sequences
using the Neighbor-Joining Method
m
1 2
4 $
m
m
i#
ion
-E/Botr
- F/lC-Cal-3
~G/UW~57
-K/UW-3!
- DAJW-3
-B/Jafi20
-H/UW-4
m
M O
jUHar-13
•C/TW-3
M1W-36
-BUW-12
-L2/434
-LI/440
-0/404
-MoPn/Niggll
TWAR-IOL-207
EA&A22-M
6BC
The values at the nodes in Figures 2-1A-B are the bootstrap confidence levels
(BCL) for the interior branch. The BCL represents the percentage of 1000
bootstrap re-samplings for which the strain to the right was separated from
the other strains.
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67
Statistical evidence for or against intraspecies and interspecies
recombination in ompA and omcB. To determine if differences in tree
topologies for ompA and omcB could be explained by intragenic
recombination, the sequence data were subjected to Sawyer's runs test using
GENECONV (Sawyer, 1989). Tables 2-2A-D present the intraspecies and
interspecies comparisons for the ompA and omcB gene sequences.
Table 2-2A. Sawyer's Runs Test Results for ompA Intraspecies Comparisons
for C. trachomatis and C. psittaci
Dataset Global
score
Global p-valuea # S.D. above
simulated
mean
S.D. of
simulations
Human C.
trachomatis
11.306 <0.0001 6.85 1.127
C. psittaci 4.541 0.038 2.03 1.142
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68
Table 2-2B. Sawyer's Runs Test Results for ompA Interspedes Comparisons
between C. trachomatis, C. pneumoniae and C. psittaci
Dataset Global
score
Global p-valuea # S.D. above
simulated
mean
S.D. of
simulations
Human C.
trachomatis and
Human
C.pneumoniae
8.290 0.002 4.33 1.117
Lower
vertebrate
animal C.
trachomatis and
Lower
vertebrate
animal C.
pneumoniae
4.171 0.033 2.40 1.107
Lower
vertebrate
animal C.
trachomatis and
C. psittaci
4.207 0.124 1.16 1.160
Lower
vertebrate
animal C.
pneumoniae and
C. psittaci
5.257 0.039 2.11 1.153
Table 2-2C. Sawyer's Runs Test Results for omcB Intraspecies Comparisons
for C. trachomatis
Dataset Global
score
Global p-valuea # S.D. above
simulated
mean
S.D. of
simulations
Human C.
trachomatis
0.989 0.582 -0.14 0.120
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69
Table 2-2D. Sawyer's Runs Test Results for omcB Interspecies Comparisons
between C. trachomatis and C. pneumoniae
Dataset Global
score
Global p-valuea # S.D. above
simulated
mean
S.D. of
simulations
Human C.
trachomatis and
Human C.
pneumoniae
0.877 0.838 -0.56 0.517
Global p-value in Tables 2-2A-D were obtained after permuting the silent
polymorphic sites in the respective dataset 10,000 times.
Based on this test, there was significant evidence for ompA intraspecies
recombination between hum an C. trachomatis strains (p<0.0001). Consistent
with the phylogenetic data, the following pairs of strains E/Bour/G /U W -57
(p=0.003), B/Jali20/E/Bour (p=0.007), Ba/Apache-2/E/Bour (p=0.007), and
LI/440/LGV-98 (p=0.023) had significant ompA gene conversion events.
OmpA intraspecies recombination between C. psittaci strains was also
supported (p=0.038) with the following pairs of strains identified N352/EAE-
S26-3 (p=0.038), 6BC/EAE- S26-3 (p=0.038), AvianC/EAE-S26-3 (p=0.038),
MnCal-10 / EAE-S26-3 (p=0.038), and EAE-A22-M/EAE-S26-3 (p=0.038). In
terms of ompA interspecies comparisons, there was significant evidence to
suggest recombination between the rodent C. trachomatis M oPn/Niggll
strain and the C. pneumoniae Horse N16 strain (p= 0.033). There was also
suggestive evidence for a barrier against recombination between C.
trachomatis rodent strains and C. psittaci strains. This latter observation was
based on the fact that the inclusion of the rodent strains with the C. psittaci
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70
strains weakened the significance of the previous findings from p=0.038 to
p=0.124. Unlike the results for ompA, there was no statistical evidence to
support recombination within omcB. In every analysis, the global score was
insignificant.
Recombination detection methods are based on the underlying
assumption that substitution rates are equal along the gene. Methods based
on distributions of polymorphic sites, such as the Sawyer's runs test, have a
higher false positive rate due to violations of this assumption than methods
that detect incompatibilities between sites and changes in local estimated
phylogenies (Wiuf, Christensen and Hein, 2001). For this reason, we
analyzed the data for compatibility using RETICULATE (Jakobsen and
Easteal, 1996). For ompA, there were large numbers of sites that were
incompatible with a single tree, both for all strains that infected humans
(neighbor similarity 0.886) and for all strains that infected lower vertebrate
mammals and birds (neighbor similarity 0.738; Figure 2-2A-B). In both cases,
the distribution was not random (p=0.0001). This provides further evidence
for recombination within the ompA gene of Chlamydia. In contrast, sites were
randomly distributed for omcB sequences from hum an hosts (p=0.211; Figure
2-2C), again suggesting no recombination within omcB.
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71
Figure 2-2A. Compatibility Matrix for the Chlamydia ompA Gene from
Human Hosts
■ n|j,s s |i|; |ii ; i
...I
Mil I
■ ■ il I
i s t i i :
■ii fl in ■ I li fci i a
■ lllh h l > ■ Ifl i |
i :m i
M 4V I Mill
m l I
I f i i H - s
m m *
e
I K r i l l - W O
™ . . . . . . . .
Hi I ai a a w — I t m ail a n • sa\ja • m m m m
ttT M 'A i Ii II r* a ii » i r i I I
um ■■
>■«« « l
aai a mm m a a
i n ; ’« ' ji
< a a
aa a "v a a
■inifilial v *• «
thbh ssar
MIVI >M C .Wlftl.VI 1.X A 1 Mill
ratal
IP II I
!■ ivi*«ia • m
• a a l i i l l
P f ' t f i f i r s
a a a aa a a ■ a
n s r E i s f I
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72
Figure 2-2B. Compatibility Matrix for the Chlamydia ompA Gene from Lower
Vertebrate Mammals and Birds
Figure 2-2C. Compatibility Matrix for the omcB Gene from Human Hosts
In the preceeding figures, white spaces represent pairs of informative sites
that are compatible with a single maximum parsimony tree while black
spaces represent those pairs that are incompatible with that tree.
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73
The ratio of average synonymous to average nonsynonymous
substitutions (ds/d N ) was calculated for ompA and omcB following the
method of Nei and Gojobori (1986). Using the same seventeen strains from
the three species, the average ds/d N was 6.7:1 for ompA and 5.7:1 for omcB.
However, when examining just the hum an C. trachomatis strains, the average
dg/dN was 6.0:1 for ompA and 1.4:1 for omcB.
Relative degree of recombination in ompA for different regions and
classes. None of the above methods can be used to detect recombination
reliably when the number of events is high. In contrast, the Index of
Association (IA ) between codons is a test statistic that can be used to test
whether polymorphic codons are in linkage equilibrium. For this reason, we
determined the IA between polymorphic codons at the ompA and omcB loci.
When all hum an C. trachomatis sequences were analyzed together, ompA
codons were in linkage disequilibrium (p< 0.001). This was also the case for
C. psittaci sequences (p<0.001). Since the degree of recombination tends to
increase with increasing similarity between strains, it was possible that
human C. trachomatis sequences were in linkage disequilibrium while
individual classes were in linkage equilibrium. To test this hypothesis, we
subdivided the sequence data into classes and repeated the analysis. For
both the B and C classes, polymorphic codons were in linkage disequilibrium
when analyzing the entire gene (p<0.001). This indicated that, overall,
recombination in the ompA gene was not frequent enough to randomize the
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74
gene or break up clonal associations between codons, thus indicating a clonal
population structure.
In order to compare the relative degree of recombination within
classes, the entire ompA gene was subdivided into regions, 20 polymorphic
codons in length, and the IA test statistic was calculated. For the B class, this
resulted in five regions of 20 codons and one region of 12 codons. For the C
class, the gene was divided into four regions of 20 codons. For every region
except for the first, IA for the B class exceeded that for the C class, as seen in
Figure 2-3.
Figure 2-3. Linkage Between Polymorphic Codons within Successive ompA
Regions, Approximately 20 Polymorphisms in Length
12
10
8
6
4
2
0
6 2 3 5 1 4
ompA region
Legend: ^ C. trachomatis C class strains and ® C. trachomatis B class strains.
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75
In fact, in region 3, codons for the C class were not in significant
linkage disequilibrium (p=0.103). These results suggest that C class strains
experienced gene conversion events to a greater degree than B class strains.
Furthermore, there was a greater degree of recombination in the
downstream half of ompA. For omcB, there was no evidence to suggest that
polymorphic codons were in linkage equilibrium indicating that the inability
to detect intragenic recombination was not a result of repeated
recombination.
Identification of breakpoints in ompA mosaics. Potential ompA
mosaics were identified as those strains detected in the GENECONV
analysis, those that clustered differently in tree topologies, and those
reported in previous studies. For each strain, the similarity against all other
strains was calculated and plotted by nucleotide position using RIP (Siepel et
al., 1995). This output was visually inspected to determine potential
breakpoints. Figures 2-4 A-D present the RIP similarity plots for G/UW-57,
D/B-120, E/Bour and LGV-98. These similarity plots provided the initial
predictions of the mosaic structure.
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Similarity t o Isolate D/B120
76
Figure 2-4A. RIP Images Depicting the Similarity of Strain D/B120 Against
all Others with Position along the ompA Gene
I % - A j
0 .9
0.8
0 .7
Sarcvgr It
0.6
0 500 1000
Position of Center of Window
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77
Figure 2-4B. RIP Images Depicting the Similarity of Strain G/UW-57 Against
all Others with Position along the ompA Gene
0.9
0.8
m 0 .7 -
SatavorD
Sarcvat E
0.6
0 500 1000
Position of Center of Window
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t o i s o l a t e s eBour
78
Figure 2-4C. RIP Images Depicting the Similarity of Sti
all Others with Position along the ompA
r — r
0,9
A
f . A . x
V r t ‘ i j]1
V l J M ■''" \r f k , ,jj
'- .V ; V '# i!
oa
0 7
A
\rK
» |K lii
1 1
0.6
f) S O D
f * m lH n r s <\t . ’tf W tfirto tM
•ain E/Bour Against
Gene
1C* JO
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79
Figure 2-4D. RIP Images Depicting the Similarity of Strain LGV-98 Against
all Others with Position along the ompA Gene
0.9 so
9
>
3
( D
I
1 0.8
B
£
1
< / >
0.7
0.6
0 1000 500
Position of Center of Window
In Figures 2-4A-D, only strains with greatest similarity to the query sequence
have been shown.
The maximum chi quared test (Maynard Smith, 1992) was used to
refine the RIP predictions and assess the statistical significance of the refined
structures. The G/UW-57, D/B-120, E/Bour, and LGV-98 strains clearly
showed mosaic structures with each cut supported at the p=0.0001 level, as
shown in Figure 2-5.
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80
Figure 2-5. Mosaic Structures for Strains E/Bour, G/UW-57, LGV-98 and
D/B-120 Predicted by the Maximum Chi-Squared Test
Ba
bl cl
G
LI
L2
LGV-98
1 1
D/B120 W SSS S m SSXSISSee^m
Each line is the ompA gene for the strain given to the left. The shading
represents the proposed contributions from parental strains to the genetic
composition of the mosaic strain, a-f are the locations of the proposed cross
over points at nucleotides 452,584, 851, 651, 794 and 547, respectively.
EEESE2 =BaC.trachomatis; = E /G C.trachomatis; ffi&S = D /IC -
Cal-8 C. trachomatis; = F C. frac/zomatis; yssm = LI C. trachomatis;
■ m = L2 C. trachomatis; and B>BBJ = divergent unknown C. trachomatis
contribution.
Table 2-3 shows the break point analysis of potential ompA mosaics.
G/UW-57 appeared to be a mosaic of strains F/IC-Cal-3 and E/Bour with
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81
two break points at nt 645, nt 651 and nt 792, nt 794. Upstream of the first
cut and downstream of the second, G/UW-57 was markedly similar to F/IC-
Cal3 and dissimilar to E/Bour. Between the cuts, the opposite was true
(Table 2-3). Strain D/B-120 appeared to be a mosaic of strain L I /440 and a
possible divergent segment of E/Bour with a single break point at nt 547, nt
567. Upstream of the cut, the E/Bour contribution was only supported at the
p=0.01 level. However, downstream of it, D/B-120 was extremely similar to
L I /440. E/Bour appeared to be a mosaic of Ba/ Apache-2, D/IC-Cal-8 and
the aforementioned G/UW -57 with three break points at nt 452, nt 485; nt
584, nt 587; and nt 851, nt 867. E/Bour was most similar to Ba/Apache-2
before the first break point, to D/IC-Cal-8 between breakpoints 1 and 2, to
G/UW-57 between break points 2 and 3, and to D/IC-Cal-8 again after the
last break point. As was originally proposed by Hayes et al. (1994), LGV-98
was a composite of LI and L2 with a single break point at nt 567. The
upstream portion of LGV-98 most likely originated from LI, while the
downstream segment was identical to L2. However, the test predicted two
upstream divisions. Due to the small number of polymorphisms, the second
division was most likely artifactual. The location of the single division agrees
exactly with that proposed by Hayes et al., (1994; nt 537 in the LGV-98
sequence corresponds to nt 567 in this alignment). Ia/IU-4168 was identical
to the la sequence originally identified to be an I/H mosaic by Lampe,
Suchland, and Stamm (1993) over all regions sequenced. Based on the results
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82
of the maximum chi squared test, Ia/IU-4168 was not a mosaic of I/H at the
0.001 significance level.
It is interesting to note that for every significant mosaic strain
analyzed, VS3 may have been involved in the gene conversion event. The
upstream division of G/UW-57 was just before VS3 and the downstream cut
within VS3. D/B120 and LGV-98 shared a presumed crossover point at nt
v
547, at the end of VS2. E/Bour had one division at the end of VS3 and
another in between VS3 and VS4. In fact, E/Bour was the only mosaic with a
cut upstream of VS3 (at the beginning of VS2). Identification of most
breakpoints just before VS3 is in agreement with the index of association
results in which the downstream half of ompA was shown to have a higher
degree of recombination than the upstream half.
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cn
0 0
Table 2-3. Break Point Analysis of Potential ompA Mosaics
ompA
mosaic
Parental
strain
Location all
cuts3
Proportional
difference1 3 before
cutl
Proportional
difference6 ” between
cutl / cut2
Proportional
difference6 ” between
cut2/cut3
Proportional
difference1 5 after last
cut
C/
UVV-57
F /
IC-Cal3
645,651
792,794
9/92 (9.8%) 17/17(100% ) 9 /4 9 (18%)
E/
Bour
654
804
90/93 (97%) 2/18 (11%) 45/47 (96%)
D/
B-120
LI/
440
547,567 28/37 (76%) 9 /5 2 (17%)
E/Bour Ba/
Apache-2
452,485
584, 587
851,867
4/19(21% ) 17/20 (85%)
D /
IC-Cal8
452,485
584, 587
851,867
7/40 (18%) 38/41 (93%) 11/53 (21%)
c/
UVV-57
452,485
584, 587
851,867
14/41 (34%) 49/53 (93%)
LGV-98 L I/440 548,567 1/14 (7%) 47/47 (100%)
L2/434 548,567 13/14 (93%) 0 /4 7 (0%)
‘ ’ Location of the cut may be represented by two numbers. Only polymorphic sites were analyzed and thus, break
point locations are not necessarily consecutive nucleotides. b Proportional difference is number of differences between
parental strain under comparison and potential recombinant divided by the total number of differences between both
parental strains and potential recombinant.
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84
Discussion
In this study, we provide comprehensive statistical analyses of
intraspecies and interspecies recombination for C. trachomatis, C. pneumoniae,
and C. psittaci at the ompA and omcB loci. These analyses revealed that
intragenic recombination at the ompA locus for C. trachomatis strains is
significant and not likely due to chance substitution events. The relative
degree of recombination for different classes and different ompA regions
were consistent with the multiple breakpoints in VS3 and VS4 that we
identified for strains D/B120, G/UW-57, E/Bour, and LGV-98. The former
three strains were first identified as recombinants in this study. Further, we
present the first evidence for intragenic recombination between C. psittaci
strains, and for interspecies recombination between a rodent C. trachomatis
strain and an equine C. pneumoniae strain at the ompA locus.
Three independent statistical methods provided strong and consistent
evidence for recombination in ompA but not in omcB. The Sawyer's runs test
requires no phylogenetic inference and is relatively unbiased by the effects
of selection. Monomorphic and amino-acid varying sites that may have
clustered under strong selection were excluded. Silent sites should be
effectively neutral. In a region that is immunodominant, however, this may
not always be the case. A potential shortcoming of the Sawyer's runs test is
that it can produce false positives when mutation rates vary along the gene
(Wiuf, Christensen, and Hein, 2001). Yet, it is unlikely that variable mutation
rates have mimicked recombination in this instance. The incompatibility
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85
method we used was less sensitive to this phenomenon (Wiuf, Christensen,
and Hein, 2001) but it also detected recombination in ompA. Further, it
should also be noted that recombination in Chlamydia is biologically plausible.
Not only has a homolog of RecA been cloned, sequenced, and characterized
for C. trachomatis (Hintz et al., 1995), but homologs of several other enzymes
in the RecBCD and RecF recombination pathways have been identified in the
complete genome sequence of the C. trachomatis D/UW -3 strain (Stephens et
al., 1998).
In contrast to ompA none of the methods used in this study detected
intragenic recombination in omcB. It is unlikely that the failure to detect
recombination was due to a high degree of recombination, since the index of
association test did not provide any evidence for recombination within omcB.
In order to explain the discordant phylogenies for ompA and omcB, there
must have been a major breakpoint somewhere in between these two
genes. In our analyses, we identified major breakpoints in the downstream
half of ompA. These results can be interpreted in at least two ways. One is
that only ompA is involved in recombination and that these breakpoints
delineate the segments involved. Another is that omcB is involved in
recombination in its entirety, and another unidentified breakpoint resides
downstream of it. This event cannot be ruled out by our analyses because
none of the methods used would have been able to detect recombination
without including the intervening sequence that encompassed the major
breakpoint(s). This latter scenario is consistent with the differences in
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86
divergence of the two genes (Fitch, Peterson, and de la Maza, 1993). If the
difference in divergence was primarily due to differences in selective
pressures, there should be the same relative divergence within species as
between species, but this was not the case. Further, the ratio of ds/d N for the
three species was nearly equal for the two genes. However, when
examining just the hum an C. trachomatis strains, the average ds/d N was over
four times as high for ompA as it was for omcB. This suggests that hum an C.
trachomatis strains evolved more quickly for omcB than for ompA and for
strains from other species. Such anomalous patterns of divergence have
been seen in other pathogenic bacteria including the Neisseria species where
it was suggested that recombination had obscured the evolutionary history
of the organism (Feil et al., 1996).
Differences in selection pressures may in part explain the relative
differences in the degrees to which intragenic and intergenic recombination
occurred at the two loci. If omcB provides an essential function to the
organism with strong functional and structural constraints, it may be under
strong stabilizing selection pressure. In this case, fitness costs attributed to
recombination within the reading frame may be too great and may in part
prevent it from occurring. On the other hand, conserved regions resulting
from strong constraints may have increased the likelihood of intergenic
recombination since the degree of homologous recombination increases
with increasing similarity between strains. Moreover, these constraints may
have provided a selective force leading to the preferential retention of one
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87
allele and so low variation. According to the "selective sweep" model
(Hudson, 1994), large regions of DNA will become fixed in areas of low
recombination, while only small areas will become fixed in areas of high
recombination. For omcB, almost the entire gene has become fixed. This
suggests that the degree of recombination m ust have been low enough to
allow a sweep and infers an upper bound for intergenic recombination in
omcB. In contrast, ompA is presumably under diversifying immune selection.
Recombination within the reading frame of ompA may actually increase
fitness and contribute to the organism's success. Since there are few to no
regions in ompA that have become fixed, there is no upper bound for the
degree of recombination that has occurred. Either recombination was too
extensive for a selective sweep or it was prevented from occurring by
diversifying selection pressures. No analyses in this study can distinguish
between these possibilities.
We provide evidence for interspecies ompA recombination between C.
trachomatis and C. pneumoniae that infect lower vertebrates. Although, it has
been recognized that interspecies transmission of C. psittaci strains occurs
between birds and hum ans in the form of psittacosis, no other evidence for
cross-species transmission exists. Interspecies mosaics have not been
identified by sequence observation, and tree topologies over multiple coding
regions are congruent with respect to species clustering. Our results are
consistent with these findings since these earlier methods were less sensitive
than the ones used in this study. Further, there may actually be a barrier to
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88
recombination between the C. trachomatis rodent strains and the C. psittaci
strains. The nucleotide differences for these species range from 39% to 41%,
that which is in excess of the presumed upper limit for homologous
recombination (Roberts and Cohan, 1993). Thus, recombination between
these species may not be feasible for lack of a viable mechanism.
Our analyses of previously identified mosaics and other well known
C. trachomatis strains provided significant evidence that strains D/B120,
G/UW-57, E/Bour, and LGV-98 were recombinants. For each of these
recombinants, several significant breakpoints just before and within VS3
were identified. These findings were in agreement with the index of
association analysis where a higher degree of recombination was found in
the downstream half of ompA. Recombination between more distantly
related strains drives clonal divergence (Guttman and Dykhuizen, 1994), and
diversity in VS3 and VS4 may contribute to the adaptation of the parasite to
changing host environments. Specifically, these mechanisms may in part be
responsible for differences in immune evasion, persistence, and tissue
tropism of the organism.
For C. trachomatis, upstream of and within VS3 are regions that elicit
T-helper cell activity (Allen, Locksley, and Stephens, 1991; Ishizaki et al.,
1992). T cell dependent antibody production presumably represents an
important evolutionary mechanism for protection against microbial
pathogens as it is conserved among vertebrate species (Hodgkin, 1997). In
turn, pathogens have developed mechanisms to vary T cell epitopes to
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89
evade this host immune response. Altered peptide antagonism is a
recognized immune escape mechanism for viruses and malaria, where T cell
epitope variants are able to eliminate or down-regulate the cell mediated
response to the index peptide (Gilbert et al., 1998; Hill et al., 1997). In this
respect, co-evolution of hosts and parasites might be likened to a molecular
arms race (Gilbert et al., 1998). For Chlamydia, in addition to evidence for the
occurrence of point mutations within T cell epitopes, our results show that a
potential mechanism exists via recombination for exchanging T cell epitopes
important for escaping immune surveillance. Immune evasion may result in
the failure of the host to clear the organism, resulting in a persistent
infection. Further, there is evidence that persistent infections are more
commonly associated with C class than B class strains despite the fact that B
and Indeterminant class strains are the most prevalent overall in genital
infections (Dean, Suchland, and Stamm, 2000). Our analyses consistently
suggest that the degree of recombination was higher for C class strains than
for B class strains. Although the sequences from the above study were not
analyzed for mosaic structures and gene conversion events, these data
suggest that recombination in ompA may generate the genetic diversity
required to evade immune surveillance, ultimately leading to persistence in
the host.
In addition to providing the genetic diversity necessary for immune
evasion, recombination within VS4 may also be important in tissue tropism.
Monoclonal anitbodies against VS1,2 and 4 neutralize chlamydial infection
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90
by inhibiting attachment (Moulder, 1991). Differential trypsin inhibition
suggests that VS2 and VS4 are critical in this process. Trypsin treatment does
not reduce attachment for serovar L2, but it dramatically reduces attachment
for serovar B. This difference may be due to the presence of trypsin-
sensitive lysine residues in VS2 and VS4 of serovar B and the absence of
these residues for serovar L2 (Hackstadt, 1999). The fact that VS4 is critical
for attachment (Hackstadt, 1999) and that there is a high frequency of
recombination within VS4 suggests that genetic diversity in this region may
contribute to serovar-specific differences in tissue tropism. Our
recombination data for serovars D/B-120 and E/Bour are suggestive of this.
Serovar D is the most prevalent serovar in rectal infections except for
serovars LI, L2, and L3. This serovar produces mild infections while
serovars LI, L2 and L3 have historically been associated with severe
lymphadenitis and proctitis (Barnes, Rompalo, and Stamm, 1987). However,
it has recently been reported that rectal infections with serovar LI are milder
and similar to those caused by serovar D (Bauwens et al., 1995). Our
analyses showed that D and LI were markedly similar within VS3 and VS4
but divergent upstream of these segments, suggesting that D inherited the
former VSs via recombination with serovar LI. Thus, recombination with
serovar LI may have allowed serovar D to more effectively invade the rectal
mucosa. Additional evidence is provided by clinical data for serovar E. It is
the most prevalent serovar in genital infections and outcompetes serovar F
for nutrients and resources in tissue culture (Jones, Williams, and van der
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91
Pol, 1998). Our analyses suggest that serovar E is a mosaic of Ba/ Apache-2,
D/IC-Cal-8, and G/UW-57. E and Ba were markedly similar upstream of
VS2. Yet, Ba is a common serovar in ocular infections and is rarely found in
genital infections. E also possesses a long shared VS4 segment with D,
another extremely prevalent serovar in genital infections. It is possible that
E was once similar to Ba in its entirety and subsequently acquired DNA from
D via recombination in VS4, which contributed to its success in the genital
mucosa. This cumulative evidence is suggestive that genetic diversity in VS4
may play a role in determining chlamydial serovar-specific differences in
tissue tropism.
The possibility that C. trachomatis C Class strains exhibit a panmictic
population structure in a region responsible for immune evasion has
important implications for the design of a multivalent DNA or protein
vaccine. A widely employed approach to the design of a chlamydial vaccine
has been to target regions of VSs conserved among strains since VSs are
likely to be surface exposed and immunodominant. Our analyses indicate
that conserved regions within VS3 and VS4 must be nearly identical such
that, within the targeted region, recombination between strains has the
effect of homogenization as opposed to diversification. In contrast, targeted
regions within VS1 and VS2 may not require the same degree of
conservation since recombination occurs to a lesser degree. We can further
refine this approach based on the fact that recombination seems to occur
primarily between strains of the same class and not between strains of
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different classes. This is suggested by the fact that the degree of
homologous recombination tends to increase with increasing similarity
between strains and, in our analyses, an increase in linkage was observed
when C class strains were analyzed separately from B class strains. The
exception to this is that the Indeterminant class strains appear to recombine
with the B class strains. For this reason, regions within VS3 and VS4 should
be nearly identical within each class while some differences between classes
may be acceptable where, for the purposes of this discussion, B and
Indeterminant classes are considered together. To be conservative, this rule
should be followed for VS1 and VS2 as well.
Following these criteria, we have identified segments in VS3 and VS4
that could be simultaneously targeted for a multi-strain vaccine. The
segment in VS3 is a hexapeptide located from nt 796 to 813. Among the C
class strains, the sequence AGTEAA is completely conserved while among
the B and Indeterminant class strains the sequence AGTDAA is conserved
except for A-*S in Ba/Apache-2 and AA->GV in L2. The segment in VS4 is a
thirteen residue peptide located from nt 982 to 1020. Among the C class
strains, the sequence DVTTLNPTIAGKG is conserved except for V->T in
A/Har-13 and A-*T in K/UW-31. Among the B and Indeterminant class
strains, the sequence DVTTLNPTIAGAG is conserved except for V-*T in
D/B120, E/Bour and LI and V-»I and A-*C in both Indeterminant dass
strains. Within this thirteen residue peptide is a nona-peptide previously
identified by Fitch, Peterson and de la Maza (1993) as being an extremely
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93
conserved segment that could be targeted for vaccine design. The
knowledge that recombination primarily occurs within classes in VS3 and
VS4 has provided criteria that has allowed us to take a less conservative
approach in identifying conserved segments, and thus, expand the repertoire
of targeted regions with considerable expectation for success.
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94
CHAPTER 3. Population-Based Genetic and
Evolutionary Analysis of Chlamydia trachomatis
Urogenital Strain Variation in the United States
Introduction
The major outer membrane protein (MOMP) of C. trachomatis has been a
major focus of research as it contains serological variant (serotype)-,
subspecies-, and species-specific epitopes (Baehr et al., 1988), has important
neutralizing determinants (Peeling and Brunham, 1991), elicits T cell help for
antibody production (Allen, Locksley, and Stephens, 1991; Su et al., 1990a),
and may play a role in the attachment and invasion of host cells and in tissue
tropism (Millman, Tavare, and Dean, 2001). Not surprisingly, MOMP has
been the focus of epidemiologic studies of the organism and has been the
primary candidate for a chlamydial vaccine. Yet, little is known about the
genetic variation of ompA, the gene that encodes MOMP, or the diversity and
evolution of MOMP epitopes among STD populations in the US and around
the world.
Our previous statistical analyses revealed recombination within ompA,
including one instance of horizontal gene transfer between Chlamydia species
(Millman, Tavare, and Dean, 2001). We found a high level of recombination
relative to substitution processes in an area that contains T cell epitopes
which are important for eliciting protective immunity (Ishizaki et al., 1992;
Stagg et al., 1993). Consequently, the genetic variation of ompA reflects the
cumulative effects of immune selection pressures, functional constraints, and
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95
other adaptive pressures that contribute to the ability of the organism to
evade immune surveillance. The possibility of recombination in this
immunogenic region strengthens the need for a quantitative assessment of
these pressures and constraints as they vary by serotype. While previous
studies have described ompA point mutations and recombination events,
adequate assessment of ompA genetic variability and stability of MOMP, and
thus the above factors, have been limited by small sample sizes, restricted
geographic sampling regions and the use of partial sequences that do not
include all VSs (Brunham et al., 1994; Frost et al., 1995; Hayes et al., 1992;
Hayes et al., 1995, Morre et al., 1998; Takourt et al., 2001; Yang, Maclean,
Brunham, 1993).
Thus, to investigate the population genetics and evolution of this
organism, we conducted a population-based study utilizing over 500 clinical
C. trachomatis STD samples from a multicentered nationwide study of STDs
and included all VSs of ompA in the analyses. The use of clinical samples
obviated many potential problems with in vitro adapted serotypes. Our
analyses provide a more comprehensive understanding of the population
genetics and evolution of C. trachomatis strains that can then be used for
robust epidemiologic studies and vaccine development.
Materials and Methods
Study Population and Specimen Collection. Consenting men and
women aged 14 to 49 years who presented to family planning and STD
clinics serving the Birmingham, Indianapolis, New Orleans, Seattle and San
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96
Francisco metropolitan areas and who met inclusion criteria (see below)
participated in a large parent study designed by the Centers for Disease
Control (CDC). The study was multi-centered, primarily cross-sectional in
design, and included enrollment into three components, designated A, B1
and C, from October 1995 to August 1997 (Black et al., 2002; Johnson et al.,
2000; W hittington et al., 2001).
Component A was designed to evaluate laboratory diagnostic
methods for C. trachomatis. Consecutive patients seen at STD and family
planning clinics were enrolled and consented unless they had taken
antibiotics within the past 30 days, were pregnant, had a hysterectomy, were
males with symptoms of urethritis, were females not receiving a pelvic
examination, or refused participation. Component B1 was designed to
evaluate risk factors for recurrent C. trachomatis infection. Women seen at
family planning clinics, health center clinics, teen clinics, and STD clinics were
consented and enrolled if they were under 34 years of age and had
laboratory documentation of C. trachomatis infection. The resulting cohort
had follow-up at four weeks and six months after the baseline visit.
Component C was designed to determine prevalence and evaluate risk
factors for C. trachomatis infection. Sentinel surveillance networks composed
of adolescent, STD, family planning and community health center clinics in
the five metropolitan areas were established. Health care providers from
individual sentinel clinics were selected from each group of clinics to
universally enroll consenting women and men who provided medical
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97
histories and underwent routine lower and upper genital tract examination.
In Birmingham, universal enrollment of consenting men and women was not
done. At this site, enrollment was limited to 50 to 60 female patients
sampled from the clinics one week per month.
Clinical specimens were obtained from all participants enrolled in the
parent study. For men, the clinician collected a urethral swab specimen for
Gram-stain to detect Neisseria gonorrhoeae and, in some cases, for N.
gonorrhoeae culture. A second urethral swab for C. trachomatis culture and
LCX or Amplicor PCR, and approximately 30 ml of the first part of a non
clean catch urine stream were obtained. Approximately 30 ml of the first
part of a non-clean catch urine stream was collected from women prior to a
complete pelvic examination and subjected to LCX or Amplicor PCR.
During the pelvic examination, debris was removed from the exocervix with
a large cotton swab and endocervical samples were collected using Dacron
swabs (Hycor Biomedical, Portland, ME). At some study centers, the first
endocervical sample was used for N. gonorrhea culture or it was used for
Gram-stain. The second cervical sample was placed in chlamydial collection
medium for subsequent C. trachomatis identification by culture, LCX, an d /o r
Amplicor PCR. Based on clinical discretion of the health care providers,
additional samples were obtained for diagnosis of concurrent STDs
according to routine clinical practice for the respective clinic.
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98
Those participants enrolled in the parent study with C. trachomatis
urethral (male) or cervical (female) infection documented by culture,
commercial Amplicor PCR test (Amplicor Test Kit, Roche Diagnostics,
Indianapolis, IN), an d /o r commercial Ligase Chain Reaction (LCR) test
(LCX; Abbott Diagnostics, Abbott Park, IL) were eligible for the current
study. The diagnostic tests were performed according to standard culture
techniques in the respective laboratory and according to manufacturer's
instructions for the commercial assays except that M4 transport medium was
used. Of those patients who were eligible, approximately 50 men and 50
women from each study center were randomly selected for inclusion.
Determ ination of ompA Genotypes. Over 1,000 clinical samples were
received from the five study centers. Of these, 532 were randomly selected
for ompA genotyping, where approximately equal numbers of specimens for
males and females (except for Birmingham, where no males were enrolled in
Component C) and for each center were randomly selected. The specimens
were from the cervix (female) or urethra (male), and were positive by
culture, LCR, or Amplicor PCR. The samples were original, nonpropagated
culture remnant samples and remnant, nonamplified LCR or Amplicor PCR
samples. DNA extraction, purification, amplification, and automated
sequencing were performed as described for omcB in Chapter 2. Except that
for ompA, the entire gene was amplified using primer pairs FII/BII or
F200/MZ2 with primer sequences given in Table 3-1. After the entire gene
was amplified, the upstream and downstream halves were re-amplified with
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99
an approximately one hundred bp central overlap between the two halves.
The two halves with the central overlap were generated in two nested
reactions using 1 jul of the amplified sample from the first reaction and
primer pair sequences shown in Table 3-1. Technicians performing the
genotyping were blinded to all clinical, demographic, geographic and
microbiologic data.
Table 3-1. OmpA Primer Sequences Used for Sequence Generation
Primer pair used to
amplify the entire
ompA gene
Primer pair used to
amplify the
upstream half of
ompA
Primer pair used to
amplify the downstream
half of ompA
FII-
5ACCACTTGGTGTG
ACGCTATCAG
BII-
5'CGGAATTGTGCAT
TTACGTGAG
MF21-
5'CGACCGCGTCTT
GAAAACAGATGT
B44-
5'CTAGATTTCATC
TTGTTCAATT
MVF3-
5'CGTGCAGCTTTG
TGGGAATGT
B44-
5'CTAGATTTCATC
TTGTTCAATT
F200 -
5' TGAAAAAACTC
TTGAAATCGGTATT
MZ2-
5'TACGGTACCTTAG
AAGCGGAATTGTGC
ATTTAC
MF100 -
5'TGTAAAACGAC
GGCCAGTGCCGT
ATTAGTG1TTGCC
GCTTTGAGT
VB3-
5'CATCGTAGTCA
ATAGAGGCAT
MVF3-
5'CGTGCAGCTTTG
TGGGAATGT
MZ2-
5'TACGGTACCTTAGA
AGCGGAATTGTGCAT
TTAC
Mixed infections were identified by the presence of two peaks at a
single nucleotide position of approximately equal intensity for a large
proportion of the gene sequenced. The sequences of the ompA genes
determined in this study have been catalogued under GenBank accession
numbers AF519807 through AF519870.
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100
Estimation of C. trachomatis Serotypes and Analysis of Putative
Recombinants. The sequence was first manually aligned against all
prototype serotype sequences (Yuan et al., 1989) using the multiple
alignment sequence editor (MASE; version 3.1, Dana-Farber Cancer Institute,
Harvard School of Public Health, [ftp.ebi.ac.uk/pub/ softw are/unix/]). The
similarity between the sequence and each prototype serotype sequence was
computed as the number of like nucleotides divided by the total number of
nucleotides under comparison. The serotype of the prototype sequence to
which the sequence was most similar provided the serotype estimate.
Statistical significance of putative recombinants and their breakpoints were
assessed by Sawyer's runs test and the maximum chi-square test as
previously described (Millman, Tavare, and Dean, 2001).
Statistical analyses. Of the 532 samples that were ompA genotyped,
twenty five were excluded due to the presence of a mixed infection, leaving
507 sequences available for analysis. Differences in chlamydial serotype
distribution by metropolitan area and by gender were determined by
permutation analysis in the following manner (Manly, 1991): For each
serotype, a 2x5 contingency table compared the number of sequences of that
serotype in each metropolitan area or gender, respectively, to the number not
of that serotype. A Pearson's chi-square homogeneity statistic was computed
for the observed data. The test statistic was recomputed after 10,000 random
reassignments of serotype, keeping the frequencies in the population
constant. The p-value was the proportion of randomizations in which the
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101
test statistic exceeded that for the observed data. The Bonferroni method was
used to adjust for multiple serotypes.
Since there is extensive evidence for recombination in the ompA gene,
phylogenetic inference and estimates of genetic distance for urogenital
strains in this study were based on simple Hamming distances (denoted by
p) without the inclusion of multiple substitution models. Tree topologies for
all strains in a serotype class were generated using the Neighbor Joining
method and MEGA (Molecular Evolutionary Genetics Analysis) version 2.1
(Kumar et al., 2001). The mean genetic distance within a serotype group was
estimated as the average of all p distances between sequences of the same
serotype for all possible sequence pairs using MEGA version 2.1 (Kumar et
al., 2001). The standard error of the estimate was generated using the
bootstrap procedure with 500 replicates and 95% confidence intervals (CIs)
were calculated in the usual fashion. The distribution of genetic distances
within a serotype group was determined by plotting a histogram of these
distances. For each serotype group, the mean proportion of synonymous
nucleotide differences per synonymous site (ps ) and the mean proportion of
nonsynonymous nucleotide differences per nonsynonymous site (pn ) were
computed between same serotype strains for all possible sequence pairs.
This was accomplished following the method of Nei and Gojobori (1986)
using MEGA version 2.1 (Kumar et al., 2001). Standard error of the estimate
and 95% CIs were determined similarly to that described above.
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102
We compared the relative prevalence of shared mutations between
strains of the same serotype group for different serotype groups. We
postulated that a serotype group with high prevalence and infrequent
incorporation of mutations within its strain pool might have undergone a
selective sweep in which strains have exhibited rapid clonal expansion. To
depict these differences graphically, we represented a mutation within a
serotype group pool as a radial spoke on a wheel with spoke length inversely
proportional to the frequency at which that mutation was present in the
serotype group pool. The radial wheel, or starburst, represented the relative
frequency of all mutations observed for that serotype group in this
urogenital US population. The location of the spoke within a 360° circle was
determined by the nucleotide location of the mutation minus 250 (nucleotide
location of the beginning of the sequence)/ 810 (total bp length of the
sequence) multiplied by 360°. For example, serotype group Z has 100
sequences and a m utation at nucleotide 250 is present in 50 of these
sequences. Hence, the radial spoke length = 1/(50/100)= 2 at 0 degrees. A
phylogenetic reconstruction was generated to establish tree topology
between serotype groups based on Hamming distances between strains of
dissimilar serotype groups for all possible sequence pairs using the Neighbor
Joining method and MEGA version 2.1. Each starburst was superimposed
onto the phylogenetic tree at the end of the branch that corresponded to the
appropriate serotype group.
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103
Results
Of the 507 study participants, 228 were men and 279 were women. Of
these, 94 were seen in the Birmingham metropolitan area, 106 in
Indianapolis, 112 in New Orleans, 88 in San Francisco and 107 in Seattle.
When comparing ompA nucleotide sequence data generated from this
population, there were 329 polymorphic sites out of the possible 810 base
pairs (bp) that were sequenced, aligned and analyzed. The 810 bps
(nucleotide 244 to 1053) included VS1 through VS4 in their entirety. In the
alignment of the urogenital strains from this study, locations of the VSs
defined by Yuan et al. (1989) were as follows: VS1 from 256-324; VS2 from
490-567; VS3 from 757-798 and VS4 from 949-1053.
Table 3-2 describes each unique sequence in this population, including
the serotype estimate for that sequence, changes with respect to its most
similar prototype sequence, and the geographic region and gender for the
strains represented by that sequence. We observed minor population
differences compared to the serovar prototype sequence with at least 25%
prevalence for serovars Ba, D, F, G, H and J (Ba: C246T, A249G, A532G,
G607, and T781G; D: C246T, A249G, A660T, and G1000A; F: G269, G:
G493A and T1036G; H: A272G and C865T; and J: T625C and G627C).
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s
Table 3-2. Genetic Description of Unique Sequences in this Population Organized by Serovar
Serovar
followed
by strain
identifier
GenBank
Accession
No.
Total No. of strains represented
No. of strains from each region
followed by gender of each*
Nucleotide change(s) from most similar prototype sequence1 *
B/IU-1226 AF063208 4 I=l;SF=3; F=3;M=1 C246T-; A249GW ; A532GL ; T781GC
Ba-CDCl AY535080 1 SF=1; M=1 C246T"; A249GC ; A532GC ; G 607^; T781GL
Ba/D-
CDC1
AY535081 1 SF=1;F=1
D-B120 X62918 18 B=l; 1=6; NO=7; S=4;
F=11;M=7
Identical to D-B120
D-IU83786 AF063197 20 B=4; 1=5; NO=2; S=7; SF=2;
F=12;M=8
C246TU ; A249GU ; A660TU
D-CDC1 AY535082 5 NO=l;S=2;SF=2; F -l; M=4 C246TU ; A249GU
D-CDC2 AY535083 14 B=2; 1=3; NO=7; S=l; SF-1;
F=7; M=7
C246TU ; A249GU ; A494GU ; A660T G1000AU
D-CDC3 AY535084 1 S*l; F=1 C 246T"; A249GU ; A660T u ; T705G u
D-CDC4 AY535085 3 B=2,-SF=1; F=1;M=2 G1000AU
D-CDC5 AY535086 1 I=1;M=1 C 246T"; A249GU ; A494GU
D-CDC6 AY535087
1 I-l-M -l
G blll^
D-CDC7 AY535088 1 I=1;M=1 C246TU ; A249GU ; G400AU ; T447G u ; A660TU
D-CDC8 AY535089 1 I=1;F=1 T246Cb G249CU ; G290AU ; G295AU ; G399Ab ; G535A0 ;
T591G0; G607CD
D-CDC9 AY535090 1 S=l; M=1 T246C u , G249CU ; T591GU
D-CDC10 AY535091 1 I=1;F=1 t246Cu ; G249CU ; A371Tb ; A441TU ; A474GU ; A494G0 ; A585C u ;
C612AD ; C614Ad ; A660T0; G1000AD
D-CDC11 AY535092 1 NO=l;F=l C984GU
D-CDC12 AY535093 1 NO=l;F=l T246CU , G249C u ; A494G u ; A660TD ; T875AU ; T963C u ; C984GU ;
G1000Ad ;T1049Ad
D-CDC13 AY535094 1 I=1;F=1 A553CU
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Table 3-2. Continued
Serovar
followed
by strain
identifier
GenBank
Accession
No.
Total No. of strains represented
No. of strains from each region
followed by gender of eadr
Nucleotide change(s) from most similar prototype sequence1 *
J-CDC6 AY535158 1 SF=1; F=1 T418GJ ;T625CJ ; G 627C *
J-CDC7 AY535159 2 SF=2; M=2 C372T; T 625C *; G 627C *
J-CDC8 AY535160 1 SF=1; M=1 G258AJ ; C372P; T625CJ ; G627C
J-CDC9 AY535161 1 SF=1; F=1 T625C’ ; G627CJ ; T855GJ ; A1006GJ ; A1041T’
Ia-IU-37538 AF063203 19 B=3; 1=5; NO=8; S=l; SF=2;
F=13;M=6
Identical to Ja-IU-37538
Ja-CDCl AY535162 1 I=1;M=1 A527G*; A559CK ; A570Gk ; A589G*
Ja-CDC2 AY535163 1 NO=l; M=1 A1012G*
Ja-CDC3 AY535164 1 S=l; M=1 A696G*; G699A*; T828C*; A1012G*; A1047CK
Ja-CDC4 AY535165 1 B=1;M=1 G268A*; T 269C *'; G699A*; T828C*
Ja-CDC5 AY535166 1 NO=l; F=1 G587T*’ ; G1037AR
K-UW31 AF056204 18 B=6; I=l;NO=8; S=l; SF=2;
F=13;M=5
Identical to K-UW31
K-CDC1 AY535167 2 NO=l; S=l; M=2 A293GL
K-CDC2 AY535168 1 NO-1; F=1 A512CL ; G541AL ; G559AL ; G563A1 ; C591TL ; G1006AL ; T1027GL
K-CDC3 AY535169 1 SF=1; M=1 A505CL ; C625T1 - ; C627GL
K-CDC4 AY535170 1 B=l; M=1 G559AL ; G563AL
K-CDC5 AY535171 2 S=1;SF=1;F=2 C625T;C627GL
K-CDC6 AY535172 1 I=1;F=1 C461AL ; T467AL ; A521GL ; T595A1 - ; C625T-; C627GL
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8
Table 3-2. Continued
Serovar
followed
by strain
identifier
GenBank
Accession
No.
Total No. of strains represented
No. of strains from each region
followed by gender of each*
Nucleotide change(s) from most similar prototype sequence8
H-CDC1 AY535137 1 S=l;F«l A272G"; C865TM
Ia-IU-4168 AF063201 55 B=12; 1=12; NO*16; S*$£F*6;
F=29;M=26
identical to Ia-ltJ-4168
Ia-Ct>Cl AY535138 1 B=l; M -l G289C1
ia - c b a AY535139 1 SF«1; M=1 C461T; A520G1 ; A521C1 ; C522T1 ; A560G1
Ia-CDC3 AY535140 1 I= 1 ;M « = 1 A521G1 ; G602T
Ia-CDC4 AY535141 1 S=l; F -l G 4 Q 0 A * ; C461Cii
Ia-CDC5 AY535142 1 I=1;F«1 T856C1
Ia-CDC6 AY535143 2 M jSF-l;F-l;M «l C461T1
Ia-CDC7 AY535144 1 I-lM -1 T418C; C456G1 ; C461T1 ; A489G‘ ; A521G1 ; A587Gi
la-CDC8 AY535145 1 NO=l; M*1 A867G';T971G‘
la-CDC9 AY535146 1 NO»l; M =1 G855C‘ ; G1027C'
Ia-CDCIO 1 NO=l; M»1 A 277G *; C758T1 ; G8S5T; A1006G1 ; G1027T; C1047A1
la-CDCll 1 SF=1; F=1 T569G'
Ia-CDC12 AY535149 1 NO=l; F=1 GSKT^TKHlA1
Ia-CDClS AY535150 1 SF=1;F»1 C664T
Ia-CDC14 AY535151 3 B=1^»F=2;F=3 A541G * ....... .......................
Ia-CDClS AY535152 1 B -l; St-1 G305T‘ ; A54lGi .......................
-CDC1 AY535153 1 I«1;F«1 T625C; G 627C *; C1047A' ...............................
-0 X 2 AY535154 1 S F * » 1 | M ® 1 Y625C,;G627C> ................ ........ ................
J-OX3 AY535155 26 B ® 7 ; 1-7; NO=3; S=5; S F * = 4 ;
F=10;M=16
T625C; G627C'
j-ctx:4 AY535156 1 S*1;F-1 T541G’ ; T625Cj f ; G627C1
J-OX3 AY535157 1 SF=1; M=1 G258C; A453G; A502G1 ; A53ld; A560T'; T625C; G627C
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Table 3-2. Continued
Serovar
followed
by strain
identifier
GenBank
Accession
No.
Total No. of strains represented
No. of strains from each region
followed by gender of each*
Nucleotide chunge(s) from most similar prototype sequence8
F-CDC6 AY535117 1 S=1;F*1 G400C1 ,
F-CDC7 AY535118 1 S»l; M=1 C267Gr ; G268A1 ' ; G269C A$36CF ; A789Tf; A9>7Gf; C1013D;
T1014Cf
F-CDC8 AY535119 1 S=l; M=1 A 6 8 8 G * '; A 738G*- ; A927Gr; A987GF
F-CDC9 AY535120 1 NO=l;M=l A536C; T537A^; A688Gr; A696G'; A72(rr; A738GF ; T756Gf;
A757Cf; G759Af; A773Cf; A789Tf; A927GF ; A987GF ; CIOITI*;
T1014Cf; G1028C?
F-CDC10 AY535121 1 G 613T*
F-CDC11 AY535122 1 B=1;F*1 G269A*; C93W
F-CDC12 AY535123 1 S-1;F»1 A738GA789l< 5 TAl029r I+
F-CDC13 AY535124 1 I-l-F -l G269Ar ;T625C*;G627CF
G-UW57 AF063199 2 I* = l; NO-1; F=1;M=1 Identical to G-UW57
G-CDQ AY535125 3 SF=3; F«l; M=2 T1036GV ’
G-CDQ AY535126 1 S=1;F*1 G269A1 * ; T1036Gg
G-CDC3 AY535127 1 SF=1; F=1 G592A1 * ; T lO S fr G ^
G-CDC4 AY535128 3 B=l; NO=2; F=1;M =2 < j493A1 * ; G 721iEu ; T1036AU
G-dDCS AY535129 1 S F = 1 ;F = * 1 G269A1 * ; G493AU ; <57210*; TIOSSAF
g-c d S> AY535130 1 SF*1; M«1 T850G0; T855GU ; T1000AG ; 110360°
G-CDO’ AY535131 1 SF=1; M=1 C 2 6 0 A '" ’ ; C509AU ; G513A°; A53?Trrfi598T'; A 9 5 5 G ; A956CG ;
A1006G°; C1028Ag; 00311°; T1036AG ; A1040C5
G-CDC8 AY535132 4 S=3; SF=1; F=1;M=3 G493AV *
g-c T x s T AY535133 1 SF*1; M=1 G269AU,<3493A U
G-CDOO AY535134 1 G493AU ; 0 7 2 1 ^ 9 2 2 0 ° ; fld36AG
G-CDCll AY535135 1 N O -j;F*l T930G°
G-Ct>Cli AY535136 1 SF*1; M*1 A1006G0
H-UW4 X16007 2 B=1,-S*1;F=2 Identical to H-UW4 1
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108
Table 3-2. Continued
Serovar
followed
by strain
identifier
GenBank
Accession
No.
Total No. of strains represented
No. of strains from each region
followed by gender of eacn*
Nucleotide change(s) from most similar prototype sequence*1
D-CDC14 AY535095 1 I=1;M=1 T246Cb , G249C u ; T477G u ; T59 tGu ; A660TU
E-Bour X52557 134 B-30; 1=29; NO=25; S=27;
SF=23; F=79;M=55
Identical to E-Bour
E-CDC1 AY535096 1 1=1; F -l G254Tt
E-CDC2 AY535097 1 S=l; F=1 G718Tf c
E-CDC3 AY535098 1 S=l; F=1 G338C*
E-CDC4 AY535099 1 S=l; F=1 G400At; A508Cf c ; A618Cf c
E-CDC5 AY535100 1 S=1;M=1 A576Cf c
E-CDC6 AY535101 1 S=1;M=1 A429Gk
E-CDC7 AY535102 1 S = 1 ;F « = 1 G958Cf c
E-CDC8 AY535103 1 S=l; F=1 G 2 8 3 A * = ; G958C*
E-CDC9 AY535104 1 SF=1;M=1 G635Cf c
E-CDC10 AY535105 1 I=1;F=1 G723Ck
E-CDC11 AY535106 1 I=1;M=1 G373Cf c ; A532Ck
E-CDC12 AY535107 1 S=1;M=1 A503Gk
E-CDC13 AY535108 1 NO=l;M=l 07181*; A795T k ; A936C*; A987Gk
E-CDCll AY535109 1 NO=l;F=l G633Af c ; C636Tk
E-CDC15 AY535110 1 SF=1$=1 C258Af c
E-CDC16 AY535111 1 S=l; M=1 A2^9Gk
F-IC-CaB X52080 62 B=ll; 1=13; NO=12; S=16;
SF=10; F=39;M=23
Identical to F-IC-Cal3
F-CDC1 AY535112 23 B=9; 1=1; NO=4; S=7;
SF=2; F=13;M=10
G269A*1 ; G 3 8 5 A * *
F-CDC2 AY535113 1 1= 1; M=1 G269A*;A1036Cf
F-CDC3 AY535114 2 S=2; M=2 C 267G *- ; C269Gk
F-CDC4 AY535115 1 NO=l; M =1 T263Ak ; G269Ak ; T314Gh
F-CDC5 AY535116 1 NO=l; M=1 G 2 5 8 C * '
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§
Table 3-2. Continued
•
A First number represents the total number of sequences found in this population with that genotype. This is followed by the metropolitan
area and gender distribution for those sequences using the fallowing key: B , Birmingham; I, Indianapolis; NO, New Orleans; S, Seattle; SF, San
Francisco; M, male; F, female. For example, we found one sequence with genotype named Ba-CDCl. This sequence was from a male study
participant from San Francisco (SF=1; M=l).
Genotype is defined by all mutations compared to its most similar prototype. Mutation is represented by the base found for the most similar
prototype, followed by the nucleotide position of the change with respect to the start site, followed by the base found for the new gentoype.
For example, C246T represents that the genotype from this study has a T at position 246, while its most similar prototype has a C at position
246.
cCompared to Ba-Apache2; ‘’ Compared to D-B120; Compared to E-Bour; ‘Compared to F-IC*Cal3; G Compared to G-UW57;
Compared to H-UW4; ‘ Compared to Ia-IU-4168; ‘ Compared to J-UW36; Compared to Ja-IU-37538; ‘ Compared to K-UW31.
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110
The distribution of serotypes was similar to other studies: Serotypes
D, E, F and la were the most prevalent; G, J, and K were intermediate; and
Ba, H, Ja and a putative Ba/D recombinant were rare (Ba, 5 (1%) of 507; D, 71
(14%) of 507; Ba/D, 1 (0.2%) of 507; E, 150 (30%) of 507; F, 98 (19%) of 507; G,
21(4%) of 507; H, 3(1%) of 507; la, 73(14%) of 507; J, 35(7%) of 507; Ja, 24 (5%)
of 507; and K, 26(5%) of 507). Of note, there were no A, B, C, Da, or I
serotypes. The putative Ba/D recombinant identified in this study had
significant mosaicism (p<0.0001) based on the Sawyer's runs test and the
maximum chi-square test. The recombinant was markedly similar to
serotype Ba before nucleotide 477, the breakpoint predicted by the maximum
chi-square test, and was markedly similar to serotype D after the breakpoint
(Figure 3-1).
Serotype distribution by metropolitan area was not homogeneous
(Table 3-3). Permutation analysis was used to test whether a serotype was
significantly associated with metropolitan area. The rare serotypes were
observed to a greater extent in San Francisco than in other metropolitan areas
with a significantly higher proportion of Ba sequences than Birmingham,
Indianapolis, and Seattle (p=0.02), and a significantly higher proportion of G
sequences than any other city (p=0.03) after adjustment for multiple
comparisons. In contrast, there was no significant difference in the
distribution of serotype by gender.
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Figure 3-1. Alignment of Ba/D Recombinant Compared to Ba and D Prototype Sequences
244 303
B/TW-5
Ba/Apache2
D/B120
TTC
. .T
CAA ATG GGT GCC AAG CCT ACA ACT
----- -----
ACT
GA.
GA.
GA.
ACA GGC AAT GCT
AG.
AG.
AG.
GTA
AC.
,c .
GCT
• • •
CCA
* ft «
-----
Da/TW-448
D/IC-Cal-8
• C.
• C.
Ba/D-CDCl
AC.
GAT
B/TW-5
304
TCC ACT CTT ACA GCA AGA GAG AAT CCT GCT TAC GGC CGA CAT ATG CAG GCT GAG
363
ATG
Ba/Apacha2
D/B120
Da/TW-448
D/IC-Cal-8
Ba/D-CDCl
B/TW-5
364
TTT ACA AAT GCC GCT TGC ATG GCA TTG AAT ATT TGG GAT CGC TTT GAT GTA TTC TGT
423
ACA
Ba/Apache2
D/B120
Da/TW-448
D/IC-Cal-8
Ba/D-CDCl
B/TW-5
424
CTA GGA GCC TCT AGC GGA TAC CTT AAA GGA AAC TCT GCT TCT TTC AAT
483
TTA GTG GOG TTA
Ba/Apach@2
D/B120 T,.
Da/TW-448 T. .
D/IC-Cal-8 T.. • * • • * • A.C • « T . . . . .T
Ba/D-CDCl
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Figure 3-1. Continued
484 543
B/TW-5 TTC GGA AAT AAT GAG AAC CAG ACT AAA------------ GTT TCA AAT GGT GCG TTT GTA
Ba/Apach@2 — .................................. A., a ...
D/B120 ..T *•• G.* •* * -*• •.A •.T •.A .AA «CG ■** GTC •.A .CG .A. .C. * *.
Da/TW-448 „.T ... G ......A . ,T ..A .AA .CG--------- GTC ..A .CG .A. .€. ...
D/IC-Cal-8 . ,T ... G ......A ..T ..A .AA .CG-----------------GTC ..A .CG .A. .C. ...
Ba/D-CDCl ..T ... G ..A . .T ..A .AA .CG------------- GTC ..A .CG .A. .C. ...
544 603
B/TW-5 CCA AAT ATG AGC TTA GAT CAA TCT GTT GTT GAG TTG TAT ACA GAT ACT GCT TTT GCG TGG
Ba/Apacha2 ............................. ....................................................
D/B120 T ...............................................A..............
Da/TW-448 ..T...............................................A..............
D/IC-Cal-8 ..T...............................................A..............
Ba/D-CDCl A . ..
604 663
B/TW-5 AGC GTC GGC GCT CGC GCA GCT TTG TGG GAA TGT GGA TGT GCA ACT TTA GGA GCT TCT TTC
Ba/Apache2 ..................................................................................
D/B120 A ...
Da/TW-448 A ...
D/IC-Cal-8......... ,.................
Ba/D-CDCl A ...
664 723
B/TW-5 CAA TAT GCT CAA TCT AAA CCT AAA GTA GAA GAA TTA AAC GTT CTC TGC AAT GCA GCA GAG
Ba/Apaeho2 ...................................... ..................................... .. ...
D/B120 ..................................................................................
Da/TW-448 ... ... ..........................................................................
D/IC-Cal-8 ......................... ........................................................
Ba/D-CDCl ............................................................................ . ...
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cn
724
Figure 3-1. Continued
783
B/TW-5
Ba/Apaeho2
D/B120
TTT ACT ATT AAT AAA CCT AAA GGG TAT GTA GGT AAG GAG TTG CCT CTT GAT CTT ACA GCA
Da/TW-448
D/IC-Cal-8
Ba/D-CDCl
784 843
B/TW-5
Ba/Apach«2
D/B120
Da/TW-448
GGA ACA GAT GCT GCG ACA GGA ACT AAG GAT GCC TCT ATT GAT TAC CAT GAA TGG CAA GCA
D/IC-Cal-8
Ba/D-CDCl
844 903
B/TW-5
Ba/Apach«2
D/B120
AST TTA GCT CTC TCT TAC AGA TTG AAT ATG TTC ACT CCT TAC ATT GGA GTT AAA TGG TCT
Da/TW-448
D/IC-Cal-8
Ba/D-CDCl
904
963
B/TW-5
Ba/Apaeha2
D/B120
Da/TW-448
D/ic-cal-8
Ba/D-CDCl
C3A GCA AGC TIT GAT GCA GAC ACG ATT CGT ATT GCT CAG CCG AAG TCA GCC GAG ACT ATC
* • • « • a
» * • • • •
• • •
a • •
• a a
• • a
• • • •.C •«T • .A
• ••C • • T ••• • »A
..C
..C
• • •
• * •
..A
..A
..A
..A
• 4 a
• « a
..T
..T
ACA
ACA
ACA
ACA
G..
G. •
G * #
G * *
..T
• • «
..T
* • *
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B/TW-5
Ba/Apache2
D/B120
Da/TW-448
D/IC-Cal-8
Ba/D-CpCl
B/TW-5
Ba/Apache2
D/BX20
Da/TW-448
D/IC-Cal-8
Ba/D-CpCl
Figure 3-1. Continued
964
TTT GAT GTT ACC ACT CTG AAC CCA ACT ATT GCT GGA GCT GGC GAT GTG AAA
1023
AC.
AC.
AC.
AC.
1024
..G ..T
•.G ..T
.*G ..T
.. G . . T
1053
ACT AGC GCA GAG GGT CAG CTC GGA
G.
G.
G.
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115
Table 3-3. Distribution of Chlamydia trachomatis Serovars by Metropolitan
Area in the United States and by Gender
Serovar Baf l D
Ba
/
D
E F Ga H la
J Ja K Total
Location
Birmingham 0 9 0 30 19 1 1 15 7 4 8 94
Indianapolis 1 21 0 32 19 2 0 16 8 6 1 106
N ew Orleans 0 19 0 27 19 4 0 20 3 10 10 112
San Francisco 4 7 1 25 12 10 0 12 11 2 4 88
Seattle 0 15 0 36 29 4 2 10 6 2 3 107
Gender
Male 2 33 1 62 42 13 0 35 21 10 9 228
Female 3 38 0 88 56 8 3 38 14 14 17 279
Total n 5 71 1 150 98 21 3 73 35 24 26 507
“ Significant differences in serovar distribution by metropolitan area were deduced by
permutation testing. San Francisco had a significantly higher proportion of Ba sequences
than Birmingham, Indianapolis, and Seattle(p=0.02) and a significantly higher proportion of
G sequences than any other city (p=0.03) after adjustment for multiple comparisons.
Figure 3-2A-C shows the phylogenetic tree topologies based on
Hamming distances for the urogenital strains in this study. For the B
seroclass (Ba, D, Da, and E) sequences, the Ba/D recombinant clustered
midway between the B and D serotype groups, and the D serotype sequences
formed two main groups differentiated by the presence/absence of
nucleotide changes at nucleotid positions 246 and 249 (D/B120, D-CDC4, D-
CDC6, D-CDC11 and D-CDC13 vs other D serotype sequences; Figure 3-2A),
which did not differentiate the D and Da subtypes.
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116
Figure 3-2A. Phylogenetic Reconstructions for the Unique ompA Gene
Sequences in the B Seroclass using the Neighbor-Joining Method
100
87
51
73
1 0 0
74
65
1 0 0
54
51
66
■ Ba-CDC1
■ M U -1226
■ BD-CDC1
■ D-CDC13
• D/B120
■ D-CDCS
■ D-CDC11
-D-CDC4
• D-CDC2
• D-CDC10
■ D-CDC12
■ D-CDC5
. 0-C DC9
■ D-CDC8
■ D-CDC14
■ D-CDC1
• D-CDC7
• D-CDC3
• M U -83786
■ E-CDC13
■ E-CDC4
■ E-CDC5
■ E-CDC2
■ E-CDC12
• E-CDC7
• E-CDC8
■ E-CDC15
• E-CDC11
■ E-CDC9
■ E-CDC10
■ E-CDC3
• E-CDC1
E-C 0C14
. B B o u r
• E-CDC6
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117
For the Intermediate seroclass (F and G), F-CDC9 and G-CDC7 clustered
apart from the rest of the sequences within their respective serotype groups
(Figure 3-2B).
Figure 3-2B. Phylogenetic Reconstructions for the Unique ompA Gene
Sequences in the Intermediate Seroclass using the Neighbor-Joining Method
5?
73
64
99
58
99
91
50
54
■ F-CD C1
■ F -C D C 1 1
• F-C D C 4
• F -C D C 13
• F -C D C 2
■ F -C D C 3
■ F/IC -C al3
■ F -C D C 10
■ F -C D C 6
• F-C D C S
■ F -C D C 12
■ F -C D C 8
■ F -C D C 7
• F -C D C 9
■ G -C D C 7
• G -C D C 3
■ G -C D C 4
• G -C D C 5
■ G -C D C 1 0
G -C D C 8
■ 6 -C D C 9
■ G/UVV57
• G -C D C 11
■ G -C D C 2
■ G -C D C 12
■ G-CD C1
• G -C D C 6
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118
In 1994, Ossewaarde et al. (1994) also reported phylogenetically distinct G
sequences that they designated as Ga subtypes. These Ga strains had T547A
and T1003G changes compared to G/UW-57. None of our G sequences
contained the T547A change, however, six (29%) of 21 G sequences contained
the T1003G change (T1036G in our alignment), confirming that these changes
are prevalent population differences not restricted to the European continent.
For the C seroclass (A, C, H, I, la, J, and Ja), the J serotype sequences
formed two main groups consistent with the J and Ja subtypes (Figure 3-2C).
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119
Figure 3-2C. Phylogenetic Reconstructions for the Unique ompA Gene
Sequences in the C Seroclass using the Neighbor-Joining Method
71
92
62
63
99
82
52
93
59
53
67
52
99
75
8 6
82
- Ia-CDC10
- Ia-CDC12
- Ia-CDC9
- Ia-CDC1
- la-CDCI 4
- Ia-CDC15
- Ia-CDC7
- Ia-CDC2
- Ia-CDC6
- I»-CDC4
- Ia-CDC3
- Ia-CDC13
- la-CDCS
. laflU-4168
- Ia-CDC11
- la-CDC8
-H/UW4
. H-CDC1
- JaflU37538
- Ja-CDC9
- Ja-CDC3
- Ja-CDC4
- Ja-CDC7
- Ja-CDC5
- J-CDC12
. J-CDC13
- J-CDC10
■ J-CDC2
- J-CDC8
• J-CDC11
■ J-CDC1
. J-CDC8
- J-CDC14
■ K-CDC2
■ K-CDC4
. K/UW31
- K-CDC1
. K-CDCS
. K-CDC3
- K-CDC5
For Figures 3-2 A-C, the values at the nodes are the bootstrap confidence levels (BCL) for the
interior branch. The BCL represents the percentage of 500 bootstrap re-samplings for which
the strain to the right was separated from the other strains.
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120
As there is extensive evidence for recombination in the ompA gene
(Millman, Tavare, and Dean, 2001), we used simple Hamming distances to
compare genetic distance estimates within serotype groups. When
comparing strains within a serotype group, serotypes E, Ba, H, la (0.001 95%
Cl 0.0006-0.002) and F (in descending order) were the most conserved, while
serotypes J, G, D and K (0.002 95% Cl 0.0009-0.003) were the most divergent
(Figure 3-3). Serovar J (0.008, 95% Cl 0.005-0.01) had a significantly higher
mean genetic distance than all serovars other than G (0.004, 95% Cl 0.002-
0.006) and D (0.003, 95% Cl 0.001-0.006); serovar E (0.0004, 95% Cl 0.0002-
0.0006) had a significantly lower mean genetic distance than all serovars
other than Ba (0.0005, 95% Cl 0-0.002), F (0.002, 95% Cl 0.0005-0.003) and H
(0.0009, 95% Cl 0-0.003); and serovar Ba had a significantly lower mean
genetic distance than serovars G and J.
Figure 3-3. Mean Hamming Distance for Each Serotype Group Based on
Hamming Distance between all Possible Pairs of Similar Serotype Sequences
0
c
re
4 - >
m
Q
CD
c
1
E
re
x
re
< U
0.014
0.012
0.01
0.008
0.006
0.004
0.002
0
j
s i
'
1 L
_ _ L - - - - - - - [ . r . . . | J . . f
i i ^ i — i— i
Ba D E F G H la
Serovar group
K
Minimum and maximum values represent lower and upper limits of the 95% Cl of the
estimate, while horizontal bar level represents the mean estimate.
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121
Using the methods of Nei and Gojobori (1986), serotype Ba had the
highest mean proportion of nonsynonymous nucleotide differences per
nonsynonymous site (pn ; Figure 3-4) among serotypes, followed by J. These
two serotypes also had the highest mean proportion of synonymous
nucleotide differences per synonymous site (ps ; Figure 3-4). Serotypes G, la
and K had pn/p s ratios greater than 1.0; all other serotypes had ratios less
than 1.0 (Figure 3-4).
The distribution of the pairwise distances within a serotype group
was primarily unimodal (Figure 3-5A; shown for serotype E). Only serotype J
had a bimodal population structure with two prevalent sequence groups
representing the J and Ja subtypes, roughly 98.5% similar (Figure 3-5B).
Serotype D had a unimodal population structure with a substantial shoulder
(Figure 3-5C); 47 sequences had two mutations, one at nt 246 (T=>C) and the
other at nt 249 (G=>C) compared to the prototype D/B-120 sequence; 24
sequences did not have these two mutations. Sequences with these two
mutations would be 99.7% similar to the D/B120 sequence. Thus, the
mutations at nt 246 and at nt 249 probably represent the differences in these
populations. It is possible that we might have detected a bimodal population
structure for serotype D if we had included additional sequence data
upstream of where our sequences began in CS1. Other studies have reported
six prevalent nucleotide changes in this region. However, even if serotype D
has a bimodal structure, the two population groups are not likely to
represent the D and Da subtypes because none of the previously reported
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122
nucleotide changes in CS1 or the prevalent changes present in our data
differentiate between the D and Da subtypes.
Figure 3-4. Mean Synonymous Mutation Rate; Nonsynonymous Mutation
Rate and Synonymous to Nonsynonymous Mutation Rate Ratio for Serotype
Groups Based on the Method of Nei and Gojobori
Synonymous Change per Synonymous Site with 95% Cl
0 .060 -
0.030 -
0 .0 0 0
Nonsynonymous Change per Nonsynonymous Site with 95% Cl
0.016 -
0 .008 -
0.000 -
Nonsynonymous to Synonymous Substitution Ratio
2.200 -
1.100 -
0.000
G Ba D E F H la J K
Serovar group
For upper and middle graphs, minimum and maximum values represent lower and upper
limits of the 95% Cl of the estimate, while horizontal bar level represents the mean estimate.
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123
Figure 3-5A. Unimodal Population Structure of Serotype E
10000 -
0 1
u
g
n
8000
4000-
2000 -
0.004 0.006 0.008 0.010 0.000 0.002
Genetic distance between sequences of serovar E
Figure 3-5B. Unimodal Population Structure with Shoulder of Serotype D
with Main and Shoulder Group Roughly 99.7% Similar
0 )
u
g
(0
1500 ua
< D
G
So 1000
o
&
G
500
0.005 0.010 0.015 0.020 0.000
Genetic distance between sequences of serovar D
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124
Figure 3-5C. Bimodal Population Structure of Serotype J with the Two Main
Groups Roughly 98.5% Similar
800 -
600 -
< u
S
V
M > 400 -
‘ 4 - 1
o
& *
e
2 200 -
cr
0 )
A
0.000 0.005 0.010 0.015 0.020 0.025
Genetic distance between sequences of serovar J
Figure 3-6 shows the degree to which ompA nucleotide changes have
been incorporated into the C. trachomatis population for different serotypes.
In this figure, starbursts represent the relative frequency of shared mutations
within a serotype group pool. The radial spoke length is inversely
proportional to the frequency of mutations so that longer spoke lengths
correspond to less frequent incorporation of mutations among similar
serotype group strains. Larger and more uniform spoke lengths within the
starburst represent both infrequent incorporation of mutations within the
serotype group and a prevalent serotype group. Serotypes in which we
observed nucleotide changes compared to the prototype that were at least
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25% prevalent in the population (Ba: C246T, A249G, A532G, G607T, and
T781G; D: C246T, A249G, A660T, and G1000A; F: G269; H: A272G and
C865T; and J: T625C and G627C) had the least uniform radial spoke lengths
while those without prevalent changes had more uniform spoke lengths (E,
la, G and K). Because of their high prevalence and a low frequency of shared
mutations, serotype groups E and la had both uniform spoke lengths and
large diameter circles.
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126
Figure 3-6. Phylogenetic Reconstructions with Starburst Superimposed at
End of Branch Corresponding to Appropriate Serotype Group
Ba
B a
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127
Discussion
Previous studies of chlamydial STDs have attempted to describe the
genetic variability of ompA by inferring the phylogeny of their population
with laboratory-maintained strains and published sequences (Stothard,
Boguslawski, and Jones; 1998). However, these studies used small
population sizes which do not reasonably approximate random ones, and the
combined populations represent strains from vastly different time periods,
since the laboratory-maintained strains were generally isolated decades
before the other strains. Phylogenetic reconstructions of ompA should only
be used to describe the short-term evolution of Chlamydia, since intra- and
intergenic recombination have previously been documented for this gene
(Fitch, Peterson, and de la Maza, 1993; Millman, Tavare, and Dean, 2001),
and recombination over time disrupts the bifurcating tree-like phylogeny
and clonal structure of the organism (Holmes, Urwin, and Maiden, 1999;
Millman, Tavare, and Dean, 2001).
We assessed the genetic variability of ompA within the context of a
large population-based study and analyzed the data using methods that
were appropriate for recombination in the gene. Consequently, the data
were analyzed without multiple substitution models so as to not introduce
an inaccurate evolutionary model. Further, we presented tree topologies
composed entirely of sequences from this study, which we used to infer the
short-term evolution of C. trachomatis. This was reasonable, as these strains
were isolated over a two-year period.
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128
The overall serotype distribution in our data was similar to other
studies of genital C. trachomatis infections with minor differences (Frost,
Deslandes, and Bourgaux-Ramoisy, 1993; Poole and Lamont, 1992; Yang,
Maclean, Brunham, 1993). In our study, there were no A, B, C, Da, or I
serotypes represented. The lack of A and C serotypes is consistent with the
universal and near universal absence, respectively, of these serotypes among
urogenital infections (Frost et al., 1995; Lan et al., 1995; Pedersen et al., 2000).
Of the three urogenital studies that reported C serotypes, two used
restriction fragment length polymorphisms (RFLP) to assign serotype. In
one study (Pedersen et al., 2000), all strains typed as C by RFLP were found
to be either F or G by sequencing, suggesting that RFLP is less reliable for
strain typing.
While Ba serotypes have regularly been found at both urogenital and
ocular sites, B serotypes have only been found twice in urogenital samples
(B/Alpha-95 (Farencena et al., 1997) and B/IU1226 (Stothard, Boguslawski,
and Jones; 1998)). When comparing these two urogenital B sequences to
prototype sequences, B/ Alpha-95 was most similar to B/TW-5, justifying its
assignment as a B. However, B/IU1226 was most similar to Ba/ Apache2, as
were the four sequences in this study. Thus, we feel that B/IU1226 should
be reassigned as a Ba, and believe that the occurrence of B sequences in
urogenital infections is indeed a rare event.
The lack of Da strains in our population contradicts a 1995 study by
Sayada et al. in which 9 (12%) of 73 urogenital strains were found to be Da by
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129
RFLP (Sayada et al., 1995). Based on sequence comparison, the D (D/B120)
and Da (Da/TW-448) subtypes can be distinguished by three nucleotide
differences, one of which encodes for an amino acid change in VS4 (G1036A
for Da/TW-448 in our alignment). None of our D sequences had this amino
acid change, and all of them were most similar to D/B120. In GenBank,
three urogenital sequences have previously been assigned Da [Da/IU-1554
(AF063209), Da/TW-488 (X62921) and Da/Ev-293 (X77365)]. However,
based on our analyses, Da/IU-1554 and Da/TW-488 sequences were most
similar to D/IC-Cal8, and Da/Ev-293 was most similar to D/B120. Only
Da/Ev-293 had the G1036A nonsynonymous change noted above. These
findings suggest that Da urogenital infections are also rare. However, the
importance of Da as a urogenital subtype should be evaluated in additional
studies that include other global regions where chlamydial STDs are
prevalent.
Finally, there were no I sequences; only la's were represented.
Recently, in other STD populations, serotype I has also been less frequently
identified (Suchland et al., 2003). This may reflect an adaptive response of
serotype I to host or antimicrobial pressure to evolve as an la. Alternatively,
there might have been an evolutionary disadvantage ascribed to serotype I
that has driven this process. Thus, these cumulative data suggest that the
occurrence of A, B, C and Da serotypes in urogenital infections are, at best,
rare events and that serotype I is being replaced by la.
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130
The Ba/D mosaic identified in this study is similar to a B/D
recombinant we previously isolated from a Tunisian trachoma patient in the
1970's (Dean et al., 1992). While both strains had similar crossover points,
three nonsynonymous mutations upstream of VS1 that were identical to
those described for B from other trachoma populations (Frost et al., 1995)
were present in the Tunisian recombinant while absent in the STD mosaic.
Thus, recombination between Ba or B and D seems to confer an evolutionary
advantage for both STD and trachoma populations. Interestingly, the Ba/D
recombinant did not have two changes that were present in all the Ba
sequences in our population (C246T - aa 60 in Frost alignment - and A249G -
aa 61 in Frost alignment) compared to B/TW-5, a trachoma strain. These
changes were also specific for the urogenital specimens in the population
described by Frost et al. (1995). Consequently, the Ba/D mosaic was
substantially more similar to B/TW-5 than the other urogenital Ba sequences
in this study, suggesting that changes upstream of nucleotide 477 are less
important for tissue specificity than the incorporation of the D sequence
downstream of 477. Moreover, there is only one change between the
urogenital Ba's in this study and B/TW-5 downstream of nucleotide 477.
This indicates that for serotype B, MOMP functional constraints are high in
this region, and thus, there is little opportunity for tissue-specific change.
We found significant differences in serotype distribution by
geographic region. While the chlamydial reservoir for the majority of strains
seems to be relatively homogeneous in the US, the rare serotypes, Ba and G,
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131
were significantly over-represented in San Francisco. Perhaps the
abundance of Ba and G serotypes in San Francisco reflects the population
dynamics of this international city that is constantly undergoing turnover
with a large immigrant population from Asia and a large homosexual
/bisexual community. In contrast, there was no significant difference in
serotype by gender, suggesting that specific serotypes do not preferentially
infect one gender.
We have provided the first quantitative assessment of the genetic
diversity within each serotype group for ompA. We found that serotypes E,
Ba, H, la and F (in descending order) were the most conserved, while J, G, D
and K were the most divergent. By comparing genetic variability, p J ps
substitution ratios and the relative degree of incorporation of substitutions in
the population, the data suggests that C. trachomatis has had occasional
selective sweeps of mutations through a serotype group or groups similar to
those described for HIV-1 (Grassly, Harvey, and Holmes, 1999). HIV-1 is
comprised of M, N, and O groups where the majority of infections are caused
by the M group, which is made up of subtypes A-J. The effective population
size of subtypes A (heterosexually transmitted) and B (transmitted via needle
sharing or male homosexual sex) based on both gag and env gene sequence
alignments were recently analyzed and found to be surprisingly smaller than
expected, similar to what we found for chlamydiae. The authors suggested
that this could be explained by genetic drift or a selective sweep of mutations
through the respective subtype populations.
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132
For Chlamydia, we found a predominantly unimodal population
structure for ompA (except for serotype J) consistent with a rapid expansion
of strains with little time for propagation of mutations. Under this model,
we expect C. trachomatis to have prevalent serotype groups with nucleotide
and amino acid changes that are infrequently shared among strains within
the same serotype. This can be seen for serotypes E and la, and less so for G
and K. The former serotypes are among the most prevalent and least
variable and are without prevalent population changes. Moreover, there is
laboratory evidence that serotype E out competes other strains for nutrients
and growth factors; this is objective evidence for rapid expansion of this
serotype group (Jones, Williams, and van der Pol B, 1998). The high pJ ps
ratios for serotypes la, G and K that also had rapid expansion indicate that,
compared to most other serotypes, they may be under a greater degree of
diversifying selection even though, for the most part, ompA has many
functional constraints and is under purifying selection. Rapid expansion of
selected serotypes may in part explain the relative replacement of I with la
and the over-abundance of serotype G in San Francisco if rapid expansion of
G has occurred with preference to geographical boundaries.
Serotypes Ba, D, F, H and J appear to have had less rapid population
growth exemplified by the presence of prevalent population changes and
greater genetic variability overall. For these serotypes, mutations have had
time to propagate throughout the population, and a lesser degree of
expansion helps to explain the lower prevalence for Ba, H and J. Moreover,
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133
they appear to be under a greater degree of purifying selection than those
with rapid expansion consistent with the lower observed pn/p s.
For serotypes D and J, the prevalent population changes observed in
this population agree with those previously reported suggesting they have
been present for at least a decade (Frost et al., 1995; Dean, Suchland, and
Stamm, 2000). We found that serotype J had a bimodal population structure,
which likely represents the J and Ja subtypes with mutations that occurred
before the two groups diverged. The prevalent population changes observed
for serotype D are also present in our Ba sequences and have previously been
ascribed to differentiate urogenital from trachoma B sequences (Frost et al.,
1995), although these changes were not universally found in our population
and, thus, appear to be nonspecific. Since the same changes for serotype D
and urogenital serotype Ba have propagated through many populations, the
evolution of these serotypes appears to be linked, either by the early
incorporation of a m utation occurring before their divergence or by
recombination events between the two.
In summary, by taking a population-based approach to the genetic
and evolutionary analysis of ompA for C. trachomatis urogenital infections in
the US, we were able to quantitatively and qualitatively assess the variation
of this gene and determine how immune selection pressures, functional
constraints, and other adaptive pressures vary among serotypes.
Importantly, our analyses provide the first evidence that the evolution of the
organism likely includes selective sweeps. This is encouraging for vaccine
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134
design in that mutations that occur during selective sweeps are unlikely to be
shared, and immune protection against recent clinical strains will be similar
to the response mounted against prototype strains. Finally, this research
provides precise genetic strain differentiation that can be applied to analyses
of phenotypic disease markers to determine if MOMP is a virulence factor for
the organism.
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135
CHAPTER 4. Population-Based Genetic Epidemiologic
Analysis of Chlamydia trachomatis Urogenital
Infections in the United States and Association with
Symptoms and Clinical Disease
Introduction
Multiple studies in Europe have found associations between serotypes
and reproductive tract signs and symptoms that were not reproducible even
though patient populations were similar. These associations were likely due
to chance since significance levels were not corrected for multiple
comparisons (Boisvert et al., 1999; Lan et al., 1995; Morre et al., 2002; van de
Laar et al., 1996; van Duynhoven et al., 1998; Workowski et al., 1994). A
recent study in the U.S. found no association between serotype and disease
manifestations, although the authors did not exclude those with co
pathogens as potential confounders in the data analyses (Geisler et al., 2003).
Two studies have evaluated the relationship between ompA genotypes and
disease severity. Variants of the F genotypes were found in one study to be
significantly associated w ith upper genital tract infection, symptoms, and
histopathology, whereas the E genotypes were more prevalent in mild,
asymptomatic cervicitis (Dean et al., 1995). In contrast, Lampe, Wong, and
Stamm (1995), using a genotyping system based only on variable segments
(VS) of ompA instead of the entire gene, found no difference in genotype
distribution among women with confirmed PID compared to women with
cervical infection who presumably were being seen for first-time infection.
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136
The inconsistency of the above findings is likely multifactorial, including
differences in study design, insufficient control for confounding, lack of
multiple comparison correction for significance levels, and small sample
sizes. In addition, insufficient sequencing of and failure to identify
polymorphisms in ompA has resulted in imprecise assignment of
serotypes/genotypes. In Chapter 3 we described the results of an analysis of
over 280,000 base pairs of the ompA gene including identification of
nucleotide and amino ad d polymorphisms within and among serotypes for
507 urogenital CT samples from five tities in the U.S.
We applied our new CT strain differentiation scheme as described in
Chapter 3 to identify CT strain types for correlations with dinical disease as
has been achieved for other hum an pathogens such as Hum an Papilloma
Virus (HPV). For example, the high-risk HPV subtypes, predominantly 16
and 18, are found in 70-78% of cervical intraepithelial neoplasia (CIN) I
lesions, in 83-89% of CIN II lesions, in 95% of squamous cell cardnomas, and
in 90% of adenocardnomas, while low-risk HPV subtypes 6 and 11 are not
significantly assodated with any type of cervical cancer. We conducted a
cross-sectional case series analysis of 344 urogenital samples from men and
women with CT infections who were part of the aforementioned population
study. Serotype, genotype and subtype variants of the infecting strain were
correlated w ith linked dinical and demographic data to investigate whether
these might be associated with recurrent infection, symptoms and outcome
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137
such as pelvic inflammatory disease (PID) and thus predict clinical
phenotype and virulence determinants.
Materials and Methods
Study Design. The cross-sectional case series described in Chapter 3
was conducted during an approximately two-year surveillance period in
collaboration with the Centers for Disease Control, Atlanta, GA. Urogenital
samples containing CT DNA were obtained as part of a larger parent study
conducted in the greater Birmingham, Indianapolis, New Orleans, San
Francisco and Seattle metropolitan areas between October of 1995 and
August of 1997. The study comprised three components designed to
evaluate: 1 ) diagnostic modalities for urogenital chlamydial infection where
females and asymptomatic males were enrolled; 2 ) risk factors for recurrent
urogenital chlamydial infection; and 3) risk factors for urogenital chlamydial
infection. The three components constitute a sentinel surveillance network
of family planning, STD and community health clinics in each metropolitan
area. Clinicians were chosen from participating sentinel clinics to collect
data for the applicable study component. For this study, standard medical,
STD, reproductive and treatment histories and demographic information as
well as results from any laboratory tests conducted to detect CT and Neisseria
gonorrhoeae, Candida albicans, Trichomonas vaginalis, Herpes simplex, HPV or
Gardnerella. Data were recorded at each site. Additional clinical and
microbiologic data were collected by extensive chart review. One hundred
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138
percent of the abstracted and nonabstracted chart data was checked by study
staff for accuracy. Patient confidentiality was maintained using patient and
specimen identification numbers. Chlamydial strain differentiation based on
ompA genotyping and estimation of serotype was described and presented in
Chapter 3.
Study population. This study included consenting patients w ith
documented CT urogenital infection seen at the aforementioned clinics.
Patients aged 14-49 years were included if they were CT positive based on
the tests used by participating clinics, which included one or more of the
following: the ligase chain reaction (LCR), Amplicor polymerase chain
reaction (PCR) and CT culture as described in Chapter 3. Patients were
excluded if they had clinical or diagnostic evidence of other STD pathogens
[Neisseria gonorrkoeae, Candida albicans, Trichomonas vaginalis, Gardnerella
(causative for bacterial vaginosis (BV)), Herpes, HPV], had taken antibiotics
within the past 30 days, were pregnant, had had a hysterectomy, or had not
received a pelvic examination. Of the thousands of patients enrolled at each
site who had urogenital CT infections, we randomly selected 507 patients, of
whom, 344 were included after excluding those patients with co-pathogen
infection or insufficient data. There was a roughly equal sex and
metropolitan area distribution for the participants in this study.
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139
Statistical Analysis. Six dichotomous outcome variables were analyzed:
1) CT-probable symptoms including any of those specific symptoms in 2-5; 2)
mucopurulent cervicitis, abnormal vaginal or urethral discharge; 3) dysuria;
4) lower abdominal pain; 5) abnormal vaginal bleeding; and 6 ) PID. Patient
complaint was used except as follows: PID was evaluated by a physician and
defined as lower abdominal pain with at least two of the following physical
signs: adnexal tenderness, cervical motion tenderness and uterine
tenderness. For mucopurulent cervicitis, vaginal or urethral discharge,
physician findings were used whenever available as they were considered to
be more accurate than patient reporting; clinician assessments were available
for all (58/ 58) individuals in Birmingham, for 81 (94.2%) of 8 6 in Seattle, for
50 (70.4%) of 71 in Indianapolis, for 9 (22.0%) of 41 in San Francisco, and for 0
(0%) of 92 in New Orleans. We handled missing covariate data by deleting
patient records from analyses that involved those variables for which the
covariate was missing. We chose to analyze only those covariates with less
than 35% missing data so that subjects with complete data would be as close
to a random sample of those in the study as possible. Out of an extensive list
of covariates for which data were originally collected the covariates that
were analyzed included gender, race/ethnicity, age, metropolitan area,
reason for visit, clinic type, usual (method used the majority of the time)
method of contraception in the last 60 days, ompA genotype (recorded as
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140
subtype as defined in Chapter 3), and ompA nucleotide positions that varied
when comparing the entire sequence set.
For the outcome variables, unadjusted analysis of each main variable
was performed using unconditional logistic regression with significance
determined using the likelihood ratio test at an alpha level of 0.05. In the
unadjusted analysis of serotype, data were too sparse to determine valid
parameter estimates. Thus, the variable was fit to a serotype term without
including the two rare serotypes, Ba and H. Covariates considered for
inclusion in multiple regression models included the main variable of
interest and any potential confounder that when included in the model
resulted in at least a ten percent change of the regression coefficient from its
baseline value. For the outcome variables that were rare (abnormal vaginal
bleeding, lower abdominal pain and PID), logistic regression models resulted
in sparse data. Fisher's two-tailed exact tests were used to test associations
for these variables. All analyses were performed using the statistical
package SAS version 6.12.
For the non-dichotomous main variables of interest- including ompA
serotype and genotype, the individual components were analyzed further
when the global analysis was significant. When this was the case, a
Bonferroni adjustment was used to account for the number of comparisons
involved. As an exception to this, we tested individual serotype associations
found in previous studies including: (a) serotype la with asymptomatic
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141
infections (Morre et al., 2000); (b) serotype D with asymptomatic infections
(Lan et al., 1995); (c) serotypes G /G a with symptomatic infections among
women (Morre et al., 2000); (d) serotype Ga with symptomatic infections
overall and among men (Morre et al., 2000); and (e) serotype F with PID
(Dean et al., 1995) using a Fisher's two-tailed exact test. Given that these
were a priori hypotheses, no corrections for multiple comparisons were
applied to these individual pairwise tests. Any ompA nucleotide position
with variation was considered an additional main variable of interest. To
determine if the nucleotide position was associated with the outcome
variable, a chi square test of homogeneity was performed using nucleotide
character states as categories, both overall and by gender. Additionally, we
tested serotypes D and J to determine the clinical relevance of prevalent
subpopulations described in Chapter 3; serotype J most likely represented
the J and Ja subtype groups, and D represented two groups differentiated by
the presence or absence of two mutations compared to the D/B120 sequence
(t246c, g249c). Significance levels were corrected with the Bonferroni method
for the total number of positions tested (5 comparisons x number of
polymorphisms).
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142
Results
Demographics. There were 180 (52%) males and 164 (48%) females,
72% of which were African American, 17% Caucasian, 4% Hispanic, 4%
Asian/Pacific Islander, 3% other, and less than 1% unknown; 75% were less
than 25 years of age (Table 4-1).
Table 4-1. Demographic Distribution for the Study Population from Five
Metropolitan Cities in the United States
Gender n (344) Percent
Male 180 52.3
Female 164 47.7
Race/Ethnicity
Caucasian 57 16.6
African American 246 71.5
Hispanic 16 4.7
Asian/Pacific
16 4.7
Other 8 2.3
Unknown 1 0.3
Age
< 15 years 5 1.5
15-20 years 143 41.6
20-25 years 116 33.7
25-30 years 48 14.0
30-35 years 2 1 6 . 1
> 35 years 1 1 3.2
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143
Table 4-1. Continued
Metropolitan area n (344) Percent
Birmingham 58 16.9
Indianapolis 69 2 0 . 1
New Orleans 90 26.2
Seattle 8 6 25.0
San Francisco 41 11.9
Risk factors for CT urogenital symptoms and outcomes. Gender, reason
for visit, and metropolitan area were significant independent predictors of
symptoms consistent with CT infection. Overall, 153 (44.5%) of 344 study
subjects reported CT-probable symptoms. Of these, 99 were males and 54
were female. In terms of specific symptoms, 141 (41%) of 344 complained of
vaginal or urethral discharge, 39 (11.3%) of 344 complained of dysuria, 12
(7.3%) of 164 women complained of lower abdominal pain, 3 (1.8%) of 164
women complained of abnormal vaginal bleeding, and 2 (1.2%) of 164
women were clinically diagnosed with PID. The relative risk for men to
report any symptoms was 2.2 times more than for women (p=0.03) and 2.8
times more to report a urethral discharge than women were to report a
vaginal discharge (p=0.007) in the adjusted analyses (Table 4-2). Those who
had contact with an individual with nongonococcal urethritis (NGU) or CT,
or who presented due to symptoms, were 14.9 (p=0.004), 4.4 (p=0.0001) and
6 . 8 (p=0 .0 0 0 1 ) times more likely to report symptoms, respectively, than those
who presented for an STD evaluation. In contrast, those who presented for
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144
family planning evaluation were 0 . 1 1 times less likely to report symptoms
than those who presented for an STD evaluation (p=0.04; Table 4-2).
Table 4-2. Adjusted Odds Ratios for Risk of Chlamydial-Probable Symptoms
and for Risk of Having M ucopurulent Cervicitis, Vaginal or Urethral
Discharge According to the Significant Independent Predictors Measured in
the Population-Based Study
Covariate
Adjusted odds
ratio (95% Cl)
Symptom
status
p-value
Sympto
m
status
Adjusted odds
ratio (95% Cl)
cervicitis or
discharge
p-value
cervicitis
or
discharge
Gender
Female 1 .0 * i.o §
Male 2.2 (1.1-4.3)* 0.03 2.8 (1.4-5.6) § 0.007
Reason for visits
STD check 1 .0 2 l.Ot
Contact CT
4.4 (1.6-11.9)
t
0 . 0 0 0 1
5.1 (1.8-14.1)
t
0 . 0 0 2
Contact NGU
14.9 (1.7-
131.1) t
0.004
8.2 (1.5-45.4)
t
0 . 0 2
Contact GC
1.28 (0.3-5.5)
t
0.74 1.6 (0.4-7.0) t 0.55
Contact other
STD
0.76 (0.3-2.3)
t
0.63
0.70 (0.2-2.2)
t
0.52
Symptomatic
6 . 8 (2.8-16.5)
t
0 . 0 0 0 1
5.0 (2.1-11.8)
t
0 . 0 0 0 2
Family
planning
0 . 1 1 (0 .0 1 -
0 .8 8 ) t
0.04
0 . 1 2 (0 .0 2 -
1 .1 ) +
0.06
Prenatal
0.46 (0.08-2.5)
t
0.36
0 . 2 (0 .0 2 -2 .0 )
+
0.18
Other reason
0.59 (0.2-1.8 )
t
0.34 0.7 (0.2-2.1) + 0.52
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145
Table 4-2. Continued
Covariate
Adjusted odds
ratio (95% Cl)
Symptom
status
p-value
Sympto
m
status
Adjusted odds
ratio (95% Cl)
cervicitis or
discharge
p-value
cervicitis
or
discharge
Race/Ethnicity
African
American
>0.05 1 . 0 1 1
Caucasian >0.05
1.28 (0.5-3.0)
1 1
0.56
Asian/Pacific
Islander
>0.05
0.11 (0.02-.65)
1 1
0.015
Hispanic >0.05
0.22 (0.05-
1 .0 ) 1 1
0.05
Other >0.05
0.75 (0.1-4.5)
1 1
0.80
* Adjusted for age, metropolitan area and reason for visit
t Adjusted for metropolitan area
i Adjusted for 'reason for visit'
§ Adjusted for metropolitan area and reason for visit
I I Adjusted for reason for visit, metropolitan area and gender
Serotype and symptoms consistent w ith CT infection. Our sample
population had the following serotype distribution: E (30%); F (20.6%); la
(14.5%); D (13.5%); J (9.9%); G (4.4%); K (4.4%); Ja (1.5%); Ba (0.6%) and H
(0.6%). None of the covariates analyzed, except for metropolitan area, were
significantly associated with serotype. In accordance with our results from
Chapter 3, there was a preponderance of Ba and G subtypes in San Francisco
compared with the other metropolitan areas.
Serotype (with categories as defined above) was not significantly
associated with CT-probable symptoms both overall and stratified by gender
for the composite variable (Table 4-3). This was the case in the unadjusted
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146
analysis and after adjusting for metropolitan area. When analyzing the four
most prevalent serotypes (D, E, F and la) alone, there was still no significant
association between serotype and CT-probable symptom (p=0.69). Similarly,
seroclass (defined as B serodass: Ba, D, Da and E; Intermediate seroclass: F
and G; C seroclass: A, C, H, I, la, J, Ja and K) or individual serotypes D, la
and G (those reported in other studies to have assotiations) were not
significantly assodated with CT-probable symptoms.
Table 4-3. Chlamydial-Probable Symptom Status across all Chlamydial
Serotypes and Stratified by Gender
Overall Men Women
Serotype n
Asympto
matic (%)
Symptomatic
(% )
Asympto
matic (%)
Symptomat
ic (%)
Asympto
matic (%)
Symptomatic
(% )
Ba 2 2 (100.0) 0(0.0) 1 (100.0) 0 (0.0) 1 (100.0) 0 (0.0)
D 47 29 (61.7) 18 (38.3) 11 (44.0) 14 (56.0) 18 (81.8) 4 (18.2)
E 103 56 (54.4) 47 (45.6) 21 (42.0) 29 (58.0) 35 (66.0) 18 (34.0)
F 71 36 (50.7) 35 (49.3) 17 (46.0) 20 (54.0) 19 (55.9) 15 (44.1)
G 15 9 (60.0) 6 (40.0) 5 (62.5) 3 (37.5) 4 (57.1) 3 (42.9)
H 2 0 (0.0) 2 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (100.0)
la 50 28 (56.0) 22 (44.0) 13 (46.4) 15 (53.6) 15 (68.2) 7 (31.8)
I
34 21 (61.8) 13 (38.2) 10 (50.0) 10 (50.0) 11 (78.6) 3 (21.4)
Ja
5 2 (40.0) 3 (60.0) 1 (25.0) 3 (75.0) 1 (100.0) 0 (0.0)
K 15 8 (53.3) 7 (46.7) 2 (28.6) 5 (71.4) 6 (75.0) 2 (25.0)
Total 344 191 (55.5) 153 (44.5) 81 (45.0) 99 (55.0) 110 (67.1) 54 (32.9)
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147
In terms of specific symptoms, there were no significant serotype
differences for vaginal or urethral discharge, dysuria, abnormal vaginal
bleeding or lower abdominal pain in this population after adjusting for
confounders. However, when stratifying by gender, women were 4.5 (95%
Cl: 1.01-20.0) times less likely to have a vaginal or urethral discharge when
infected with serotype D compared to others (p=0.03). Clinically diagnosed
PID was a rare event; it occurred in 2 (1.4%) of 147 women evaluated. Both
women were infected with serotype F. Based on a Fisher's two-tailed exact
test, women with PID were significantly more likely to be infected with
serotype F than any other serotype (p=0.046; OR = infinity) in this
population.
OmpA variant positions and symptom status. In order to determine if
there were individual variant orrnpA positions (not associated with serotype)
that were associated with the outcome variables, we tested each variant
nucleotide position in ompA. At the nucleotide level, we found 330
polymorphic sites within the ompA gene analyzed. For women in this
population, five variable ompA positions were significantly associated with
abnormal vaginal bleeding after adjusting for multiple comparisons (nt 240,
nt 294, nt 398, nt 590, nt 606). When stratifying by serotype, it was evident
that, for the woman infected with serotype D, these five positions and two
additional positions (nt 289 and nt 534; Table 4-4) were significantly
associated with abnormal vaginal bleeding. However, none of these
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148
positions was responsible for the division of serotype D into two different
subpopulations (two groups differentiated by the presence or absence of
changes w ith respect to the D/B120 sequence at nt 246 and nt 249; Table 4-4).
Table 4-4 shows the odds ratios by nucleotide character states with low and
high risk for variable ompA positions found to be significantly associated
with abnormal vaginal bleeding.
Table 4-4. Odds Ratios for Abnormal Vaginal Bleeding According to
Character States at Specific Variable ompA Positions
Nucleotide
Position
Character state
with increased
risk
Character state
with decreased
risk
Odds ratio p-value
Women infected
with any
serotype,
including D
241 C G Infinity 2 .0 ' 23
295 A T /G Infinity 1 .6 4 8
399 A G Infinity 5.8'26
591 G C /T Infinity
2 . 7 4 8
607 C G Infinity 5.8'26
Women infected
with only
serotype D
289 A G Infinity 1 .6 ' 5
534 A G Infinity 1 .6 ' 5
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149
Discussion
This is the first study that couples a very large gene sequencing effort
with clinical and demographic data to investigate the genetic epidemiology
of CT STDs among diverse communities in the United States. We provided
increased genetic and statistical precision for strain discrimination to
examine whether serotype as defined by ompA sequence or specific ompA
polymorphisms can predict clinical phenotype and whether these comprise
virulence determinants for the organism. The study was designed to include
a broad population base in terms of geographical location, spectrum of
clinics, and demographics of the populations served.
We first evaluated risk factors for symptoms consistent with
urogenital CT infection among CT-positive individuals and found gender,
reason for visit and metropolitan area were significant independent
predictors. Earlier studies have not excluded multiple infections, and since
the inclusion of those with multiple infections biases the odds ratios, this
study provides a more accurate measure of symptoms that are attributable to
CT infection than was reported in earlier studies.
In terms of gender, it may be possible in part to explain the difference
in the reported odds ratio seen between this and other studies, where men
were found to be more symptomatic than women with an average odds
ratios of 3.0 compared to 2.2 in the present study (Marrazzo et al., 1997; Pabst
et al., 1992; Smith et al., 1978; Stamm et al., 1984). Since asymptomatic men
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150
only were included in the first component of the parent study, which
comprised 44% of the study data, this represented a selection bias for
analysis of gender and symptoms. The second and third components of the
parent study included men and women according to symptom status. Given
these facts, the reported odds ratio for gender and symptom status in this
study is somewhat biased.
Race was not an independent predictor of symptom status in this
population, which may be due to the fact that our CT-positive study
population was 71.5% African American. Since black race is a risk factor for
CT infection, one would expect a significantly higher percentage of African
Americans and lower percentage of Caucasians among CT infected cases.
This was the case compared to the parent study, which was composed of all
comers with and without CT infection.
We tested whether the current serotype classification of CT based on
MOMP is predictive of clinical manifestations. We supposed that if there
was no difference in clinical presentation for different serotypes that
virulence determinants could still be present at individual amino acid
positions that do not correlate with serotype. This latter point is supported
by the fact that additional chlamydial serotypes have been identified based
on ompA nucleotide changes in variable segments (VS) with subsequent
recognition of the corresponding epitopes by new monoclonal antibodies
(MAbs) or combinations of existing Mabs (Lampe, Suchland, and Stamm,
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151
1993; Wang and Grayston, 1991). For this reason, we also tested whether
specific variable ompA nucleotide positions were associated with clinical
signs or symptoms, with gender, and with serotype differentiation.
In the multiple logistic regression analysis, we found no association of
serotype with the outcome variable that was a composite of chlamydial
probable symptoms. This result is not in agreement with recent studies
based on serotype (Boisvert et al., 1999; Dean et al., 1985; Lan et al., 1995;
Morre et al., 2000; van de Laar et al., 1996; van Duynhoven et al., 1998;
Workowski et al., 1994), which reported discrepant associations. For
example, Morre et al. (2000) found associations between serotype la and
asymptomatic individuals; serotype Ga and symptomatic individuals; and
serotypes G /G a and symptomatic women. In contrast, Lan et al. (1995)
found an association only between serotype D and asymptomatic
individuals. This is interesting because both studies utilized Dutch
populations and measured serotype by RFLP. However, in all of these
studies, each serotype was tested separately without correction for multiple
comparisons. If such corrections had been made, most if not all of the
associations would not have been significant and could likely be explained
by chance. In contrast, in this study we tested this relationship globally with
a logistic regression model. Since there was no association revealed for the
global test (i.e., 1 0 x 2 analysis of serotype associated with symptom), we did
not test individual serotype associations (i.e., 2 x 2 analysis of serotype E
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152
associated with symptom) except for those reported in other studies (D, F, G,
Ga and la).
In terms of specific symptoms, when we stratified by gender, we did
find a significant association between serotype D and the absence of vaginal
discharge, albeit the confidence interval was wide. We also found a strong
and significant association between PID and serotype F, however it was only
based on two patients with PID diagnoses. Despite the rarity of PID in this
population, the probability of finding only one serotype in both cases out of
the 1 0 possible serotypes that could cause an upper genital tract infection is
low and would be unexpected due solely to chance. While there may be an
association between serotype F and PID supported by this and our earlier
work, there are inherent difficulties in the measurement of PID, and most
studies have been hampered by this fact. Silent PID is prevalent and
unmeasured by noninvasive approaches. Further, clinical assessments of the
diagnosis vary widely between observers. Moreover, since PID is associated
with prior infection or persistence, a prospective study is needed to account
for persistent or multiple infections that may occur over the period of time
required for the complication to develop. The possible association between
serotype F and PID, then, requires longitudinal measurement of outcome
and serotype as well as confirmation of the outcome by invasive
measurement. Yet, this type of study would be extremely difficult to do.
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153
We also tested independently whether ompA polymorphism(s)
predicts clinical manifestations. We found five positions among women
with any serotype, including serotype D, and seven positions among women
infected with serotype D that were significantly associated with abnormal
vaginal bleeding (Table 4-4). On inspection, these seven nt positions
represented the unique differences of a D sequence that is very divergent
compared to its most similar reference serotype. Thus, the association was
solely driven by one sequence. Two women with abnormal vaginal bleeding
who were infected with serotypes other than D (i.e., E and J) did not have
any of these changes. While it is possible that these changes result in
abnormal vaginal bleeding only when the infecting serotype is D, we think it
unlikely. We also tested both serotype D and J separately to ascertain
whether there was any clinical relevance to the population differences we
reported in Chapter 3. Since none of the positions associated with abnormal
bleeding were responsible for the division of serotypes D or J into their
respective subtype populations, there were no differences in clinical signs
and symptoms that could be ascribed to these population differences.
Three theoretical explanations for the inability to detect a global
association between clinical findings and serotype or genotype, if one does
exist, include lack of power, nondifferential misclassification, and inadequate
measurement of confounding. This study was sufficiently powered for the
four most prevalent serotypes - D, E, F and la - for which we had 80% power
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154
to detect a relative risk as low as 2.2. However, the power was not sufficient
for the rare serotypes, Ba and H, or the outcome PID. For Ba and H, 1,100
and 8,600 CT infected individuals would have been needed to detect relative
risks of 5.0 and 2.0, respectively, with 80% power at an alpha level of 0.05.
Thus, we cannot rule out a difference for the rare serotypes due to lack of
power or for the rare outcomes in the study including PID.
There was essentially no misdassification in the exposure variables
since the definition of serotype and genotype was extremely objective.
Except for PID, we think it unlikely that nondifferential misdassification of
outcome variables explains the inability to find an assodation. Since, by
definition, dysuria and lower abdominal pain are purely subjective variables,
there is minimal chance for misdassification despite wide differences in pain
perception. However, as stated earlier, there is considerable misdassification
for PID since the diagnosis is based on noninvasive clinical criteria with
considerable interobserver variation. For the outcome of vaginal or urethral
discharge, physidan finding was used 58% of the time. Of note is that
vaginitis due to Candida, trichomonas, or BV was not induded with vaginal
discharge since each was exduded based on microbiologic testing. While
there is some interobserver variation in diagnosing vaginal or urethral
discharge, physidan finding (along with microbiologic testing) was
considered to be far more accurate than patient reporting and was used,
when available, as the gold standard. For data where both physidan finding
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155
and patient complaint were available, we calculated the sensitivity and
specificity of patient reporting compared to our gold standard. In this study,
we found patient reporting to be highly specific (94.1%) but not sensitive
(45.6%); patients tended to underreport the complaint compared to physician
findings.
It is likely that other outcome variables such as abnormal vaginal
bleeding were also underreported when present and not over-reported when
absent. For example, a woman may underreport bleeding if she attributes it
to her own cycle or spotting between periods or hormonal therapy. The
estimate of specificity for abnormal discharge in this study was quite high
and we expect the estimate of specificity for abnormal vaginal bleeding to be
similar. Thus, it is unlikely that nondifferential misdassification biased the
risk ratio substantially because a lack in sensitivity in the response variable
produces no bias in the risk ratio while a lack in spedfidty does (Rothman
and Greenland, 1998).
Co-pathogen infection was an exdusion criterion of this study since it
would confound the assodations between clinical findings and CT.
However, co-pathogens such as Mycoplasma genitalium were not routinely
measured unless there was clinical suspicion of the infection. Although
these pathogens are less common than CT, this would result in some
misdassification of confounder status. Further, the sensitivities of most STD
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156
diagnostic assays are not 1 0 0 %, which would also contribute some
misdassification and confounding.
The cross-sectional nature of this study did not provide data on the
natural history of chlamydial urogenital infections with respect to symptom
profile, which may result in misdassification of the outcome. Thus, same-
serotype infedions may produce different symptoms at different stages of
infection, but we had no information on how long it had been from the initial
infection to actual enrollment in the study. Only a prospective study could
elucidate these relationships, and the hum an subjects consideration and
logistics would be prohibitive.
In summary, we have provided evidence from a large multicentered
STD study that there are no differences at the serotype or single nudeotide
level that result in important differences in selected dinical presentation,
disease, or disease outcome other than perhaps for PID, vaginal bleeding and
genotypes Ba and G, where the latter genotypes were rare. In these
instances, much larger studies with suffident patients with PID and
abnormal vaginal bleeding would be required to more clearly define the
assodations. While various MOMP determinants and genetic differences
have been reported to be important in host immune response (Allen,
Locksley, and Stephens, 1991; Morrison et al., 1990; Peeling and Branham,
1991; Su et al., 1990) and tissue tropism supported by the data we present in
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157
Chapter 2, they do not appear to contribute to virulence or pathogenicity of
the organism.
A strain-typing approach that includes a Multi-Locus Sequence
Typing (MLST) scheme might provide the discriminatory power for
identifying the strains that correlate with clinical phenotype (Selander et al.,
1986). Other chlamydial genes or proteins that have been identified through
comparative genomics may significantly contribute to enhancing our
understanding of clinical phenotypes. These include Hsp60, which has been
associated with immunopathogenic responses in hum an fallopian tube
tissues (Patton, Sweeney, and Kuo, 1994; Toye et al., 1993; Wagar et al., 1993),
cy totoxin genes with homology to a large clostridial cytotoxin B (Belland et
al., 2001), partial tryptophan operon proteins (TrpB/A) that may allow for
resistance to interferon gamma through indole rescue (Fehlner-Gardiner et
al., 2002), Type III secretion system proteins that may disrupt signal
transduction pathways (Bavoil and Hsia, 1998), and the chlamydial protease-
or proteasome-like activity factor (CPAF). It is the continued efforts in these
exciting new avenues of research that will enable development of a
classification system for CT that is clinically prognostic and will enhance our
ability to design effective therapeutic interventions to prevent or ameliorate
the devastating sequelae of CT.
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158
BIBLIOGRAPHY
Alexander JJ. 1968. Separation of protein synthesis in meningopneumonitis
agent from that in L cells by differential susceptibility to
cycloheximide. J Bacteriol; 95:327-332.
Allen JE, Locksley RM, Stephens RS. 1991. A single peptide from the major
outer membrane protein of Chlamydia trachomatis elicits T cell help for
the production of antibodies to protective determinants. J Immunol;
147: 674-679.
Allen JE, Stephens RS. 1989. Identification by sequence analysis of two-site
posttranslational processing of the cysteine-rich outer membrane
protein 2 of Chlamydia trachomatis serovar L2. J Bacteriol; 171: 285-291.
Amann R, Springer N, Schonhuber W, Ludwig W, Schmid EN, Muller KD,
Michel R. 1997. Obligate intracellular bacterial parasites of
acanthamoebae related to Chlamydia spp. Appl Environ Microbiol; 63:
115-121.
Baehr W, Zhang YZ, Joseph T, Su H, Nano FE, Everett KD, Caldwell HD.
1988. Mapping antigenic domains expressed by Chlamydia trachomatis
major outer membrane protein genes. Proc Natl Acad Sri USA; 85:
4000-4004.
Bandea Cl, Kubota K, Brown TM, Kilmarx PH, Bhullar V, Yanpaisarn S,
Chaisilwattana P, Siriwasin W, Black CM. 2001. Typing of Chlamydia
trachomatis strains from urine samples by amplification and
sequencing the major outer membrane protein gene (ompl). Sex
Transm Infect; 77: 419-422.
Bannantine JP, Stamm WE, Suchland RJ, Rockey DD. 1998. Chlamydia
trachomatis IncA is localized to the inclusion membrane and is
recognized by antisera from infected humans and primates. Infect
Immun; 66:6017-6021.
Barbour AG, Amano K, Hackstadt T, Perry L, Caldwell HD. 1982. Chlamydia
trachomatis has penidllin-binding proteins but not detectable muramic
arid. J Bacteriol; 151: 420-428.
Bames RC, Katz BP, Rolfs RT, Batteiger B, Caine V, Jones RB. 1990.
Quantitative culture of endocervical Chlamydia trachomatis. J Clin
Microbiol; 28: 774-780.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
159
Barnes RC, Rompalo AM, Stamm WE. 1987. Comparison of Chlamydia
trachomatis serovars causing rectal and cervical infections. J Infect Dis;
156: 953-958.
Barnes RC, Suchland RJ, Wang SP, Kuo CC, Stamm WE. 1985. Detection of
multiple serovars of Chlamydia trachomatis in genital infections. J
Infect Dis; 152: 985-989.
Barry 3rd CE, Brickman TJ, Hackstadt T. 1993. Hc-l-mediated effects on
DNA structure: a potential regulator of chlamydial development. Mol
Microbiol; 9: 273-283.
Barry 3rd CE, Hayes SF, Hackstadt T. 1992. Nucleoid condensation in
Escherichia coli that express a chlamydial histone homolog. Science;
256: 377-379.
Batteiger BE, Fraiz J, Newhall WJ, Katz BP, Jones RB. 1989a. Association of
recurrent chlamydial infection with gonorrhea. J Infect Dis; 159: 661-
669.
Batteiger BE, Lennington W, Newhall WJ, Katz BP, Morrison HT, Jones RB.
1989b. Correlation of infecting serovar and local inflammation in
genital chlamydial infections. J Infect Dis; 160: 332-336.
Batteiger BE, Rank RG, Bavoil PM, Soderberg LS. 1993. Partial protection
against genital reinfection by immunization of guinea-pigs with
isolated outer-membrane proteins of the chlamydial agent of guinea-
pig inclusion conjunctivitis. J Gen Microbiol; 139: 2965-2972.
Bauwens JE, Lampe MF, Suchland RJ, Wong K, Stamm WE. 1995. Infection
with Chlamydia trachomatis lymphogranuloma venereum serovar LI in
homosexual m en with proctitis: Molecular analysis of an unusual
case cluster. Clin Infect Dis; 20: 576-581.
Bavoil PM, Hsia RC. 1998. Type IH secretion in Chlamydia: a case of deja vu?
Mol Microbiol; 28: 860-862.
Bavoil P, Ohlin A, Schachter J. 1984. Role of disulfide bonding in outer
membrane structure and permeability in Chlamydia trachomatis. Infect
hnmun; 44:479-485.
Beatty WL, Morrison RP, Byrne GI. 1995. Reactivation of persistent
Chlamydia trachomatis infection in cell culture. Infect Immun; 63:199-
205.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
160
Belland RJ, Sridmore MA, Crane DD, Hogan DM, Whitmire W, McClarty G,
Caldwell HD. 2001. Chlamydia trachomatis cytotoxicity associated
with complete and partial cytotoxin genes. Proc Natl Acad Sci USA;
98:13984-13989.
Belland RJ, Zhong G, Crane DD, Hogan D, Sturdevant D, Sharma J, Beatty
WL, Caldwell HD. 2003. Genomic transcriptional profiling of the
developmental cycle of Chlamydia trachomatis. Proc Natl Acad Sd
USA; 100: 8478-8483.
Belunis Q , Mdluli KE, Raetz CR, Nano FE. 1992. A novel 3-deoxy-D-
manno-octulosonic acid transferase from Chlamydia trachomatis
required for expression of the genus-spedfic epitope. J Biol Chem;
267:18702-18707.
Birkelund S, Johnsen H, Christiansen G. 1994. Chlamydia trachomatis serovar
L2 induces protein tyrosine phosphorylation during uptake by HeLa
cells. Infect Immun; 62:4900-4908.
Birkelund S, Lundemose AG, Christiansen G. 1988. Chemical cross-linking
of Chlamydia trachomatis. Infect Immun; 56:654-659.
Birkelund S, Lundemose AG, Christiansen G. 1989. Characterization of
native and recombinant 75-kilodalton immunogens from Chlamydia
trachomatis serovar L2. Infect Immun; 57:2683-2690.
Black CM, Marrazzo J, Johnson RE, Hook 3rd EW, Jones RB, Green TA,
Schachter J, Stamm WE, Bolan G, St Louis ME, Martin DH. 2002. Head-
to-head multicenter comparison of DNA probe and nucleic add
amplification tests for Chlamydia trachomatis infection in women
performed with an improved reference standard. J Clin Microbiol; 40:
3757-3763.
Blythe MR, Katz BP, Batteiger BE, Ganser JA, Jones RB. 1992. Recurrent
genitourinary chlamydial infections in sexually active female
adolescents. J Pediatr; 121:487-493.
Boisvert JF, Koutsky LA, Suchland RJ, Stamm WE. 1999. Clinical features of
Chlamydia trachomatis rectal infection by serovar among homosexually
active men. Sex Trans Dis; 26: 392-398.
Bose SK, Liebhaber H. 1979. Deoxyribonucleic add synthesis, cell cycle
progression, and division of Chlamydia-infected HeLa 229 cells. Infect
Immun; 24:953-957.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
161
Brade H, Brade L, Nano FE, 1987. Chemical and serological investigations
on the genus-specific lipopolysaccharide epitope of Chlamydia. Proc
Natl Acad Sd USA; 84:2508-2512.
Brade L, Nano FE, Schlecht S, Schramek S, Brade H. 1987. Antigenic and
immunogenic properties of recombinants from Salmonella typhimurium
and Salmonella minnesota rough mutants expressing in their
lipopolysaccharide a genus-specific chlamydial epitope. Infect
Immun; 55:482-486.
Braun J, Laitko S, Trehame J, Eggens U, Wu P, Distler A, Sieper J. 1994.
Chlamydia pneumoniae— a new causative agent of reactive arthritis and
undifferentiated oligoarthritis. Ann Rheum Dis; 53:100-105.
Brickman TJ, Barry 3rd CE, Hackstadt T. 1993. Molecular cloning and
expression of HctB encoding a strain-variant chlamydial histone-like
protein with DNA-binding activity. J Bacteriol; 175:4274-4281.
Branham RC. 1999. Hum an Immunity to Chlamydiae. In: Chlamydia:
Intracellular Biology, Pathogenesis, and Immunity. Stephens RS,
editor. Washington, D.C.: ASM Press; pp. 211-238.
Branham RC, Kimani J, Bwayo J, Maitha G, Maclean I, Yang C, Shen C,
Roman S, Nagelkerke NJ, Cheang M, Plummer FA. 1996. The
epidemiology of Chlamydia trachomatis within a sexually transmitted
diseases core group. I Infect Dis; 173: 950-956.
Branham R, Yang C, Maclean I, Kimani J, Maitha G, Plummer F. 1994.
Chlamydia trachomatis from individuals in a sexually transmitted
disease core group exhibit frequent sequence variation in the major
outer membrane protein (ompl) gene. J Clin Invest; 94:458-463.
Byrne GI. 1976. Requirements for ingestion of Chlamydia psittaci by mouse
fibroblasts (L cells). Infect Immun; 14:645-651.
Byrne GI. 1978. Kinetics of phagocytosis of Chlamydia psittaci by mouse
fibroblasts (L cells): separation of the attachment and ingestion stages.
Infect Immun; 19:607-612.
Byrne GI, Moulder JW. 1978. Parasite-spedfied phagocytosis of Chlamydia
psittaci and Chlamydia trachomatis by L and HeLa cells. Infect Immun;
19: 598-606.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
162
Campbell LA, Kuo CC, Grayston JT. 1990. Structural and antigenic analysis
of Chlamydia pneumoniae. Infect Immun; 58: 93-97.
Carter MW, Al-Mahdawi SAH, Giles IG, Trehame JD, Ward ME, Clarke IN.
1991. Nucleotide sequence and taxonomic value of the major outer
membrane protein gene of Chlamydia pneumoniae IOL-207. J Gen
Microbiol; 137:465-475.
Cates W Jr, Wasserheit JN. 1991. Genital chlamydial infections:
Epidemiology and reproductive sequelae. Am J Obstet Gynecol; 164:
1771-1781.
Centers for Disease Control and Prevention. 1993. Recommendations for the
preventions and management of Chlamydia trachomatis infections.
Morbid Mortal Weekly Rep; 42:1-36.
Centers for Disease Control and Prevention. 1997. Chlamydia trachomatis
genital infections - United States, 1995. Morbid Mortal Weekly Rep;
46:193-198.
Centers for Disease Control and Prevention. 1998. Compendium of
measures to control Chlamydia psittaci infection among humans
(psittacosis) and pet birds (avian chlamydiosis). Morbid Mortal
Weekly Rep; 47:1-14.
Chen JC, Stephens RC. 1997. Chlamydia trachomatis glycosaminoglycan-
dependent and independent attachment to eukaryotic cells. Microb
Pathog; 22:23-30.
Chi EY, Kuo CC, Grayston JT. 1987. Unique ultrastructure in the elementary
body of Chlamydia sp. strain TWAR. J Bacteriol; 169:3757-3763.
Christiansen G, Ostergaard L, Birkelund S. 1997. Molecular biology of the
Chlamydia pneumoniae surface. Scand J Infect Dis Suppl; 104:5-10.
Chungue E, Soulier J, Philippon G, Wang SP. 1994. Immunotypes of
Chlamydia trachomatis isolated from genital tract specimens in Tahiti.
European Journal of Clinical Microbiology & Infectious Diseases; 13:
436-438.
Clarke IN, Ward ME, Lambden PR. 1988. Molecular cloning and sequence
analysis of a developmentally regulated cysteine-rich outer membrane
protein from Chlamydia trachomatis. Gene; 71:307-314.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
163
Clausen JD, Christiansen G, Holst HU, Birkelund S. 1997. Chlamydia
trachomatis utilizes the host cell microtubule network during early
events of infection. Mol Microbiol; 25:441-449.
Coles AM, Allan I, Pearce JH. 1990. The nucleotide and derived amino add
sequence of the omcB gene of Chlamydia trachomatis serovar E. Nucleic
A dds Res; 18: 6713.
Collett BA, Newhall WJ, Jersild RA Jr, Jones RB. 1989. Detection of surface-
exposed epitopes on Chlamydia trachomatis by immune electron
microscopy. J Gen Microbiol; 135: 85-94.
Danilition SL, Maclean IW, Peeling R, Winston S, Brunham RC. 1990. The
75-kilodalton protein of Chlamydia trachomatis: a member of the heat
shock protein 70 family? Infect Immun; 58:189-196.
Davis CH, Wyrick PB. 1997. Differences in the assodation of Chlamydia
trachomatis serovar E and serovar E2 with epithelial cells in vitro may
reflect biological differences in vivo. Infect Immun; 65:2914-2924.
Dean D. 1997. Chlamydia trachomatis Sexually Transmitted Diseases. In:
Diagnostic Pathology of Infectious Diseases. Stamford, CT: Appleton
and Lange; pp. 473-490.
Dean D. 1999a. Chlamydial Infections. In: Pathology of Infectious Diseases.
Connor DH, Chandler FW, Schwartz DA, Manz HJ, Lack EE, editors.
Stamford, CT: Appleton and Lange; pp. 473-497.
Dean D. 1999b. Trachoma. In: Pathology of Infectious Diseases. Connor
DH, Chandler FW, Schwartz DA, Manz HJ, Lack EE, editors.
Stamford, CT: Appleton and Lange; pp. 498-507.
Dean D. 2002. Pathogenesis of Chlamydial Ocular Infections. In: Duane's
Foundations of Clinical Ophthalmology. Tasman W, Jaeger EA, editors.
Philadelphia, PA: Lippincott Williams & Wilkins, vol. 2, chapter 77; pp.
1- 20.
Dean D, Millman K. 1997. Molecular and Mutation Trends Analyses of ompl
Alleles for Serovar E of Chlamydia trachomatis: Implications for the
Immunopathogenesis of Disease. J Clin Invest; 99:475-483.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
164
Dean D, Oudens EM, Padian N, Bolan G, Schachter J. 1995. Major outer
membrane protein variants of Chlamydia trachomatis are associated
with severe upper genital tract infections and histopathology in San
Francisco. J Infect Dis; 172:1013-1022.
Dean D, Patton M, Stephens RS. 1991. Direct sequence evaluation of the
major outer membrane protein gene variant regions of Chlamydia
trachomatis subtypes D', T, and L2'. Infect Immun; 59:1579-1582.
Dean D, Schachter J, Dawson C, Stephens RS. 1992. Comparison of the
major outer membrane protein sequence variant regions of B/Ba
isolates: A molecular epidemiologic approach to Chlamydia trachomatis
infections. J Infect Dis; 166: 383-392.
Dean D, Suchland RJ, Stamm WE. 2000. Evidence for long term cervical
persistence of Chlamydia trachomatis by ompl genotyping. J Infect Dis;
182:909-916.
de la Maza LM, Fiedler TJ, Carlson EJ, Markoff BA, Peterson EM. 1991.
Sequence diversity of the 60-kilodalton protein and of a putative 15-
kilodalton protein between the trachoma and lymphogranuloma
venereum biovars of Chlamydia trachomatis. Infect Immun; 59:1196-
1201.
Douglas AL, Saxena NK, Hatch TP. 1994. Enhancement of in vitro
transcription by addition of cloned, overexpressed major sigma factor
of Chlamydia psittaci 6BC. J Bacteriol; 176: 3033-3039. Erratum in: J
Bacteriol; 176:4196.
Engel JN, Ganem D. 1990. A polymerase chain reaction-based approach to
cloning sigma factors from eubacteria and its application to the
isolation of a sigma-70 homolog from Chlamydia trachomatis. J
Bacteriol; 172:2447-2455.
Everett KDE, Andersen AA, Plaunt M, Hatch TP. 1991. Cloning and
sequence analysis of the major outer membrane protein gene of
Chlamydia psittaci 6BC. Infect Immun; 59: 2853-2855.
Everett KDE, Bush RM, Andersen AA. 1999. Emended description of the
order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and
Simkaniaceae fam. nov., each containing one monotypic genus, revised
taxonomy of the family Chlamydiaceae, including a new genus and five
new species, and standards for the identification of organisms. Int J
Syst Bacteriol; 49: 415-440.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
165
Everett KDE, Bush RM, Andersen AA. 2000. Phylogenetic analysis of five
coding genes support the new chlamydial taxonomy. In: The
Proceedings of the Fourth Meeting of the European Society for
Chlamydia Research. Saikku P, editor. Helsinki, Finland: Universitas
Helsingiensis; pp. 5.
Everett KDE, Desiderio DM, Hatch TP. 1994. Characterization of lipoprotein
EnvA in Chlamydia psittaci 6BC. J Bacteriol; 176:6082-6087.
Everett KDE, Hatch TP. 1991. Sequence analysis and lipid modification of
the cysteine-rich envelope proteins of Chlamydia psittaci 6BC. J
Bacteriol; 173: 3821-3830.
Everett KDE, Hatch TP. 1995. Architecture of the cell envelope of Chlamydia
psittaci 6BC. J Bacteriol; 177: 877-882.
Fahr MJ, Douglas AL, Xia W, Hatch TP. 1995. Characterization of late gene
promoters of Chlamydia trachomatis. J Bacteriol; 177:4252-4260.
Fahr MJ, Sriprakash KS, Hatch TP. 1992. Convergent and overlapping
transcripts of the Chlamydia trachomatis 7,5-kb plasmid. Plasmid; 28:
247-257.
Farencena A, Comanducci M, Donati M, Ratti G, Cevenini R. 1997.
Characterization of a new isolate of Chlamydia trachomatis which lacks
the common plasmid and has properties of biovar trachoma. Infect
Immun; 65:2965-2969.
Fawaz FS, van Ooij C, Homola E, Mutka SC, Engel JN. 1997. Infection with
Chlamydia trachomatis alters the tyrosine phosphorylation an d /o r
localization of several host cell proteins including cortactin. Infect
Immun; 65: 5301-5308.
Fehlner-Gardiner C, Roshick C, Carlson JH, Hughes S, Belland RJ, Caldwell
HD, McClarty G. 2002. Molecular basis defining hum an Chlamydia
trachomatis tissue tropism. A possible role for tryptophan synthase. J
Biol Chem; 277: 26893-26903.
Feil E, Zhou J, Maynard Smith J, Spratt BG. 1996. A comparison of the
nucleotide sequence of the adk and recA genes of pathogenic and
commensal Neisseria species: Evidence for extensive interspecies
recombination within adk. J Mol Evol; 43: 631-640.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
166
Fielder TJ, Pal S, Peterson EM, de la Maza LM. 1991a. Sequence of the gene
encoding the major outer membrane protein of the mouse
pneumonitis biovar of Chlamydia trachomatis. Gene; 106:137-138.
Fielder TJ, Peterson EM, de la Maza LM. 1991b. Nucleotide sequence of
DNA encoding the major outer membrane protein of Chlamydia
trachomatis serovar L3. Gene; 101:159-60.
Fitch WM, Peterson EM, de la Maza LM. 1993. Phylogenetic analysis of the
outer-membrane-protein genes of Chlamydiae, and its implication for
vaccine development. Mol Biol Evol; 10: 892-913.
Fortenberry JD, Evans DL. 1989. Routine screening for genital Chlamydia
trachomatis in adolescent females. Sex Trans Dis; 16:168-172.
Friis RR. 1972. Interaction of L cells and Chlamydia psittaci: entry of the
parasite and host responses to its development. J Bacteriol; 110:706-
721.
Frost EH, Deslandes S, Bourgaux-Ramoisy D. 1993. Chlamydia trachomatis
serovars in 435 urogenital specimens typed by restriction endonuclease
analysis of amplified DNA. J Infect Dis; 168:497-501.
Frost EH, Deslandes S, Gendron D, Bourgauz-Ramoisly P, Bourgaux P. 1995.
Variation outside variable segments of the major outer membrane
protein distinguishes trachoma from urogenital isolates of the same
serovar of Chlamydia trachomatis. Genitourin Med; 71:18-23.
Fukushi H, Hirai K. 1992. Proposal of Chlamydia pecorum sp. nov. for
Chlamydia strains derived from ruminants. International J of
Systematic Bacteriology; 42:306-308.
Garrett AJ, Harrison MJ, Manire GP. 1974. A search for the bacterial
mucopeptide component, muramic add, in Chlamydia. J Gen
Microbiol; 80:315-318.
Gaydos CA, Bobo L, Welsh L, Hook EW 3rd , Vistidi R, Quinn TC. 1992.
Gene typing of Chlamydia trachomatis by polymerase chain reaction
and restriction endonudease digestion. Sex Trans Dis; 19: 303-8.
Geisler WM, Suchland RJ, W hittington WL, Stamm WE. 2003. The
relationship of serovar to clinical manifestations of urogenital
Chlamydia trachomatis infedion. Sex Trans Dis; 30:160-165.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
167
Geisler WM, Whittington WL, Suehland RJ, Stamm WE. 2002.
Epidemiology of anorectal chlamydial and gonococcal infections
among men having sex w ith men in Seattle: utilizing serovar and
auxotype strain typing. Sex Trans Dis; 29:189-195.
Gerbase AC, Rowley JT, Heymann DH, Berkley SF, Piot P. 1998. Global
prevalence and incidence estimates of selected curable STDs. Sex
Transm Infect; 74: S12-16.
Giannikopoulou P, Bini L, Simitsek PD, Pallini V, Vretou E. 1997. Two-
dimensional electrophoretic analysis of the protein family at 90 kDa of
abortifacient Chlamydia psittaci Electrophoresis; 18:2104-2108.
Gilbert SC, Plebanski M, Gupta S, Morris J, Cox M, Aidoo M, Kwiatkowski
D, Greenwood BM, Whittle HC, Hill AV. 1998. Association of
malaria parasite population structure, HLA, and immunological
antagonism. Science; 279:1173-1177.
Girjes A, Carrick F, Lavin M. 1994. Remarkable sequence relatedness in the
DNA encoding the major outer membrane protein of Chlamydia
psittaci (koala type I) and Chlamydia pneumoniae. Gene; 138:139-142.
Grassly NC, Harvey PH, Holmes EC. 1999. Population dynamics of HIV-1
inferred from gene sequences. Genetics; 151:427-438.
Grayston JT, Kuo CC, Campbell LA, Wang SP. 1989. Chlamydia pneumoniae
sp. nov. for Chlamydia sp. strain TWAR. Int J Syst Bacteriol; 39: 88-90.
Grayston JT, Wang SP. 1975. New knowledge of chlamydiae and the
diseases they cause. J Infect Dis; 132: 87-105.
Grim wood J, Olinger L, Stephens RS. 2001. Expression of Chlamydia
pneumoniae polymorphic membrane protein family genes. Infect
Immun; 69:2383-2389.
Grimwood J, Stephens RS. 1999. Computational analysis of the polymorphic
membrane protein superfamily of Chlamydia trachomatis and Chlamydia
pneumoniae. Microb Comp Genomics; 4:187-201.
Guttman DS, Dykhuizen DE. 1994. Clonal divergence in Escherichia coli as a
result of recombination, not mutation. Science; 266:1380-1383.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
168
Hackstadt T. 1999. Cell Biology. In: Chlamydia: Intracellular Biology,
Pathogenesis, and Immunity. Stephens RS, editor. Washington, D.C.:
ASM Press; p.101-138.
Hackstadt T, Baehr W, Yuan Y. 1991. Chlamydia trachomatis developmentally
regulated protein is homologous to eukaryotic histone H I. Proc Natl
Acad Sci USA; 88: 3937-3941.
Hackstadt T, Caldwell HD. 1985. Effect of proteolytic cleavage of surface-
exposed proteins on infectivity of Chlamydia trachomatis. Infect
Immun; 48:546-551.
Hackstadt T, Fischer ER, Scidmore MA, Rockey DD, Heinzen RA. 1997.
Origins and functions of the chlamydial inclusion. Trends Microbiol;
5:288-293.
Hackstadt T, Scidmore MA, Rockey DD. 1995. Lipid metabolism in
Chlamydia trachomatis-miected cells: directed trafficking of Golgi-
derived sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci
USA; 92:4877-4881.
Hatch TP. 1975a. Competition between Chlamydia psittaci and L cells for host
isoleudne pools: a limiting factor in chlamydial multiplication. Infect
Immun; 12:211-220.
Hatch TP. 1975b. Utilization of L-cell nucleoside triphosphates by Chlamydia
psittaci for ribonucleic acid synthesis. J Bacteriol; 122:393-400.
Hatch TP. 1988. Metabolism of Chlamydia. In: Microbiology of Chlamydia.
Barron AL, editor. Boca Raton, Fla: CRC Press; pp. 97-109.
Hatch TP. 1999. Developmental biology. In: Chlamydia: Intracellular
Biology, Pathogenesis, and Immunity. Stephens RS, editor.
Washington, D.C.: ASM Press; pp. 29-68.
Hatch TP, Al-Hossainy E, Silverman JA. 1982. Adenine nucleotide and
lysine transport in Chlamydia psittaci. J Bacteriol; 150: 662-670.
Hatch TP, Allan I, Pearce JH. 1984. Structural and polypeptide differences
between envelopes of infective and reproductive life cycle forms of
Chlamydia spp. J Bacteriol; 157:13-20.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
169
Hatch TP, Miceli M, Sublett JE. 1986. Synthesis of disulfide-bonded outer
membrane proteins during the developmental cycle of Chlamydia
psittaci and Chlamydia trachomatis. J Bacteriol; 165: 379-385.
Hatch TP, Vance DW Jr, Al-Hossainy E. 1981. Attachment of Chlamydia
psittaci to formaldehyde-fixed and unfixed L cells. J Gen Microbiol;
125:273-283.
Hayes LJ, Bailey RL, Mabey DC, Clarke IN, Pickett MA, Watt PJ, Ward ME.
1992. Genotyping of Chlamydia trachomatis from a trachoma-endemic
village in the Gambia by a nested polymerase chain reaction:
identification of strain variants. J Infect Dis; 166:1173-1177.
Hayes LJ, Clarke IN. 1990. Nucleotide sequence of the major outer
membrane protein gene of Chlamydia trachomatis strain A/SA1/OT.
Nucleic Acids Res; 18: 6136.
Hayes LJ, Pecharatana S, Bailey RL, Ham pton TJ, Pickett MA, Mabey DC,
Watt PJ, W ard ME. 1995. Extent and kinetics of genetic change in the
ompl gene of Chlamydia trachomatis in two villages with endemic
trachoma. J Infect Dis; 172:268-272.
Hayes LJ, Pickett MA, Conlan JW, Ferris S, Everson JS, Ward ME, Clarke IN.
1990. The major outer-membrane proteins of Chlamydia trachomatis
serovars A and B: intra-serovar amino acid changes do not alter
specificities of serovar- and C subspecies-reactive antibody-binding
domains. J Gen Microbiol; 136:1559-1566.
Hayes LJ, Yearsley P, Treharne JD, Ballard RA, Fehler GH, Ward ME. 1994.
Evidence for naturally occurring recombination in the gene encoding
the major outer membrane protein of lymphogranuloma venereum
isolates of Chlamydia trachomatis. Infect Immun; 62:5659-5663.
Heinzen RA, Scidmore MA, Rockey DD, Hackstadt T. 1996. Differential
interaction with endocytic and exocytic pathways distinguish
parasitophorous vacuoles of Coxiella burnetii and Chlamydia
trachomatis. Infect Immun; 64: 796-809.
Herring AJ, Tan TW, Baxter S, Inglis NF, Dunbar S. 1989. Sequence analysis
of the major outer membrane protein gene of an ovine abortion strain
of Chlamydia psittaci. FEMS Microbiol Lett; 65:153-158.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
170
Heuer D, Brinkmann V, Meyer TF, Szczepek AJ. 2003. Expression and
translocation of chlamydial protease during acute and persistent
infection of the epithelial HEp-2 cells with Chlamydophila (Chlamydia)
pneumoniae. Cell Microbiol; 5:315-322.
Hill AV, Jepson A, Plebanski M, Gilbert SC. 1997. Genetic analysis of host-
parasite co-evolution in hum an malaria. In Series B: Biological
Sciences. London: Philosophical Trans Roy Soc London; pp. 1317-
1325.
Hillis SD, Nakashima AK, Marchbanks P, Addiss, DG, Davis J. 1994. Risk
factors for recurrent Chlamydia trachomatis infections in women. Sex
Trans Dis; 21: S137-138.
Hintz NJ, Ennis DG, Liu WF, Larsen SH. 1995. The recA gene of Chlamydia
trachomatis: Cloning, sequence, and characterization in Escherichia coli.
FEMS Microbiol Lett; 127:175-180.
Hodgkin PD. 1997. An antigen valence theory to explain the evolution and
organization of the humoral immune response. Immunol Cell Biol;
75: 604-618.
Hodinka RL, Davis CH, Choong J, Wyrick PB. 1988. Ultrastructural study of
endocytosis of Chlamydia trachomatis by McCoy cells. Infect Immun;
56:1456-1463.
Hodinka RL, Wyrick PB. 1986. Ultrastructural study of mode of entry of
Chlamydia psittaci into L-929 cells. Infect Immun; 54:855-863.
Holmes EC, Ur win R, Maiden MC. 1999. The influence of recombination on
the population structure and evolution of the hum an pathogen Neisseria
meningitidis. Mol Biol Evol; 16: 741-749.
Horn M, Wagner M, Muller B C D , Schmid EN, Fritsche TR, Schleifer B C H ,
Michel R. 2000. Neochlamydia hartmannellae gen. nov., sp. nov.
(Parachlamydiaceae), an endoparasite of the amoeba Hartmannella
vermiformis. Microbiology; 146:1231-1239.
Hsia RC, Pannekoek Y, Ingerowski E, Bavoil PM. 1997. Type III secretion
genes identify a putative virulence locus of Chlamydia. Mol Microbiol;
25:351-359.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
171
Hudson R. 1994. How can low levels of DNA sequence variation in regions
of the Drosophila genome with low recombination rates be explained?
Proc Natl Acad Sd USA; 91: 6815-6818.
Ingalls RR, Rice PA, Qureshi N, Takayama K, Lin JS, Golenbock DT. 1995.
The inflammatory cytokine response to Chlamydia trachomatis infection
is endotoxin mediated. Infect Immun; 63:3125-3130.
Ishizaki M, Allen JE, Beatty PR, Stephens RS. 1992. Immune spetifitity of
murine T-cell lines to the major outer membrane protein of Chlamydia
trachomatis. Infect Immun; 60: 3714-3718.
Jakobsen IB, Easteal S. 1996. A program for calculating and displaying
compatibility matrices as an aid in determining reticulate evolution in
molecular sequences. CABIOS; 12:291-295.
Jenkin HM. 1960. Preparation and properties of cell walls of the agent of
meningopneumonitis. J Bacteriol; 80:639-647.
Johnson RE, Green TA, Schachter J, Jones RB, Hook 3rd EW, Black CM,
Martin DH, St Louis ME, Stamm WE. 2000. Evaluation of nudeic ad d
amplification tests as reference tests for Chlamydia trachomatis infections
in asymptomatic men. J Clin Microbiol; 38: 4382-4386.
Jones RB, Mammel JB, Shepard MK, Fisher RR. 1986. Recovery of Chlamydia
trachomatis from the endometrium of women at risk for chlamydial
infections. Am J Obstet Gynecol; 155:35-39.
Jones RB, Williams JA, van der Pol B. 1998. Competitive growth of serovars
E and F combined in mixed tissue culture infections. In: Chlamydia
Infections, Proceedings of the Ninth International Symposium on
Hum an Chlamydial Infection. Stephens RS, Byrne GI, Christiansen G,
Clarke IN, Grayston JT, Rank RG, Ridgway GL, Saikku P, Schachter J,
Stamm WE, editors. San Frandsco, CA: International Chlamydia
Symposium; pp. 523-526.
Jordan IK, Makarova KS, Wolf YI, Koonin EV. 2000. Gene conversions in
genes encoding outer-membrane proteins in H. pylori and C.
pneumoniae. Trends in Genetics; 17: 7-10.
Jurstrand M, Falk L, Fredlund H, Lindberg M, Olcen P, Andersson S, Persson
K, Albert J, Backman A. 2001. Characterization of Chlamydia
trachomatis ompl genotypes among sexually transmitted disease
patients in Sweden. J Clin Microbiol; 39:3915-3919.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
172
Kahane S, Gonen R, Sayada C, Elion J, Friedman MG. 1993. Description and
partial characterization of a new Chlamydia-like microorganism.
FEMS Microbiol Lett; 126:203-208.
Kalman S, Mitchell W, Marathe R, Lammel C, Fan J, Hyman RW, Olinger L,
Grimwood J, Davis RW, Stephens RS. 1999. Comparative genomes of
Chlamydia pneumoniae and C. trachomatis. Nat Genet; 21: 385-389.
Kaltenbbck B, Schmeer N, Schneider R. 1997. Evidence for numerous ompl
alleles of porcine Chlamydia trachomatis and novel chlamydial species
obtained by PCR. J Clin Microbiol; 35:1835-1841.
Karam GH, Martin DH, Flotte TR, Bonnarens FO, Joseph JR, Mroczkowski
TF, Johnson WD. 1986. Asymptomatic Chlamydia trachomatis
infections among sexually active men. J Infect Dis; 154: 900-903.
Karunakaran KP, Noguchi Y, Read TD, Cherkasov A, Kwee J, Shen C,
Nelson CC, Branham RC. 2003. Molecular analysis of the multiple
GroEL proteins of Chlamydiae. J Bacteriol; 185:1958-1966.
Kaul R, Hoang A, Yau P, Bradbury EM, Wenman WM. 1997. The
chlamydial EUO gene encodes a histone Hl-specific protease. J
Bacteriol; 179:5928-5934.
Kawa DE, Stephens RS. 2002. Antigenic topology of chlamydial PorB
protein and identification of targets for immune neutralization of
infectivity. J Immunol; 168: 5184-5191.
Kent GP, Harrison HR, Berman SM. 1988. Screening for Chlamydia
trachomatis infection in a sexually transmitted disease clinic:
comparison of diagnostic tests with clinical and historical risk factors.
Sex Trans Dis; 15: 51-57.
Kimura M. 1980. A simple method for estimating evolutionary rate of base
substitutions through comparative studies of nucleotide sequences. J
Mol Evol; 16:111-120.
Koehler JE, Burgess RR, Thompson NE, Stephens RS. 1990. Chlamydia
trachomatis RNA polymerase major sigma subunit. Sequence and
structural comparison of conserved and unique regions with
Escherichia coli sigma 70 and Bacillus subtilis sigma 43. J Biol Chem; 265:
13206-13214. Erratum in: J Biol Chem; 265: 21390.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
173
Kubo A, Stephens RS. 2000. Characterization and functional analysis of
PorB, a Chlamydia porin and neutralizing target. Mol Microbiol;
38:772-780.
Kumar S, Tamura K, Jakobsen IB, Nei M. 2001. MEGA2: molecular
evolutionary genetics analysis software. Bioinformatics; 17:1244-1245.
Kuo CC, Chen HH, Wang SP, Grayston JT. 1986. Identification of a new
group of Chlamydia psittaci strains called TWAR. J Clin Microbiol; 24:
1034-1037.
Kuo CC, Chi EY. 1987. Ultrastructural study of Chlamydia trachomatis
surface antigens by immunogold staining with monoclonal
antibodies. Infect Immun; 55:1324-1328.
Kuo CC, Grayston T. 1976. Interaction of Chlamydia trachomatis organisms
and HeLa 229 cells. Infect Immun; 13:1103-1109.
Lambden PR, Everson JS, W ard ME, Q arke IN. 1990. Sulfur-rich proteins of
Chlamydia trachomatis: developmentally regulated transcription of
polycistronic mRNA from tandem promoters. Gene; 87:105-112.
Lampe MF, Suchland RJ, Stamm WE. 1993. Nucleotide sequence of the
variable domains within the major outer membrane protein gene from
serovariants of Chlamydia trachomatis. Infect Immun; 61:213-9.
Lampe MF, Wong KG, Stamm WE. 1995. Sequence conservation in the
major outer membrane protein gene among Chlamydia trachomatis
strains isolated from the upper and lower urogenital tract. J Infect
Dis; 172: 589-592.
Lan J, Meijer CJ, van den Hoek AR, Ossewaarde JM, Walboomers JM, van
den Brule AJ. 1995. Genotyping of Chlamydia trachomatis serovars
derived from heterosexual partners and a detailed genomic analysis of
serovar F. Genitourin Med; 71: 299-303.
Lawn AM, Blyth WA, Taveme J. 1973. Interactions of TRIC agents with
macrophages and BHK-21 cells observed by electron microscopy. J
Hyg; 71: 515-528.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
174
Lin JS, Donegan SP, Heeren TC, Greenberg M, Flaherty EE, Haivanis R, Su
XH, Dean D, Newhall WJ, Knapp JS, Sarafian SK, Rice RJ, Morse SA,
Rice PA. 1998. Transmission of Chlamydia trachomatis and Neisseria
gonorrhoeae among men with urethritis and their female sex partners.
J Infect Dis; 178:1707-1712.
Longbottom D, Russell M, Dunbar SM, Jones GE, Herring AJ. 1998.
Molecular cloning and characterization of the genes coding for the
highly immunogenic cluster of 90-kilodalton envelope proteins from
the Chlamydia psittaci subtype that causes abortion in sheep. Infect
Immun; 66:1317-24.
Longbottom D, Russell M, Jones GE, Lainson FA, Herring AJ. 1996. ^
Identification of a multigene family coding for the 90 kDa proteins of
the ovine abortion subtype of Chlamydia psittaci. FEMS Microbiol Lett;
142:277-281.
Lundemose AG, Birkelund S, Larsen PM, Fey SJ, Christiansen G. 1990.
Characterization and identification of early proteins in Chlamydia
trachomatis serovar L2 by two-dimensional gel electrophoresis. Infect
Immun; 58:2478-2486.
Lycke I, Lowhagen GB, Hallhagen G, Johannisson G, Ramstedt K. 1980. The
risk of transmission of genital C. trachomatis infections is less than that
of genital Neisseria gonorrhoeae infection. Sex Trans Dis; 7: 6-10.
Magder LS, Harrison HR, Ehret JM, Anderson TS, Judson FN. 1988. Factors
related to genital Chlamydia trachomatis and its diagnosis by culture in
a sexually transmitted disease clinic. Am J Epidmiol; 128:298-308.
Manly BFJ. 1991. Randomization, bootstrap and Monte Carlo methods in
biology. 2nd ed. London: Chapman and Hall; pp. 1-399.
Marin BVO, Gomez CA, Hearst N. 1993. Multiple heterosexual partners and
condom use among Hispanics and Non-Hispanic Whites. Fam Plann
Perspect; 25:170-174.
Marrazzo JM, White CL, Krekeler B, Celum CL, Lafferty WE, Stamm WE,
Handsfield HH. 1997. Community-based urine screening for Chlamydia
trachomatis with a ligase chain reaction assay. Ann Intern Med; 127:
796-803.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
175
Mathews SA, Sriprakash KS. 1994. The RNA polymerase of Chlamydia
trachomatis has a flexible sequence requirement at the -10 and -35
boxes of its promoters. J Bacteriol; 176:3785-3789.
Matsumoto A. 1982a. Surface projections of Chlamydia psittaci elementary
bodies as revealed by freeze-deep-etching. J Bacteriol; 151:1040-1042.
Matsumoto A. 1982b. Electron microscopic observations of surface
projections on Chlamydia psittaci reticulate bodies. J Bacteriol; 150:358-
364.
Maynard Smith J. 1992. Analyzing the mosaic structure of genes. JM ol
Evol; 34:126-129.
McClarty G. 1994. Chlamydiae and the biochemistry of intracellular
parasitism. Trends Microbiol; 2:157-164.
McClarty G. 1999. Metabolism. In: Chlamydia: Intracellular Biology,
Pathogenesis, and Immunity. Stephens RS, editor. Washington, D.C.:
ASM Press; pp. 69-100.
McClarty G, Tipples G. 1991. In situ studies on incorporation of nucleic add
precursors into Chlamydia trachomatis DNA. J Bacteriol; 173:4922-4931.
Meijer A, Ossewaarde JM. 1998. Broad range Chlamydia PCR detects
previously unrecognized Chlamydia sequences: a new genus in the
family Chlamydiaceae? In: Chlamydia Infections. Proceedings of the
Ninth International Symposium on Hum an Chlamydial Infection.
Stephens RS, Byrne GI, Christiansen G, Clarke IN, Grayston JT, Rank
RG, Ridgway GL, Saikku P, Schachter J, Stamm WE, editors. San
Frandsco, CA: International Chlamydia Symposium; pp. 523-526.
Meijer A, Roholl PJM, Ossewaarde JM. 2000. Use of the broad range PCR
assay for the identification and dassification of bacteria in the order
Chlamydiales. In: The Proceedings of the Fourth Meeting of the
European Society for Chlamydia Research. Saikku P, editor. Helsinki,
Finland: Universitas Helsingiensis; p 9.
Melgosa MP, Kuo CC, Campbell LA. 1993. Sequence analysis of the major
outer membrane protein gene of Chlamdia pneumoniae. Infect Immun;
59:2195-2199.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
176
Millman KL, Tavare S, Dean D. 2001. Recombination in the ompA gene but
not the omcB gene of Chlamydia contributes to serovar-spedfic
differences in tissue tropism, immune surveillance, and persistence of
the organism. J Bacteriol; 183: 5997-6008.
Moncan T, Eb F, Orfila J. 1990. Monoclonal antibodies in serovar
determination of 53 Chlamydia trachomatis isolates from Amiens,
France. Res Microbiol; 141:695-701.
Montfort WR, Weichsel A. 1997. Thymidylate synthase: structure,
inhibition, and strained conformations during catalysis. Pharmacol
Ther; 76: 29-43.
Morre SA, Munk C, Persson K, Kruger-Kjaer S, van Dijk R, Meijer CJ, van
Den Brule AJ. 2002. Comparison of three commerdally available
peptide-based immunoglobulin G (IgG) and IgA assays to
microimmunofluorescence assay for detection of Chlamydia trachomatis
antibodies. J Clin Microbiol; 40:584-587.
Morre SA, Ossewaarde JM, Lan J, van Doomum GJ, Walboomers JM,
MacLaren DM, Meijer CJ, van den Brule AJ. 1998. Serotyping and
genotyping of genital Chlamydia trachomatis isolates reveal variants of
serovars Ba, G, and J as confirmed by ompl nucleotide sequence
analysis. J Clin Microbiol; 36:345-351.
Morre SA, Rozendaal L, van Valkengoed IG, Boeke AJ, van Voorst Vader PC,
Schirm J, de Blok S, van Den Hoek JA, van Doornum GJ, Meijer CJ,
van Den Brule AJ. 2000. Urogenital Chlamydia trachomatis serovars in
men and women with a symptomatic or asymptomatic infection: an
assodation with clinical manifestations? J Clin Microbiol; 38:2292-
2296.
Morrison RP, Belland RP, Lyng K, Caldwell FED. 1989. Chlamydial disease
pathogenesis. The 57-K-kD chlamydial hypersensitivity is a stress
response protein. J Exp Med; 169:663-675.
Morrison RP, Manning DS, Caldwell FID. 1992. Immunology of Chlamydia
trachomatis infections: Immunoprotective and immimopathogenetic
responses. In: Advances in Host Defense Mechanisms, Vol 8, Sexually
transmitted diseases. Gallin JI, Fauci AS, Quinn TC, editors. New
York, NY: Raven Press; pp. 57-84.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
177
Morrison RP, Su H, Lyng K, Yuan Y. 1990. The Chlamydia trachomatis hyp
operon is homologous to the groE stress response operon of
Escherichia coli. Infect Immun; 58:2701-2705.
Moulder JW. 1970. Glucose metabolism of L cells before and after infection
with Chlamydia psittaci. J Bacteriol; 104:1189-1196.
Moulder JW. 1991. Interaction of chlamydiae and host cell in vitro.
Microbiol Rev; 55:143-190.
Moulder JW, Novosel DL, Officer JE. 1963. Inhibition of the growth of
agents of the psittacosis group by D-cycloserine and its specific
reversal by D-alanine. J Bacteriol; 85: 707-711.
Mygind PH, Christiansen G, Birkelund S. 1998. Topological analysis of
Chlamydia trachomatis L2 outer membrane protein 2. J Bacteriol; 180:
5784-5787.
Mygind PH, Christiansen G, Roepstorff P, Birkelund S. 2000. Membrane
proteins PmpG and PmpH are major constituents of Chlamydia
trachomatis L2 outer membrane complex. FEMS Microbiol Lett;
186:163-169.
Nei M, Gojobori T. 1986. Simple methods for estimating the numbers of
synonymous and nonsynonymous nucleotide substitutions. Mol Biol
Evol; 3:418-426.
Newhall WJ. 1987. Biosynthesis and disulfide cross-linking of outer
membrane components during the growth cycle of Chlamydia
trachomatis. Infect Immun; 55:162-168.
Newhall WJ. 1988. Macromolecular and antigenic composition of
chlamydiae. In: Microbiology of chlamydiae. Barron AL, editor. Boca
Raton, Fla: CRC Press Inc; pp. 47-70.
Newhall WJ, Jones RB. 1983. Disulfide-linked oligomers of the major outer
membrane protein of chlamydiae. J Bacteriol; 154: 998-1001.
Nigg C. 1942. An unidentified virus which produces pneumonia and
systemic infection in mice. Science; 95:49-50.
Nurminen M, Leinonen M, Saikku P, Makela PH. 1983. The genus-spedfic
antigen of Chlamydia: resemblance to the lipopolysaccharide of enteric
bacteria. Sdence; 220:1279-1281.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
178
Ojrius DM, Degani H, Mispelter J, Dautiy-Varsat A. 1998. Enhancement of
ATP levels and glucose metabolism during an infection by Chlamydia.
NMR studies of living cells. J Biol Chem; 273: 7052-7058.
Oriel D, Ridgway GL. 1982. Genital Infection by Chlamydia trachomatis. E.
Arnold, editor. London, United Kingdom.
Ossewaarde JM, Rieffe M, de Vries A, Derksen-Nawrocki RP, Hooft HJ, van
Doornum JG, van Loon AM. 1994. Comparison of two panels of
monoclonal antibodies for determination of Chlamydia trachomatis
serovars. J Clin Microbiol; 32:2968-2974.
Pabst KM, Reichart CA, Knud-Hansen CR, Wasserheit JN, Quinn TC, Shah
K, Dallabetta G, Hook EW 3rd. 1992. Disease prevalence among women
attending a sexually transmitted disease clinic varies with reason for
visit. Sex Transm Dis; 19: 88-91.
Pal S, Theodor I, Peterson EM, de la Maza LM. 1997a. Immunization with
an acellular vaccine consisting of the outer membrane complex of
Chlamydia trachomatis induces protection against a genital challenge.
Infect Immun; 65:3361-3369.
Pal S, Theodor I, Peterson EM, de la Maza LM. 1997b. Monoclonal
immunoglobulin A antibody to the major outer membrane protein of
the Chlamydia trachomatis mouse pneumonitis biovar protects mice
against a chlamydial genital challenge. Vaccine; 15:575-582.
Patton DL, Kuo CC. 1989. Histopathology of Chlamydia trachomatis salpingitis
after primary and repeated reinfections in the monkey subcutaneous
pocket model. J Reprod Fertil; 85:647-656.
Patton DL, Sweeney YT, Kuo CC. 1994. Demonstration of delayed
hypersensitivity in Chlamydia trachomatis salpingitis in monkeys: a
pathogenic mechanism of tubal damage. J Infect Dis; 169: 680-683.
Pedersen LN, Kjaer HO, Moller JK, Orntoft TF, Ostergaard L. 2000. High-
resolution genotyping of Chlamydia trachomatis from recurrent
urogenital infections. J Clin Microbiol; 38:3068-3071.
Peeling RW, Branham RC. 1991. Neutralization of Chlamydia trachomatis:
kinetics and stoichiometry. Infect Immun; 59:2624-2630.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
179
Perara E, Ganem D, Engel JN. 1992. A developmentally regulated
chlamydial gene with apparent homology to eukaryotic histone HI.
Proc Natl Acad Sd USA; 89:2125-2129.
Perkins HR, Allison AC. 1963. Cell-wall constituents of rickettsiae and
psittacosis-lymphogranuloma organisms. J Gen Microbiol; 30:469-
480.
Peterson EM, Markoff BA, de la Maza LM. 1990. The major outer membrane
protein nudeotide sequence of Chlamydia trachomatis, serovar E.
Nudeic Acids Res; 18:3414.
Petersen LB, Birkelund S, Christiansen G. 1996. Purification of recombinant
Chlamydia trachomatis histone Hl-like protein Hc2, and comparative
functional analysis of H cl and Hc2. Mol Microbiol; 20:295-311.
Pickett MA, Everson JS, Clarke IN. 1988. Chlamydia psittaci ewe abortion
agent: Complete nudeotide sequence of the major outer membrane
protein gene. FEMS Microbiol. Lett; 55: 229-234.
Pickett MA, Ward ME, Clarke IN. 1987. Complete nucleotide sequence of
the major outer membrane protein gene from Chlamydia trachomatis
serovar LI. FEMS Microbiol Lett; 42:185-190.
Plaunt MR, Hatch TP. 1988. Protein synthesis early in the developmental
cyde of Chlamydia psittaci. Infect Immun; 56:3021-3025.
Poole E, Lamont I. 1992. Chlamydia trachomatis serovar differentiation by
direct sequence analysis of the variable segment 4 region of the major
outer membrane protein gene. Infect Immun; 60:1089-1094.
Prain CJ, Pearce JH. 1989. Ultrastructural studies on the intracellular fate of
Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and
Chlamydia trachomatis (strain lymphogranuloma venereum 434):
modulation of intracellular events and relationship with endocytic
mechanism. J Gen Microbiol; 135:2107-2123.
Quinn TC, Gaydos C, Welsh L, Bobo L, Rompalo A, Hook 3rd E, Visddi R.
1994. Reassessment of Chlamydia trachomatis transmission by
polymerase chain reaction among 460 sexual partners. In:
Chlamydial infections. Orfila J, Byrne GI, Chernesky MA, Grayston
JT, Jones RB, Ridgway GL, Saikku P, Schachter J, Stamm WE, Stephens
RS, editors. Bologna, Italy: Sodeta Editrice Esculapio; pp. 21-24.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
180
Ramstedt K, Forssman L, Giesecke J, Johannisson G. 1991. Epidemiologic
characteristics of two different populations of women with C.
trachomatis infection and their male partners. Sex Trans Dis; 18:205-
210.
Raulston JE, Davis CH, Schmiel DH, Morgan MW, Wyrick PB. 1993.
Molecular characterization and outer membrane association of a
Chlamydia trachomatis protein related to the hsp70 family of proteins. J
Biol Chem; 268:23139-23147. Erratum in: J Biol Chem; 269:10184.
Raulston JE, Paul TR, Knight ST, Wyrick PB. 1998. Localization of Chlamydia
trachomatis heat shock proteins 60 and 70 during infection of a hum an
endometrial epithelial cell line in vitro. Infect Immun; 66:2323-2329.
Read TD, Branham RC, Shen C, Gill SR, Heidelberg JF, White O, Hickey EK,
Peterson J, Utterback T, Berry K, Bass S, Linher K, Weidman J, Khouri
H, Craven B, Bowman C, Dodson R, Gwinn M, Nelson W, DeBoy R,
Kolonay J, McClarty G, Salzberg SL, Eisen J, Fraser CM. 2000.
Genome sequences of Chlamydia trachomatis MoPn and Chlamydia
pneumoniae AR39. Nucleic Acids Res; 28:1397-1406.
Reichard P. 1993. From RNA to DNA, why so many ribonucleotide
reductases? Science; 260:1773-1777.
Reichard P. 1997. The evolution of ribonucleotide reduction. Trends
Biochem Sci; 22: 81-85.
Reynolds DJ, Pearce JH. 1990. Characterization of the cytochalasin D-
resistant (pinocytic) mechanisms of endocy tosis utilized by
chlamydiae. Infect Immun; 58: 3208-3216.
Ricci S, Cevenini R, Cosco E, Comanducci M, Ratti G, Scarlato V. 1993.
Transcriptional analysis of the Chlamydia trachomatis plasmid pCT
identifies temporally regulated transcripts, anti-sense RNA and sigma
70-selected promoters. Mol Gen Genet; 237:318-326.
Ricci S, Ratti G, Scarlato V. 1995. Transcriptional regulation in the Chlamydia
trachomatis pCT plasmid. Gene; 154: 93-98.
Ridderhof JC, Barnes RC. 1989. Fusion of inclusions following
superinfection of HeLa cells by two serovars of Chlamydia trachomatis.
Infect Immun; 57:3189-3193.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
181
Roberts MS, Cohan FM. 1993. The effect of DNA sequence divergence on
sexual isolation in Bacillus. Genetics; 134:401-408.
Rockey DD, Heinzen RA, Hackstadt T. 1995. Cloning and characterization
of a Chlamydia psittaci gene coding for a protein localized in the
inclusion membrane of infected cells. Mol Microbiol; 15: 617-626.
Rockey DD, Rosquist JL. 1994. Protein antigens of Chlamydia psittaci present
in infected cells but not detected in the infectious elementary body.
Infect Immun; 62:106-112.
Rothman KJ, Greenland S. 1998. Precision and validity of studies. In:
Modern Epidemiology. Philadelphia: Lippincott-Raven; pp. 127-132.
Rurangirwa FR, Dilbeck PM, Crawford TB, McGuire TC, McElwain TF. 1999.
Analysis of the 16S rRNA gene of micro-organism WSU 86-1044 from
an aborted bovine foetus reveals that it is a member of the order
Chlamydiales: proposal of Waddliaceae fam. nov., Waddlia chondrophila
gen. nov., sp. nov. Int J Syst Bacteriol; 49: 577-581.
Saikku P. 1999. Epidemiology of Chlamydia pneumoniae in atherosclerosis.
Am. Heart J; 138: S500-S503.
Saikku P, Wang SP. 1987. Chlamydia trachomatis immunotypes in Finland.
ACTA Pathol Microbiol Immunol Scand; 95:131-134.
Saikku P, Wang SP, Kleemola M, Brander E, Rusanen E, Grayston JT. 1985.
An epidemic of mild pneumonia due to an unusual strain of
Chlamydia psittaci. J Infect Dis; 151: 832-839.
Saitou N, Nei M. 1987. The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol Biol Evol; 4: 406-425.
Sawyer SA. 1989. Statistical tests for detecting gene conversion. Mol Biol
Evol; 6: 526-538.
Sayada C, Denamur E, Elion J. 1992. Complete sequence of the major outer
membrane protein-encoding gene of Chlamydia trachomatis serovar Da.
Gene; 120:129-130.
Sayada C, Vretou E, Orfila J, Elion J, Denamur E. 1995. Heterogeneity
within the first constant segment of the major outer membrane protein
gene in Chlamydia trachomatis serovar D /D a distinguishes 2 lineages.
C R Acad Sd HI; 318: 943-949.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
182
Schachter J. 1999. Infection and Disease Epidemiology. In: Chlamydia:
Intracellular Biology, Pathogenesis, and Immunity. Stephens RS,
editor. Washington, D.C.: ASM Press; pp. 139-170.
Schachter J, Grayston JT. 1998. Epidemiology of hum an chlamydial
infections. In: Chlamydial Infections. Proceedings of the Ninth
International Symposium on Hum an Chlamydial Infection. Stephens
RS, Byrne GI, Christiansen G, Clarke IN, Grayston JT, Rank RG,
Ridgway GI, Saikku P, Schachter J, Stamm WE, editors. San Francisco,
CA: International Chlamydia Symposium; pp. 3-10.
Schachter J, Stoner E, Moncada J. 1983. Screening for chlamydial infections
in women attending family planning clinics. West J Med; 138:375-
379.
Schechter EM. 1966. Synthesis of nucleic acid and protein in L cells infected
with the agent of meningopneumonitis. J Bacteriol; 91: 2069-2080.
Schmiel DH, Wyrick PB. 1994. Another putative heat-shock gene and
aminoacyl-tRNA synthetase gene are located upstream from the grpE-
like and dnaK-like genes in Chlamydia trachomatis. Gene; 145:57-63.
Scidmore MA, Fischer ER, Hackstadt T. 1996. Sphingolipids and
glycoproteins are differentially trafficked to the Chlamydia trachomatis
inclusion. J Cell Biol; 134:363-374.
Seidman SN, Mosher WD, Aral SO. 1992. Women with multiple sexual
partners: United States, 1988. Am J Public Health; 82:1388-1394.
Selander RK, Caugant DA, Ochman H, Musser JM, Gilmour MN, W hittam
IS. 1986. Methods of multilocus enzyme electrophoresis for bacterial
population genetics and systematics. Appl Environ Microbiol; 51: 873-
884.
Shaw AC, Christiansen G, Roepstorff P, Birkelund S. 2000. Genetic
differences in the Chlamydia trachomatis tryptophan synthase alpha-
subunit can explain variations in serovar pathogenesis. Microbes
Infect; 2: 581-592.
Shaw AC, Vandahl BB, Larsen MR, Roepstorff P, Gevaert K,
Vandekerckhove J, Christiansen G, Birkelund S. 2002.
Characterization of a secreted Chlamydia protease. Cell Microbiol; 4:
411-424.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
183
Shirai M, Hirakawa H, Kimoto M, Tabucbi M, Kishi F, Chichi K, Shiba T, Ishii
K, Hattori M, Kuhara S, Nakazawa T. 2000. Comparison of whole
genome sequences of Chlamydia pneumoniae J138 from Japan and
CWL029 from USA. Nucleic A dds Res; 28:2311-2314.
Siepel AC, Halpern AL, Macken C, Korber BTM. 1995. A computer program
designed to screen rapidly for HTV type 1 intersubtype recombinant
sequences. AIDS Res Hum Retrovir; 11:1413-1416.
Smith TF, Weed LA, Pettersen GR, O'Brien PC. 1978. A comparison of
genital infections caused by Chlamydia trachomatis and by Neisseria
gonorrhoeae. Am J Clin Pathol; 70:333-336.
Sneddon JM, Wenman WM. 1985. The effect of ions on the adhesion and
internalization of Chlamydia trachomatis by HeLa cells. Can J
Microbiol; 31:371-374.
Spratt BG, Bowler LD, Zhang QY, Zhou J, Smith JM. 1992. Role of
interspedes transfer of chromosomal genes in the evolution of penicillin
resistance in pathogenic and commensal Neisseria spedes. J Mol Evol;
34:115-125.
Sriram S, Mitchell W, Stratton C. 1998. Multiple sclerosis assodated with
Chlamydia pneumoniae infedion of the CNS. Neurology; 50: 571-572.
Stagg A J, Elsley WA, Pickett MA, Ward ME, Knight SC. 1993. Primary
hum an T-cell responses to the major outer membrane protein of
Chlamydia trachomatis. Immunology; 79:1-9.
Stamm WE, Koutsky LA, Benedetti JK, Jourden JL, Brunham RC, Holmes
KK. 1984. Chlamydia trachomatis urethral infections in men. Prevalence,
risk factors, and dinical manifestations. Ann Intern Med; 100:47-51.
Stamm WE, Wagner KF, Amsel R, Alexander ER, Turck M, Counts GW,
Holmes KK. 1980. Causes of the acute urethral syndrome in women.
N Engl J Med; 303: 409-415.
Stephens RS. 1994. Molecular mimicry and Chlamydia trachomatis infection
of eukaryotic cells. Trends Microbiol; 2: 99-101.
Stephens RS, Kalman S, Lammel C, Fan J, Marathe R, Aravind L, Mitchell W,
Olinger L, Tatusov RL, Zhao Q, Koonin EV, Davis RW. 1998.
Genome sequence of an obligate intracellular pathogen of humans:
Chlamydia trachomatis. Science; 282: 754-759.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
184
Stephens RS, Mullenbach G, Sanchez-Pescador R, Agabian N. 1986.
Sequence analysis of the major outer membrane protein gene from
Chlamydia trachomatis serovar L2. J Bacteriol; 168:1277-1282.
Stills HF, Fox JG, Paster BJ, Dewhirst FE. 1991. A "new" Chlamydia sp. strain
SFPD isolated from transmissable proliferative ileitis in hamsters.
Microbiol Ecol Health Dis; 4: S99.
Storey C, Lusher M, Yates P, Richmond S. 1993. Evidence for Chlamydia
pneumoniae of non-human origin. J Gen Microbiol; 139:2621-2626.
Stothard DR, Boguslawski G, Jones RB. 1998. Phylogenetic analysis of the
Chlamydia trachomatis major outer membrane protein and examination
of potential pathogenic determinants. Infect Immun; 66: 3618-3625.
Su H, Morrison RP, Watkins NG, Caldwell HD. 1990a. Identification and
characterization of T helper cell epitopes of the major outer membrane
protein of Chlamydia trachomatis. J Exp Med; 172:203-212.
Su H, Watkins NG, Zhang YX, Caldwell HD. 1990b. Chlamydia trachomatis-
host cell interactions: role of the chlamydial major outer membrane
protein as an adhesin. Infect Immun; 56:2094-2100.
Suchland RJ, Eckert LO, Hawes SE, Stamm WE. 2003. Longitudinal
Assessment of Infecting Serovars of Chlamydia trachomatis in Seattle
Public Health Clinics: 1988-1996. Sex Trans Dis; 30: 357-361.
Takourt B, de Barbeyrac B, Khyatti M, Radouani F, Bebear C, Dessus-Babus
S, Benslimane A. 2001. Direct genotyping and nucleotide sequence
analysis of VS1 and VS2 of the Ompl gene of Chlamydia trachomatis
from Moroccan trachomatous specimens. Microbes Infect; 3:459-466.
Tan M, Gaal T, Gourse RL, Engel JN. 1998. Mutational analysis of the
Chlamydia trachomatis rRNA PI promoter defines four regions
important for transcription in vitro. J Bacteriol; 180:2359-2366.
Tan M, Wong B, Engel JN. 1996. Transcriptional organization and
regulation of the dnaK and groE operons of Chlamydia trachomatis. J
Bacteriol; 178: 6983-6990.
Tan TW, Herring AJ, Anderson IE, Jones GE. 1990. Protection of sheep
against Chlamydia psittaci infection with a subcellular vaccine
containing the major outer membrane protein. Infect Immun; 58:
3101-3108.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
185
Tanner MA, Harris JK, Pace NR. 1999. Molecular phylogeny of Chlamydia
and relatives. In: Chlamydia: Intracellular biology, pathogenesis and
immunity. Stephens RS, editor. Washington, D.C.: ASM Press; pp. 1-
8.
Tanzer RJ, Longbottom D, Hatch TP. 2001. Identification of polymorphic
outer membrane proteins of Chlamydia psittaci 6BC. Infect Immun;
69:2428-2434.
Tao S, Kaul R, Wenman WM. 1991. Identification and nucleotide sequence
of a developmentally regulated gene encoding a eukaryotic histone
Hl-like protein from Chlamydia trachomatis. J Bacteriol; 173:2818-2822.
Taraska T, Ward DM, Ajioka RS, Wyrick PB, Davis-Kaplan SR, Davis CH,
Kaplan J. 1996. The late chlamydial inclusion membrane is not
derived from the endocy tic pathway and is relatively deficient in host
proteins. Infect Immun; 64:3713-3727.
Thylefors B, Negrel AD, Pararajasegaram R, Dadzie KY. 1995. Global data
on blindness. Bull. W.H.O.; 73:115-121.
Ting LM, Hsia RC, Haidaris CG, Bavoil PM. 1995. Interaction of outer
envelope proteins of Chlamydia psittaci GPIC with the HeLa cell
surface. Infect Immun; 63:3600-3608.
Tipples G, McClarty G. 1993. The obligate intracellular bacterium Chlamydia
trachomatis is auxotrophic for three of the four ribonucleoside
triphosphates. Mol Microbiol; 8:1105-1114.
Tipples G, McClarty G. 1995. Cloning and expression of the Chlamydia
trachomatis gene for CTP synthetase. J Biol Chem; 270: 7908-7914.
Todd WJ, Caldwell HD. 1985. The interaction of Chlamydia trachomatis with
host cells: ultrastructural studies of the mechanism of release of a
biovar II strain from HeLa 229 cells. J Infect Dis; 151:1037-1044.
Toye B, Laferriere C, Claman P, Jessamine P, Peeling R. 1993. Association
between antibody to the chlamydial heat-shock protein and tubal
infertility. J Infect Dis; 168:1236-1240.
Vandahl BB, Birkelund S, Demol H, Hoorelbeke B, Christiansen G,
Vandekerckhove J, Gevaert K. 2001. Proteome analysis of the
Chlamydia pneumoniae elementary body. Electrophoresis; 22:1204-
1223.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
186
van de Laar MJ, van Duynhoven YT, Fennema JS, Ossewaarde JM, van den
Brule AJ, van Doomum GJ, Coutinho RA, van den Hoek JA. 1996.
Differences in clinical manifestations of genital chlamydial infections
related to serovars. Genitourin Med; 72: 261-265.
van Duynhoven YT, Ossewaarde JM, Derksen-Nawrocki RP, van der
Meijden WI, van de Laar MJ. 1998. Chlamydia trachomatis genotypes:
correlation with clinical manifestations of infection and patients’
characteristics. J Clin Infect Dis; 26:314-322.
van Ooij C, Apodaca G, Engel J. 1997. Characterization of the Chlamydia
trachomatis vacuole and its interaction with the host endocytic
pathway in HeLa cells. Infect Immun; 65: 758-766.
Visddi RP, Bobo L, Hook EW, Quinn TC. 1993. Transmission of Chlamydia
trachomatis among sex partners assessed by polymerase chain reaction.
J Infect Dis; 168: 488-492.
Wagar EA, Schachter J, Bavoil P, Stephens RS. 1990. Differential human
serologic response to two 60,000 molecular weight Chlamydia
trachomatis antigens. J Infect Dis; 162: 922-927.
Wagenvoort JH, Suchland RJ, Stamm WE. 1988. Serovar distribution of
urogenital Chlamydia trachomatis strains in The Netherlands.
Genitourin Med; 64:159-161.
Wang SP, Grayston JT. 1982. Micro immunofluorescence antibody
responses in Chlamydia trachomatis infection. A review. In:
Chlamydial infections. Mardh PA, Holmes KK, Oriel JD, Piot P,
Schachter J, editors. Amsterdam: Elsevier Biomedical; pp. 301-316.
Wang SP, Grayston JT. 1991. Three new serovars of Chlamydia trachomatis:
Da, la, and L2a. J Infect Dis; 163:403-405.
Wang SP, Grayston JT, Gale JL. 1973. Three new immunologic types of
trachoma-indusion conjunctivitis organisms. J Immunol; 110: 873-879.
Wang SP, Kuo CC, Barnes RC, Stephens RS, Grayston JT. 1985.
Immunotyping of Chlamydia trachomatis with monoclonal antibodies. J
Infect Dis; 152:791-800.
Ward ME, Murray A. 1984. Control mechanisms governing the infectivity of
Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis. J
Gen Microbiol; 130:1765-1780.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
187
Watson MW, al-Mahdawi S, Lamden PR, Clarke IN. 1990. The nudeotide
sequence of the 60 kDa cysteine rich outer membrane protein of
Chlamydia pneumoniae strain IOL-207. Nudeic A dds Res; 17: 5299.
Watson MW, Clarke IN, Everson JS, Lambden PR. 1995. The CrP operon of
Chlamydia psittaci and Chlamydia pneumoniae. Microbiology; 141:2489-
2497.
Watson MW, Lambden PR, Clarke IN. 1990. The nudeotide sequence of the
60 kDa cysteine rich outer membrane protein of Chlamydia psittaci
strain EAE / A22 / M. Nucleic Acids Res; 18:5300.
Watson MW, Lambden PR, Ward ME, Clarke IN. 1989. Chlamydia
trachomatis 60 kDa cysteine rich outer membrane protein: sequence
homology between trachoma and LGV biovars. FEMS Microbiol Lett;
53:293-297.
Weinstock H, Dean D, Bolan G. 1995. Chlamydia trachomatis infedions. In:
Infedious Disease Clinics of North America: Sexually transmitted
Diseases in the AIDS era: Part II. Cohen MS, Hook 3rd EW, Hitchcock
PJ, editors. Philadelphia, PA: W.B. Sauders Company; pp. 797-820.
Weiss, E. 1950. The effed of antibiotics on agents of the psittacosis-
lymphogranuloma group. I. The effect of penicillin. J Infect Dis; 87:
249-263.
Westrom, L. 1975. Effect of acute pelvic inflammatory disease on infertility.
Am J Obstet Gynecol; 121:707-713.
Westrom L, Bengtsson LPH, and M ardh P -A . 1981. Inddence, trends, and
risks of edopic pregnancy in a population of women. Br Med J; 282:
15-19.
W hittington WL, Kent C, Kissinger P, Oh MK, Fortenberry JD, Hillis SE,
Litchfield B, Bolan GA, St Louis ME, Farley TA, Handsfield HH. 2001.
Determinants of persistent and recurrent Chlamydia trachomatis
infection in young women: results of a multicenter cohort study. Sex
Trans Dis; 28:117-123.
Wiuf C, Christensen T, Hein J. 2001. A simulation study of the reliability of
recombination detection methods. Mol Biol Evol; 18:1929-1939.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
188
Wolner-Hanssen P, Kiviat NB, Holmes KK. 1990. Atypical pelvic
inflammatory disease: subacute, chronic, or subdinical upper genital
tract infection in women. In: Sexually transmitted diseases. 2nd ed.
Holmes KK, Mardh P-A, Sparling PF, Wiesner PJ, editors. San
Francisco: McGraw-Hill Information Services; pp. 615-620.
Workowski KA, Stevens CE, Suchland RJ, Holmes KK, Eschenbach DA,
Pettinger MB, Stamm WE. 1994. Clinical manifestations of genital
infection due to Chlamydia trachomatis in women: differences related to
serovar. Clin Infect Dis; 19:756-760.
Workowski KA, Suchland RJ, Pettinger MB, Stamm WE. 1992. Association
of genital infection with specific Chlamydia trachomatis serovafs and
race. J Infect Dis; 166:1445-1449.
Wylie JL, Berry JD, McClarty G. 1996. Chlamydia trachomatis CTP synthetase:
molecular characterization and developmental regulation of
expression. Mol Microbiol; 22:631-642.
Wylie JL, Hatch GM, McClarty G. 1997. Host cell phospholipids are
trafficked to and then modified by Chlamydia trachomatis. J Bacteriol;
179: 7233-7242.
Wylie JL, Wang LL, Tipples G, McClarty G. 1996. A single point mutation
in CTP synthetase of Chlamydia trachomatis confers resistance to
cyclopentenyl cytosine. J Biol Chem; 271:15393-15400.
Wyllie S, Ashley RH, Longbottom D, Herring AJ. 1998. The major outer
membrane protein of Chlamydia psittaci functions as a porin-like ion
channel. Infect Immun; 66: 5202-5207.
Wyrick PB, Brownridge EA. 1978. Growth of Chlamydia psittaci in
macrophages. Infect Immun; 19:1054-1060.
Wyrick PB, Choong J, Davis CH, Knight ST, Royal MO, Maslow AS, Bagnell
CR. 1989. Entry of genital Chlamydia trachomatis into polarized hum an
epithelial cells. Infect Immun; 57: 2378-2389.
Yang CL, Maclean I, Branham RC. 1993. DNA sequence polymorphism of
the Chlamydia trachomatis ompl gene. J Infect Dis; 168:1225-1230.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
189
Yuan Y, Zhang YX, Watkins NG, Caldwell HD. 1989. Nucleotide and
deduced amino acid sequences for the four variable domains of the
major outer membrane proteins of the 15 Chlamydia trachomatis
serovars. Infect Immun; 57:1040-1049.
Zhang D, Yang X, Berry J, Shen C, McClarty G, Brunham RC. 1997. DNA
vaccination with the major outer-membrane protein gene induces
acquired immunity to Chlamydia trachomatis (mouse pneumonitis)
infection. J Infect Dis; 176:1035-1040.
Zhang JP, Stephens RS. 1992. Mechanism of C. trachomatis attachment to
eukaryotic host cells. Cell; 69: 861-869.
Zhang YX, Fox JG, Ho Y, Zhang L, Stills HF Jr, Smith TF. 1993. Comparison
of the major outer-membrane protein (MOMP) gene of mouse
pneumonitis (MoPn) and hamster SFPD strains of Chlamydia
trachomatis with other Chlamydia strains. Mol Biol Evol; 10:1327-1342.
Zhang YX, Morrison SG, Caldwell HD. 1990. The nucleotide sequence of
major outer membrane protein gene of Chlamydia trachomatis serovar
F. Nucleic Acids Res; 18:1061.
Zhang YX, Morrison SG, Caldwell HD, BaehrW. 1989. Cloning and
sequence analysis of the major outer membrane protein genes of two
Chlamydia psittaci strains. Infect Immun; 57:1621-1625.
Zhong G, Fan P, Ji H, Dong F, Huang Y. 2001. Identification of a chlamydial
protease-like activity factor responsible for the degradation of host
transcription factors. J Exp Med; 193: 935-942.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Millman, Kim Lori (author)

Core Title Molecular and genetic epidemiology of Chlamydia trachomatis in the United States

Degree Doctor of Philosophy

Degree Program Epidemiology

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