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Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis., Myeloid cells are implicated in the innate immune and inflammatory response during infection with Chlamydia trachomatis. Here the authors show the evasion of the neutrophil response to infection and concomitant induction of sterile immunity via the purinergic P2X7 receptor.

The Chlamydia trachomatis plasmid and inclusion membrane protein CT135 are virulence factors in the pathogenesis of murine female genital tract infection. To determine if these virulence factors play a similar role in female nonhuman primates, we infected pig-tailed macaques with the same C. trachomatis strains shown to be important in the murine model. Wild-type C. trachomatis and its isogenic mutant strain deficient in both plasmid and CT135 were used to infect macaques. Macaques were given primary and repeated cervicovaginal challenges with the wild-type and mutant strains. The infection rate, infection duration, and antibody response were similar among macaques infected with both strains. Unexpectedly, colposcopy, laparoscopy, and histologic analysis revealed no substantial genital tract pathology following either primary or repeated cervicovaginal challenges. Cytokine analysis of cervicovaginal secretions from both challenged groups revealed low concentrations of interleukin 1β (IL-1β) and elevated levels of the interleukin 1 receptor agonist (IL-1RA). We propose that an imbalance of IL-1β and IL-1RA in macaques is the reason for the mild inflammatory responses observed in infected urogenital tissues. Thus, understanding the pathobiology of chlamydial infection requires a better understanding of host epigenetic and chlamydial genetic factors. Our findings also have implications for understanding the high frequency of asymptomatic infections in humans.

We studied infection and immunity of hysterectomized mice infected with Chlamydia muridarum and Chlamydia trachomatis to determine if there were differences between these species in their ability to infect vaginal squamous epithelial cells in vivo independently of proximal upper genital tract tissues. We found that C. muridarum readily colonized and infected vaginal squamous epithelial cells, whereas C. trachomatis did not. Primary infection of the vaginal epithelium with C. muridarum produced infections of a duration longer than that reported for normal mice. Infection resulted in an inflammatory response in the vagina characterized by neutrophils and infiltrating submucosal plasma cells consisting primarily of T cells. Despite the delayed clearance, rechallenged C. muridarum -infected mice were highly immune. Mice vaginally infected with C. muridarum produced serum and vaginal wash antibodies and an antigen-specific gamma interferon-dominated Th1-biased T cell response. By comparison, mice vaginally infected with C. trachomatis exhibited transient low-burden infections, produced no detectable tissue inflammatory response, and failed to seroconvert. We discuss how these marked differences in the biology of vaginal infection between these otherwise genetically similar species are possibly linked to pathogen-specific virulence genes and how they may influence pathology and immunity in the upper genital tract.

Chlamydia trachomatis is an obligate intracellular pathogen characterized by a unique biphasic developmental cycle that alternates between infectious and non-infectious organisms. Chlamydial ChxR is a transcriptional activator that has been implicated in the regulation of the development cycle. We used a reverse genetics approach to generate three chxR null mutants. All three mutants grew normally in cultured mammalian cells. Whole genome sequencing identified SNPs in other genes; however, none of the mutated genes were common to all three ChxR null mutants arguing against a genetic compensatory mechanism that would explain the non-essential in vitro growth phenotype. Comparative proteomics identified five proteins, CT005, CT214, CT565, CT694 and CT695, that were significantly downregulated in all ChxR null mutants. This group includes established inclusion membrane and type III secreted proteins. ChxR transcriptional regulation of these genes was confirmed by qRT-PCR. Importantly, while ChxR null mutants exhibited no growth deficiencies in in vitro, they did show significant differences in in vivo growth using a mouse genital tract model. Collectively, our findings demonstrated that ChxR is a transcriptional activator that regulates the expression of virulence genes whose functions are restricted to in vivo infection.

Chlamydia muridarum and Chlamydia trachomatis, mouse and human strains, respectively, have been used to study immunity in a murine model of female genital tract infection. Despite evidence that unique genes of these otherwise genomically similar strains could play a role in innate immune evasion in their respective mouse and human hosts, there have been no animal model findings to directly support this conclusion. Here, we infected C57BL/6 and adaptive immune-deficient Rag1(-/-) female mice with these strains and evaluated their ability to spontaneously resolve genital infection. Predictably, C57BL/6 mice spontaneously cleared infection caused by both chlamydial strains. In contrast, Rag1(-/-) mice which lack mature T and B cell immunity but maintain functional innate immune effectors were incapable of resolving C. muridarum infection but spontaneously cleared C. trachomatis infection. This distinct dichotomy in adaptive and innate immune-mediated clearance between mouse and human strains has important cautionary implications for the study of natural immunity and vaccine development in the mouse model.

Background Rectal sexually transmitted infections (STIs) may increase HIV susceptibility in men who have sex with men (MSM), and Chlamydia trachomatis is prevalent among HIV-positive MSM. To study STIs and HIV infection in MSM, we first evaluated whether cynomolgus macaques can sustain both C. trachomatis and SHIVSF162p3 infections. Methods Four SHIVSF162p3-positive male cynomolgus macaques were used (n = 3 rectally inoculated with 106 IFU; n = 1 control). Systemic and rectal SHIV RNA levels and cytokines were measured by real-time PCR and Luminex assays, respectively. Results Macaques were successfully Chlamydia infected. Rectal SHIV shedding (P = 0.02 χ2) and levels of G-CSF, IL-1ra, IL-6, IL-8, IFN-γ, and TNF-α (P ≤ 0.01, Mann–Whitney) in rectal secretions increased following infection. Conclusions These pilot data successfully demonstrate rectal C. trachomatis–SHIV coinfection in cynomolgus macaques and suggest the feasibility of a rectal C. trachomatis model for SHIV susceptibility and biomedical prevention studies in the context of rectal STIs.

Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1 , - 2 , - 6 , and - 8 are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3 and multiple chromosomal genes, including the glycogen synthase gene glgA , that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmid's role in chlamydial pathogenesis at the molecular level.

In cynomolgus macaques, ocular infection with a live trachoma strain lacking the conserved 7.5-kb plasmid induced no ocular pathology but facilitated solid or partial protection from subsequent infection with a virulent strain of trachoma., Blinding trachoma is an ancient neglected tropical disease caused by Chlamydia trachomatis for which a vaccine is needed. We describe a live-attenuated vaccine that is safe and efficacious in preventing trachoma in nonhuman primates, a model with excellent predictive value for humans. Cynomolgus macaques infected ocularly with a trachoma strain deficient for the 7.5-kb conserved plasmid presented with short-lived infections that resolved spontaneously without ocular pathology. Multiple infections with the attenuated plasmid-deficient strain produced no inflammatory ocular pathology but induced an anti-chlamydial immune response. Macaques vaccinated with the attenuated strain were either solidly or partially protected after challenge with virulent plasmid-bearing organisms. Partially protected macaques shed markedly less infectious organisms than controls. Immune correlates of protective immunity were not identified, but we did detect a correlation between MHC class II alleles and solid versus partial protection. Epidemiological models of trachoma control indicate that a vaccine with this degree of efficacy would significantly reduce the prevalence of infection and rates of reinfection, known risk factors which drive blinding disease.

Background Sexually transmitted infections (STIs) are associated with an increased risk of HIV infection. To model the interaction between STIs and HIV infection, we evaluated the capacity of the pigtail macaque model to sustain triple infection with Trichomonas vaginalis, Chlamydia trachomatis, and SHIVSF162P3.

The obligate intracellular bacterial pathogen Chlamydia trachomatis causes an estimated 92 million new cases of sexually transmitted infection each year worldwide [1]. Lasting and durable control of these infections, and prevention of the deleterious consequences of infection, will require the development of a vaccine. A limitation to the development of a vaccine relates to the incomplete understanding of the natural history of human chlamydial genital infection. Diagnosis of infection mandates treatment, and thus it is not fully understood which responses are necessary for protective immunity and when during the course of infection those responses mature and become effective. Therefore, Chlamydia infection of animals—primarily mice—has been used as a surrogate to study the natural history of genital infection and the development of adaptive immunity. Two mouse models of infection have been used extensively to study chlamydial genital infection and immunity: one model uses the mouse pathogen, Chlamydia muridarum; and the other uses the human pathogen, C. trachomatis. The need for adaptive immunity in resolving murine C. muridarum infection is unequivocal [2], but the role of adaptive immunity in resolving murine C. trachomatis infection is ambiguous [3, 4], even though primary infection elicits adaptive immune responses and a level of resistance to reinfection appears to develop [5–10]. In this study we sought to directly examine the contribution of adaptive immunity (CD4+ T cells) in resolving primary murine C. trachomatis genital infection. Because previous studies have clearly demonstrated that C. trachomatis genital infection of mice is highly dependent on mouse strain [5, 11], it was crucial to choose a mouse strain that supported a robust infection that persisted for sufficient time to allow for the development of adaptive immune responses. Accordingly, we chose to use the C3H/HeJ strain of mice. Mice of the C3H background have been shown to be susceptible to C. trachomatis genital infection, and the C3H/HeJ strain in particular has impaired innate immunity due to a nonfunctional Toll-like receptor (TLR) 4 [12]. Those combined attributes should favor the development of an infection of sufficient intensity and duration to delineate the role of adaptive immunity in resolving C. trachomatis genital infection. We show here that CD4+ T cell–mediated adaptive immunity is not required to control primary murine C. trachomatis genital infection.

Chlamydia trachomatisis a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine.C. trachomatispolymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved amongC. trachomatisstrains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.

Chlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1 to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene., IMPORTANCE Chlamydia’s obligate intracellular life style requires both entry into and exit from host cells. Virulence factors that function in exiting are unknown. The chlamydial inclusion is stabilized late in the infection cycle by F-actin. A prerequisite of chlamydial exit is its ability to disassemble actin from the inclusion. We show that chlamydial plasmid-free organisms, and also a plasmid gene protein 4 (pgp4) null mutant, do not disassociate actin from the inclusion and fail to exit cells. We further provide evidence that Pgp4-regulated exit is dependent on the chlamydial type III secretion system. This study is the first to define a genetic mechanism that functions in chlamydial lytic exit from host cells. The findings also have practical implications for understanding why plasmid-free chlamydiae are highly attenuated and have the ability to elicit robust protective immune responses.

Chlamydia trachomatis is an obligate intracellular epitheliotropic bacterial pathogen of humans. Infection of the eye can result in trachoma, the leading cause of preventable blindness in the world. The pathophysiology of blinding trachoma is driven by multiple episodes of reinfection of conjunctival epithelial cells, producing an intense chronic inflammatory response resulting in submucosal tissue remodeling and scarring. Recent reports have shown that infection with trachoma organisms lacking the cryptic chlamydial plasmid is highly attenuated in macaque eyes, a relevant experimental model of human trachoma infection. To better understand the molecular basis of plasmid-mediated infection attenuation and the potential modulation of host immunity, we conducted transcriptional profiling of human epithelial cells infected with C. trachomatis plasmid-bearing (A2497) and plasmid-deficient (A2497P − ) organisms. Infection of human epithelial cells with either strain increased the expression of host genes coding for proinflammatory (granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage colony-stimulating factor [MCSF], interleukin-6 [IL-6], IL-8, IL-1α, CXCL1, CXCL2, CXCL3, intercellular adhesion molecule 1 [ICAM1]), chemoattraction (CCL20, CCL5, CXCL10), immune suppression (PD-L1, NFKB1B, TNFAIP3, CGB), apoptosis (CASP9, FAS, IL-24), and cell growth and fibrosis (EGR1 and IL-20) proteins. Statistically significant increases in the levels of expression of many of these genes were found in A2497-infected cells compared to the levels of expression in A2497P − -infected cells. Our findings suggest that the chlamydial plasmid plays a focal role in the host cell inflammatory response to infection and immune avoidance. These results provide new insights into the role of the chlamydial plasmid as a chlamydial virulence factor and its contributions to trachoma pathogenesis.

Chlamydia trachomatis is an obligate intracellular mucosotropic pathogen of significant medical importance. It is the etiological agent of blinding trachoma and bacterial sexually transmitted diseases, infections that afflict hundreds of millions of people globally. The C. trachomatis polymorphic membrane protein D (PmpD) is a highly conserved autotransporter and the target of broadly cross-reactive neutralizing antibodies; however, its role in host-pathogen interactions is unknown. Here we employed a targeted reverse genetics approach to generate a pmpD null mutant that was used to define the role of PmpD in the pathogenesis of chlamydial infection. We show that pmpD is not an essential chlamydial gene and the pmpD null mutant has no detectable deficiency in cultured murine cells or in a murine mucosal infection model. Notably, however, the pmpD null mutant was significantly attenuated for macaque eyes and cultured human cells. A reduction in pmpD null infection of human endocervical cells was associated with a deficiency in chlamydial attachment to cells. Collectively, our results show that PmpD is a chlamydial virulence factor that functions in early host-cell interactions. This study is the first of its kind using reverse genetics to evaluate the contribution of a C. trachomatis gene to disease pathogenesis.

Sexually transmitted infection (STI) can increase the risk of human immunodeficiency virus (HIV) infection by 2–5-fold [1–5]. It is unclear whether this association is largely attributable to sexual or behavioral practices or to an underlying STI-associated biological mechanism. Human studies of STI coinfection and HIV susceptibility are often precluded because of study size, confounding behaviors, and ethical considerations. The use of a macaque model of STI and HIV coinfection would help to address these questions and also provide a model for preclinical testing of biomedical HIV prevention strategies. Because of the high STI prevalence among individuals at high risk for HIV infection [3, 4, 6], rigorous testing of these strategies in the context of STIs would help to ensure broad efficacy of prevention methods among varying populations. A current focus of HIV prevention research is identifying effective preexposure prophylaxis (PrEP) strategies for high-risk groups. PrEP with Truvada was recently approved by the Food and Drug Administration for use in men who have sex with men (MSM), HIV-discordant couples, and other high-risk populations (eg, injection drug users) [7, 8]. However, some PrEP strategies in women have shown limited efficacy (as in the CAPRISA cohort) or no efficacy (as in the VOICE and FemPrEP cohorts) [9–11]. In addition to addressing adherence issues, it is also important to investigate biological factors that could lower PrEP efficacy in women, such as reproductive hormone levels, coinfections, and inflammation in the female genital tract. We and others have used female pig-tailed macaques to study topical and systemic PrEP efficacy [12, 13] and HIV susceptibility factors unique to the female host [14]. These macaques model human female reproductive tract physiology, have year-round lunar menstrual cycles, and can be vaginally infected with simian/HIV (SHIV) without progestin treatment. In these macaques, using a repeat, low-dose SHIV challenge model, we showed that susceptibility to SHIV infection is inconsistent throughout the menstrual cycle and peaks during the late-luteal and menses phases [14, 15]. With repeated low-dose exposures in pig-tailed macaques, we can carefully study factors affecting HIV susceptibility by evaluating completed menstrual cycles during virus exposures (as opposed to challenges required for infection) to account for the varying susceptibility a woman experiences throughout her menstrual cycle [13, 16–18]. We recently established a pig-tailed macaque model of concomitant STI and HIV infection [19], providing an effective system with which to assess effects of both STIs and hormonal changes on susceptibility to vaginal SHIV transmission. Studies of macaques with concomitant STI and HIV infection are limited, with only Crostarosa et al reporting on the effect of herpes simplex virus type 2 (HSV-2) infection on SHIV susceptibility in Depo-Provera-treated rhesus macaques [20]. Yet a macaque STI model could be used to conclusively assess whether these infections do in fact increase the risk of HIV infection, to investigate associated mechanisms, and to rigorously assess HIV prevention methods. The study described here uses our previously developed pig-tailed macaque triple infection STI-SHIV model [19] to evaluate the effect of non-ulcerative STIs (ie, Chlamydia trachomatis and Trichomonas vaginalis infections) on SHIV susceptibility. Patton et al have established C. trachomatis infection models in several macaque species, investigated Chlamydia and SHIV coinfection in pig-tailed macaques, and have also studied T. vaginalis infection in pig-tailed macaques [21–24]. C. trachomatis and T. vaginalis were also selected because infections with these pathogens are epidemiologically associated with an increased risk of HIV infection and are highly prevalent among individuals at high risk for HIV [3, 6, 25, 26]. From an early report by Laga et al, women with Chlamydia or Trichomonas infections were almost 4 and 2 times as likely, respectively, to become HIV-positive [3]. These pathogens induce tissue inflammation and may also cause abrasions to the genital tract epithelium, potentially facilitating access of virus to target cells [27–30]. Cell populations involved in clearing STIs are also targets for HIV infection (and for simian immunodeficiency virus [SIV] and SHIV infections), particularly in the case of C. trachomatis and T. vaginalis [30–34]. It is hypothesized these combined factors increase susceptibility to mucosal HIV transmission. This current study incorporates methods developed in our laboratory [19] and the Patton laboratory and reports increased susceptibility to SHIV infection among macaques coinfected with C. trachomatis and T. vaginalis, introduces possible mechanisms for enhanced susceptibility in vaginal transmission, and indicates a definitive role for these coinfections in the risk of HIV infection, apart from sexual or behavioral practices alone. Moreover, this study establishes a macaque model of genital tract inflammation for studies of high-risk HIV transmission and prevention.

Trachoma, caused by the obligate intracellular organism Chlamydia trachomatis, is the world’s leading cause of preventable blindness for which a vaccine is needed. We have previously shown that a plasmid-deficient live-attenuated trachoma vaccine delivered ocularly to macaques elicited either solid or partial protective immunity against a virulent ocular challenge. Solidly protected macaques shared the same MHC class II alleles implicating CD4+ T cells in superior protective immunity. Understandably, we sought to define T cell immune correlates in these animals to potentially improve vaccine efficacy. In this study, following a 2-y resting period, these macaques were boosted i.m. with the live-attenuated trachoma vaccine and their peripheral T cell anamnestic responses studied. Both solidly and partially protected macaques exhibited a CD4+ and CD8+ T cell anamnestic response following booster immunization. CD8+ but not CD4+ T cells from solidly protected macaques proliferated against soluble chlamydial Ag. We observed a more rapid T cell inflammatory cytokine response in tears of solidly protected animals following ocular rechallenge. Most notably, depletion of CD8+ T cells in solidly protected macaques completely abrogated protective immunity. Collectively, our findings support the conclusion that CD8+ T cells play an important but unexpected role in live-attenuated trachoma vaccine-mediated protective immunity.

Chlamydia trachomatis is the most common cause of human bacterial sexually transmitted infections and is the world's leading cause of infectious preventable blindness. The chlamydial 7.5-kb plasmid and chromosomal gene CT135 have been shown to be important virulence factors in both nonhuman primate and mouse infection models. Chlamydia trachomatis plasmid-deficient urogenital isolates and a predicted CT135 null mutant have been evaluated independently in the female mouse genital tract model and both have been shown to reduce infectivity and virulence. However, these attenuating phenotypes have not been evaluated collectively in the murine model. Here, we test the infectivity of C. trachomatis serovar D strains in the mouse model that are plasmid-deficient, CT135 disrupted, or possess a combination of these attenuating genotypes. We find that the presence of the plasmid results in infections with higher infectious burdens, whereas CT135 facilitates a more protracted or chronic infection. Not unexpectedly, a combination of these genetic deficiencies resulted in a strain with enhanced infection attenuation characteristics.

Chlamydia trachomatis and C. muridarum are human and mouse pathogens respectively, which show high conservation of gene order and content. Both species contain a common 7.5 kb plasmid that is an important virulence factor. Recently described transformation systems have been used to characterize C. trachomatis L2 plasmid gene functions; however, similar studies have not been reported for C. trachomatis ocular tropic serovar A or the mouse strain, C. muridarum. Here, we have conducted genetic experiments with C. trachomatis serovar A and C. muridarum and report the following; (i) successful transformation of C. muridarum and C. trachomatis serovar A is restricted to a shuttle vector with a C. muridarum, or C. trachomatis serovar A plasmid backbone, respectively, ii) transformation of plasmid-deficient C. muridarum with the C. muridarum based shuttle vector complemented glycogen accumulation and inclusion morphology, and (iii) C. muridarum plasmid encoded Pgp4 is a regulator of chromosomal (glgA) and plasmid (pgp3) virulence genes. In summary, our findings show a previously unrecognized and unexpected role for the chlamydial plasmid in its transformation tropism and confirm the plasmids regulatory role of virulence genes in C. muridarum.

Chlamydia trachomatisis a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model ofC. trachomatisinfection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivatedin vitroprior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiplein vitropassages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrastingin vivovirulence characteristics, the strains exhibited no differences in the results ofin vitroinfectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.

Introduction Previous studies demonstrated that a Chlamydia trachomatis plasmid negative ocular strain (A/P-) resulted in attenuation of infection and pathology, paired with immune stimulation that produced protective immunity in a monkey ocular model. Expanding upon these findings, we assessed a plasmid negative genital strain (D/P-) in the pigtailed macaque model of chlamydial reproductive tract infection. Infection and pathology were compared to a plasmid positive genital strain (D/P+). Methods Groups of six macaques were cervically challenged with C. trachomatis D/P+ or D/P-. All animals were followed for infection, circulating antibody, local immunity, and tissue inflammation. Upon spontaneous clearance of cervical infection, each animal underwent repeated challenges with matched strains to drive upper reproductive tract disease. The same strains were similarly compared in the macaque ocular infection model. Results Similar rates and duration of chlamydial genital infection were documented in both the P+ and P- challenged macaques. Likewise serum and local antibodies were similar. Tissue inflammation graded by gross observation during laparoscopic procedures and by tissue histology yielded no discernible patterns to disease pathology between P+ and P- strains. Because A/P+ and A/P- ocular strains in macaques exhibited dramatic differences in infectivity and pathology in the eye, we ocularly challenged animals with D/P+ and D/P-. Unexpectedly, no differences in infectivity or pathology were observed between the D/P+ and D/P- strains; each produced similar infection kinetics with ocular disease characterised by conjunctival hyperemia and follicle formation. Conclusions Unlike the ocular strain, the plasmid negative genital strain is not attenuated in either genital or ocular macaque infection models. This suggests that genetic determinants unrelated to the plasmid play a dominant role in the pathogenesis of urogenital strains. Disclosure of interest statement This research was supported by NIH Contract Numbers HHSN266200700013C and HHSN272201400016C, and by the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health through Grant Number P51 OD010425 Washington National Primate Research Centre. No pharmaceutical grants were received in the development of this study.

Chlamydia trachomatis is an important human pathogen causing both ocular and sexually transmitted disease. Recently, we identified CT135 as an important virulence determinant in a mouse infection model. Results from CEL 1 digestion assays and sequencing analyses indicated that CT135 was much more polymorphic in high in vitro passage reference serovars than it was in clinical strains that had undergone limited passaging. Herein, we used targeted next-generation sequencing of the CT134-135 locus, from reference strains and clinical isolates, enabling accurate discovery of single nucleotide polymorphisms and other population genetic variations. Our results indicate that CT134 is stable in all C. trachomatis serovars examined. In contrast, CT135 is highly polymorphic in high-passaged reference ocular and non-LGV genital serovars, with the majority of the mutations resulting in gene disruption. In low-passaged ocular clinical isolates, CT135 was frequently disrupted, whereas in genital clinical isolates CT135 was intact in almost all instances. When a serovar K isolate, with an intact CT134 and CT135, was subjected to serial passage in vitro CT134 remained invariable, while numerous gene interrupting mutations rapidly accumulated in CT135. Collectively, our data indicate that, for genital serovars, CT135 is under strong positive selection in vivo, and negative selection in vitro.