Haematopoietic innate interleukin 17A production drives immunopathology in female mouse genital Chlamydia muridarum infection.

Chlamydia trachomatis infection is the leading cause of bacterial urogenital infection and has been demonstrated to drive inflammation and scarring of the reproductive tract. Recent studies have identified key triggers of proinflammatory adaptive immune responses driven by innate leukocytes and epithelia driving immunopathology. Utilizing chimeric mouse models, we investigated the definitive source and role of IL17 and IL17 signalling receptors during early Chlamydia muridarum infection of the female urogenital tract. Bone marrow transplants from wild-type (WT) and IL17A-/- mice to recipients demonstrated equivocal infection kinetics in the reproductive tract, but interestingly, adoptive transfer of IL17A-/- immune cells to WT recipients resulted in no infertility, suggesting a haematopoietic (as opposed to tissue) source of IL17 driving immunopathology. To further delineate the role of IL17 in immunopathology, we infected WT and IL17 receptor A (IL17RA)-/- female mice and observed a significant reduction in immunopathology in IL17RA-/- mice. WT bone marrow transplants to IL17RA-/- recipient mice prevented hydrosalpinx, suggesting signalling through IL17RA drives immunopathology. Furthermore, early chemical inhibition of IL17 signalling significantly reduced hydrosalpinx, suggesting IL17 acts as an innate driver of disease. Early during the infection, IL17 was produced by γδ T cells in the cervico-vagina, but more importantly, by neutrophils at the site of infertility in the oviducts. Taken together, these data suggest innate production of IL17 by haematopoietic leukocytes drives immunopathology in the epithelia during early C. muridarum infection of the female reproductive tract.

Irgm proteins attenuate inflammatory disease in mouse models of genital Chlamydia infection.

Chlamydiae are obligate intracellular bacterial pathogens that may cause genital pathology via induction of destructive host immune responses. Human-adapted Chlamydia trachomatis causes inflammatory disease in human hosts but is easily cleared in mice, and mouse-adapted Chlamydia muridarum establishes a productive and pathogenic infection in murine hosts. While numerous anti-chlamydial host resistance factors have been discovered in mice and humans alike, little is known about host factors promoting host fitness independent of host resistance. Here, we show that interferon-inducible immunity-related GTPase M (Irgm) proteins function as such host factors ameliorating infection-associated sequalae in the murine female genital tract, thus characterizing Irgm proteins as mediators of disease tolerance. Specifically, we demonstrate that mice deficient for all three murine Irgm paralogs (pan-Irgm-/-) are defective for cell-autonomous immunity to C. trachomatis, which correlates with an early and transient increase in bacterial burden and sustained hyperinflammation in vivo. In contrast, upon infection of pan-Irgm-/- mice with C. muridarum, bacterial burden is unaffected, yet genital inflammation and scarring pathology are nonetheless increased, demonstrating that Irgm proteins can promote host fitness without altering bacterial burden. Additionally, pan-Irgm-/- mice display increased granulomatous inflammation in genital Chlamydia infection, implicating Irgm proteins in the regulation of granuloma formation and maintenance. These findings demonstrate that Irgm proteins regulate pathogenic immune responses to Chlamydia infection in vivo, establishing an effective infection model to examine the immunoregulatory functions and mechanisms of Irgm proteins. IMPORTANCE: In response to genital Chlamydia infection, the immune system mounts a proinflammatory response to resist the pathogen, yet inflammation must be tightly controlled to avoid collateral damage and scarring to host genital tissue. Variation in the human IRGM gene is associated with susceptibility to autoinflammatory diseases but its role in ameliorating inflammatory diseases caused by infections is poorly defined. Here, we use mice deficient for all three murine Irgm paralogs to demonstrate that Irgm proteins not only provide host resistance to Chlamydia infections but also limit associated inflammation in the female genital tract. In particular, we find that murine Irgm expression prevents granulomatous inflammation, which parallels inflammatory diseases associated with variants in human IRGM. Our findings therefore establish genital Chlamydia infection as a useful model to study the roles for Irgm proteins in both promoting protective immunity and limiting pathogenic inflammation.

Elimination of Chlamydia muridarum from the female reproductive tract is IL-12p40 dependent, but independent of Th1 and Th2 cells.

Chlamydia vaccine approaches aspire to induce Th1 cells for optimal protection, despite the fact that there is no direct evidence demonstrating Th1-mediated Chlamydia clearance from the female reproductive tract (FRT). We recently reported that T-bet-deficient mice can resolve primary Chlamydia infection normally, undermining the potentially protective role of Th1 cells in Chlamydia immunity. Here, we show that T-bet-deficient mice develop robust Th17 responses and that mice deficient in Th17 cells exhibit delayed bacterial clearance, demonstrating that Chlamydia-specific Th17 cells represent an underappreciated protective population. Additionally, Th2-deficient mice competently clear cervicovaginal infection. Furthermore, we show that sensing of IFN-γ by non-hematopoietic cells is essential for Chlamydia immunity, yet bacterial clearance in the FRT does not require IFN-γ secretion by CD4 T cells. Despite the fact that Th1 cells are not necessary for Chlamydia clearance, protective immunity to Chlamydia is still dependent on MHC class-II-restricted CD4 T cells and IL-12p40. Together, these data point to IL-12p40-dependent CD4 effector maturation as essential for Chlamydia immunity, and Th17 cells to a lesser extent, yet neither Th1 nor Th2 cell development is critical. Future Chlamydia vaccination efforts will be more effective if they focus on induction of this protective CD4 T cell population.

BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

Regulation of chlamydial spreading from the small intestine to the large intestine by IL-22-producing CD4+ T cells.

Following an oral inoculation, Chlamydia muridarum descends to the mouse large intestine for long-lasting colonization. However, a mutant C. muridarum that lacks the plasmid-encoded protein pGP3 due to an engineered premature stop codon (designated as CMpGP3S) failed to do so even following an intrajejunal inoculation. This was because a CD4+ T cell-dependent immunity prevented the spread of CMpGP3S from the small intestine to the large intestine. In the current study, we found that mice deficient in IL-22 (IL-22-/-) allowed CMpGP3S to spread from the small intestine to the large intestine on day 3 after intrajejunal inoculation, indicating a critical role of IL-22 in regulating the chlamydial spread. The responsible IL-22 is produced by CD4+ T cells since IL-22-/- mice were rescued to block the CMpGP3S spread by donor CD4+ T cells from C57BL/6J mice. Consistently, CD4+ T cells lacking IL-22 failed to block the spread of CMpGP3S in Rag2-/- mice, while IL-22-competent CD4+ T cells did block. Furthermore, mice deficient in cathelicidin-related antimicrobial peptide (CRAMP) permitted the CMpGP3S spread, but donor CD4+ T cells from CRAMP-/- mice were still sufficient for preventing the CMpGP3S spread in Rag2-/- mice, indicating a critical role of CRAMP in regulating chlamydial spreading, and the responsible CRAMP is not produced by CD4+ T cells. Thus, the IL-22-producing CD4+ T cell-dependent regulation of chlamydial spreading correlated with CRAMP produced by non-CD4+ T cells. These findings provide a platform for further characterizing the subset(s) of CD4+ T cells responsible for regulating bacterial spreading in the intestine.

Chlamydia muridarum infection causes bronchointerstitial pneumonia in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice.

The murine bacterial pathogen Chlamydia muridarum (Cm) has been used to study human Chlamydia infections in various mouse models. CD4+ T-cells, natural killer cells, and interferon-gamma (IFN-γ)-mediated immunity are important to control experimentally induced Cm infections. Despite its experimental use, natural infection by Cm has not been documented in laboratory mice since the 1940s. In 2022, the authors reported the discovery of natural Cm infections in numerous academic institutional laboratory mouse colonies around the globe. To evaluate the impact of Cm infection in severely immunocompromised mice, 19 NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were cohoused with Cm shedding, naturally infected immunocompetent mice and/or their soiled bedding for 4 weeks and subsequently euthanized. Clinical disease, characterized by lethargy, dyspnea, and weight loss, was observed in 11/19 NSG mice, and 16/18 NSG mice had neutrophilia. All mice exhibited multifocal to coalescing histiocytic and neutrophilic bronchointerstitial pneumonia (17/19) or bronchiolitis (2/19) with intraepithelial chlamydial inclusions (CIs). Immunofluorescence showed CIs were often associated with bronchiolar epithelium. CIs were frequently detected by immunohistochemistry in tracheal and bronchiolar epithelium (19/19), as well as throughout the small and large intestinal epithelium without lesions (19/19). In a subset of cases, Cm colonized the surface epithelium in the nasopharynx (16/19), nasal cavity (7/19), and middle ear canal (5/19). Endometritis and salpingitis with intraepithelial CI were identified in a single mouse. These findings demonstrate that Cm infection acquired through direct contact or soiled bedding causes significant pulmonary pathology and widespread intestinal colonization in NSG mice.

A novel chimeric recombinant FliC-Pgp3 vaccine promotes immunoprotection against Chlamydia muridarum infection in mice.

The Pgp3 subunit vaccine elicits immune protection against Chlamydia trachomatis infection, but additional adjuvants are still required to enhance its immunoprotective efficacy. Flagellin can selectively stimulate immunity and act as an adjuvant. In this research, the FliC-Pgp3 recombinant was successfully expressed and purified. Tri-immunization with the FliC-Pgp3 vaccine in Balb/C mice induced rapid and persistent germinal center B-cell response and Tfh differentiation, promoting a significantly higher IgG antibody titer compared to the Pgp3 group. FliC-Pgp3 immunization primarily induced Th1-type cellular immunity, leading to higher levels of IFN-γ, TNF-α, and IL-2 secreted by CD4+ T cells than in Pgp3-vaccinated mice. Chlamydia muridarum challenge results showed that FliC-Pgp3-vaccinated mice exhibited more rapid clearance of Chlamydia muridarum colonization in the lower genital tract, ensuring a lower hydrosalpinx rate and cumulative score. Histological analysis showed reduced dilation and inflammatory infiltration in the oviduct and uterine horn of FliC-Pgp3-vaccinated mice compared to the PBS and Pgp3 control. Importantly, tri-immunization with FliC-Pgp3 effectively activated CD4+ T cells and dendritic cells, as confirmed by the adoptive transfer, resulting in better immune protection in recipient mice. In summary, the novel FliC-Pgp3 chimeric is hoped to be a novel vaccine with improved immunoprotection against Chlamydia muridarum.

IL-23 receptor signaling licenses group 3-like innate lymphoid cells to restrict a live-attenuated oral Chlamydia vaccine in the gut.

An IFNγ-susceptible mutant of Chlamydia muridarum is attenuated in pathogenicity in the genital tract and was recently licensed as an intracellular Oral vaccine vector or intrOv. Oral delivery of intrOv induces transmucosal protection in the genital tract, but intrOv itself is cleared from the gut (without shedding any infectious particles externally) by IFNγ from group 3-like innate lymphoid cells (ILC3s). We further characterized the intrOv interactions with ILC3s in the current study, since the interactions may impact both the safety and efficacy of intrOv as an oral Chlamydia vaccine. Intracolonic inoculation with intrOv induced IFNγ that in return inhibited intrOv. The intrOv-IFNγ interactions were dependent on RORγt, a signature transcriptional factor of ILC3s. Consistently, the transfer of oral intrOv-induced ILC3s from RORγt-GFP reporter mice to IFNγ-deficient mice rescued the inhibition of intrOv. Thus, IFNγ produced by intrOv-induced ILC3s is likely responsible for inhibiting intrOv, which is further supported by the observation that oral intrOv did induce significant levels of IFNγ-producing LC3s (IFNγ+ILC3s). Interestingly, IL-23 receptor knockout (IL-23R-/-) mice no longer inhibited intrOv, which was accompanied by reduced colonic IFNγ. Transfer of oral intrOv-induced ILC3s rescued the IL-23R-/- mice to inhibit intrOv, validating the dependence of ILC3s on IL-23R signaling for inhibiting intrOv. Clearly, intrOv induces intestinal IFNγ+ILC3s for its own inhibition in the gut, which is facilitated by IL-23R signaling. These findings have provided a mechanism for ensuring the safety of intrOv as an oral Chlamydia vaccine and a platform for investigating how oral intrOv induces transmucosal protection in the genital tract.

Preclinical screen for protection efficacy of chlamydial antigens that are immunogenic in humans.

To search for subunit vaccine candidates, immunogenic chlamydial antigens identified in humans were evaluated for protection against both infection and pathology in a mouse genital tract infection model under three different immunization regimens. The intramuscular immunization regimen was first used to evaluate 106 chlamydial antigens, which revealed that two antigens significantly reduced while 11 increased genital chlamydial burden. The two infection-reducing antigens failed to prevent pathology and 23 additional antigens even exacerbated pathology. Thus, intranasal mucosal immunization was tested next since intranasal inoculation with live Chlamydia muridarum prevented both genital infection and pathology. Two of the 29 chlamydial antigens evaluated were found to prevent genital infection but not pathology and three exacerbate pathology. To further improve protection efficacy, a combinational regimen (intranasal priming + intramuscular boosting + a third intraperitoneal/subcutaneous boost) was tested. This regimen identified four infection-reducing antigens, but only one of them prevented pathology. Unfortunately, this protective antigen was not advanced further due to its amino acid sequence homology with several human molecules. Two pathology-exacerbating antigens were also found. Nevertheless, intranasal mucosal priming with viable C. muridarum in control groups consistently prevented both genital infection and pathology regardless of the subsequent boosters. Thus, screening 140 different chlamydial antigens with 21 repeated multiple times in 17 experiments failed to identify a subunit vaccine candidate but demonstrated the superiority of viable chlamydial organisms in inducing immunity against both genital infection and pathology, laying the foundation for developing a live-attenuated Chlamydia vaccine.

IL-21/IL-21R Promotes the Pro-Inflammatory Effects of Macrophages during C. muridarum Respiratory Infection.

Interleukin-21 and its receptors (IL-21/IL-21R) aggravate chlamydial lung infection, while macrophages (Mφ) are one of the main cells infected by chlamydia and the main source of inflammatory cytokines. Therefore, it is particularly important to study whether IL-21/IL-21R aggravates chlamydia respiratory infection by regulating Mφ. Combined with bioinformatics analysis, we established an IL-21R-deficient (IL-21R-/-) mouse model of Chlamydia muridarum (C. muridarum) respiratory tract infection in vivo, studied C. muridarum-stimulated RAW264.7 by the addition of rmIL-21 in vitro, and conducted adoptive transfer experiments to clarify the association between IL-21/IL-21R and Mφ. IL-21R-/- mice showed lower infiltration of pulmonary total Mφ, alveolar macrophages, and interstitial macrophages compared with WT mice following infection. Transcriptomic analysis suggested that M1-related genes are downregulated in IL-21R-/- mice and that IL-21R deficiency affects the Mφ-mediated inflammatory response during C. muridarum infection. In vivo experiments verified that in IL-21R-/- mice, pulmonary M1-type CD80+, CD86+, MHC II+, TNFα+, and iNOS+ Mφ decreased, while there were no differences in M2-type CD206+, TGF-ß+, IL-10+ and ARG1+ Mφ. In vitro, administration of rmIL-21 to C. muridarum-stimulated RAW264.7 cells promoted the levels of iNOS-NO and the expression of IL-12p40 and TNFα, but had no effect on TGFß or IL-10. Further, adoptive transfer of M1-like bone marrow-derived macrophages derived from IL-21R-/- mice, unlike those from WT mice, effectively protected the recipients against C. muridarum infection and induced relieved pulmonary pathology. These findings help in understanding the mechanism by which IL-21/IL-21R exacerbates chlamydia respiratory infection by promoting the proinflammatory effect of Mφ.

Neisseria gonorrhoeae Coinfection during Chlamydia muridarum Genital Latency Does Not Modulate Murine Vaginal Bacterial Shedding.

Chlamydia trachomatis and Neisseria gonorrhoeae are the most frequently reported agents of bacterial sexually transmitted disease worldwide. Nonetheless, C. trachomatis/N. gonorrhoeae coinfection remains understudied. C. trachomatis/N. gonorrhoeae coinfections are more common than expected by chance, suggesting C. trachomatis/N. gonorrhoeae interaction, and N. gonorrhoeae infection may reactivate genital chlamydial shedding in women with latent (quiescent) chlamydial infection. We hypothesized that N. gonorrhoeae would reactivate latent genital Chlamydia muridarum infection in mice. Two groups of C. muridarum-infected mice were allowed to transition into genital latency. One group was then vaginally inoculated with N. gonorrhoeae; a third group received N. gonorrhoeae alone. C. muridarum and N. gonorrhoeae vaginal shedding was measured over time in the coinfected and singly infected groups. Viable C. muridarum was absent from vaginal swabs but detected in rectal swabs, confirming C. muridarum genital latency and consistent with the intestinal tract as a C. muridarum reservoir. C. muridarum inclusions were observed in large intestinal, but not genital, tissues during latency. Oviduct dilation was associated with C. muridarum infection, as expected. Contradicting our hypothesis, N. gonorrhoeae coinfection did not reactivate latent C. muridarum vaginal shedding. In addition, latent C. muridarum infection did not modulate recovery of vaginal viable N. gonorrhoeae. Evidence for N. gonorrhoeae-dependent increased C. muridarum infectivity has thus not been demonstrated in murine coinfection, and the ability of C. muridarum coinfection to potentiate N. gonorrhoeae infectivity may depend on actively replicating vaginal C. muridarum. The proportion of mice with increased vaginal neutrophils (PMNs) was higher in N. gonorrhoeae-infected than in C. muridarum-infected mice, as expected, while that of C. muridarum/N. gonorrhoeae-coinfected mice was intermediate to the singly infected groups, suggesting latent C. muridarum murine infection may limit PMN response to subsequent N. gonorrhoeae infection. IMPORTANCE Our work builds upon the limited understanding of C. muridarum/N. gonorrhoeae coinfection. Previously, N. gonorrhoeae infection of mice with acute (actively replicating) vaginal C. muridarum infection was shown to increase recovery of viable vaginal N. gonorrhoeae and vaginal PMNs, with no effect on C. muridarum vaginal shedding (R. A. Vonck et al., Infect Immun 79:1566-1577, 2011). It has also been shown that chlamydial infection of human and murine PMNs prevents normal PMN responses, including the response to N. gonorrhoeae (K. Rajeeve et al., Nat Microbiol 3:824-835, 2018). Our findings show no effect of latent genital C. muridarum infection on the recovery of viable N. gonorrhoeae, in contrast to the previously reported effect of acute C. muridarum infection, and suggesting that acute versus latent C. muridarum infection may have distinct effects on PMN function in mice. Together, these studies to date provide evidence that Chlamydia/N. gonorrhoeae synergistic interactions may depend on the presence of replicating Chlamydia in the genital tract, while chlamydial effects on vaginal PMNs may extend beyond acute infection.

Induction of Transmucosal Protection by Oral Vaccination with an Attenuated Chlamydia.

Chlamydia muridarum has been used to study chlamydial pathogenesis because it induces mice to develop hydrosalpinx, a pathology observed in C. trachomatis-infected women. We identified a C. muridarum mutant that is no longer able to induce hydrosalpinx. In the current study, we evaluated the mutant as an attenuated vaccine. Following an intravaginal immunization with the mutant, mice were protected from hydrosalpinx induced by wild-type C. muridarum. However, the mutant itself productively colonized the mouse genital tract and produced infectious organisms in vaginal swabs. Nevertheless, the mutant failed to produce infectious shedding in the rectal swabs following an oral inoculation. Importantly, mice orally inoculated with the mutant mounted transmucosal immunity against challenge infection of wild-type C. muridarum in the genital tract. The protection was detected as early as day 3 following the genital challenge infection and the orally immunized mice were protected from any significant pathology in the upper genital tract. However, the same orally immunized mice failed to prevent the colonization of wild-type C. muridarum in the gastrointestinal tract. The transmucosal immunity induced by the oral mutant was further validated in the airway. The orally vaccinated mice were protected from both lung infection and systemic toxicity caused by intranasally inoculated wild-type C. muridarum although the same mice still permitted the gastrointestinal colonization by the wild-type C. muridarum. These observations suggest that the mutant C. muridarum may be developed into an intracellular oral vaccine vector (or IntrOv) for selectively inducing transmucosal immunity in extra-gut tissues.

Prophylactic and therapeutic vaccination protects sperm health from Chlamydia muridarum-induced abnormalities.

Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the rampant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFß were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220+) B cells and mature (CD138+) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Homologues of the Chlamydia trachomatis and Chlamydia muridarum Inclusion Membrane Protein IncS Are Interchangeable for Early Development but Not for Inclusion Stability in the Late Developmental Cycle.

Chlamydia trachomatis is an obligate intracellular bacterium, which undergoes a biphasic developmental cycle inside a vacuole termed the inclusion. Chlamydia-specific effector proteins embedded into the inclusion membrane, the Inc proteins, facilitate inclusion interaction with cellular organelles. A subset of Inc proteins engages with specific host factors at the endoplasmic reticulum (ER)-inclusion membrane contact site (MCS), which is a discrete point of contact between the inclusion membrane and the endoplasmic reticulum (ER). Here, we report that the C. trachomatis Inc protein CTL0402/IncSCt is a novel component of the ER-inclusion MCS that specifically interacts with and recruits STIM1, a previously identified host component of the ER-inclusion MCS with an unassigned interacting partner at the inclusion membrane. In comparison, the Chlamydia muridarum IncS homologue (TC0424/IncSCm) does not interact with or recruit STIM1 to the inclusion, indicating species specificity. To further investigate IncS function and overcome the recently reported early developmental defect of the incS mutant, we achieved temporal complementation by expressing IncS exclusively during the early stages of the developmental cycle. Additionally, we used allelic exchange to replace the incSCt open reading frame with incSCm in the C. trachomatis chromosome. Inclusions harboring either of these strains progressed through the developmental cycle but were STIM1 negative and displayed increased inclusion lysis 48 h postinfection. Expression of incSCt in trans complemented these phenotypes. Altogether, our results indicate that IncS is necessary and sufficient to recruit STIM1 to C. trachomatis inclusion and that IncS plays an early developmental role conserved in C. trachomatis and C. muridarum and a late role in inclusion stability specific to C. trachomatis. IMPORTANCE Obligate intracellular pathogens strictly rely on the host for replication. Specialized pathogen-encoded effector proteins play a central role in sophisticated mechanisms of host cell manipulation. In Chlamydia, a subset of these effector proteins, the inclusion membrane proteins, are embedded in the membrane of the vacuole in which the bacteria replicate. Chlamydia encodes 50 to 100 putative Inc proteins. Many are conserved among species, including the human and mouse pathogens Chlamydia trachomatis and Chlamydia muridarum, respectively. However, whether the function(s) of Inc proteins is indeed conserved among species is poorly understood. Here, we characterized the function of the Inc protein IncS conserved in C. trachomatis and C. muridarum. Our work reveals that a single effector protein can play multiple functions at various stages of the developmental cycle. However, these functions are not necessarily conserved across species, suggesting a complex evolutionary path among Chlamydia species.

Influx of podoplanin-expressing inflammatory macrophages into the genital tract following Chlamydia infection.

Genital Chlamydia trachomatis infection remains a major health issue as it causes severe complications including pelvic inflammatory disease, ectopic pregnancy and infertility in females as a result of infection-associated chronic inflammation. Podoplanin, a transmembrane receptor, has been previously reported on inflammatory macrophages. Thus, strategies that specifically target podoplanin might be able to reduce local inflammation. This study investigated the expression level and function of podoplanin in a C. trachomatis infection model. C57BL/6 mice infected with the mouse pathogen Chlamydia muridarum were examined intermittently from days 1 to 60 using flow cytometry analysis. Percentages of conventional macrophages (CD11b+ CD11c- F4/80+ ) versus inflammatory macrophages (CD11b+ CD11c+ F4/80+ ), and the expression of podoplanin in these cells were investigated. Subsequently, a podoplanin-knockout RAW264.7 cell was used to evaluate the function of podoplanin in C. trachomatis infection. Our findings demonstrated an increased CD11b+ cell volume in the spleen at day 9 after the infection, with augmented podoplanin expression, especially among the inflammatory macrophages. A large number of podoplanin-expressing macrophages were detected in the genital tract of C. muridarum-infected mice. Furthermore, analysis of the C. trachomatis-infected patients demonstrated a higher percentage of podoplanin-expressing monocytes than that in the noninfected controls. Using an in vitro infection in a transwell migration assay, we identified that macrophages deficient in podoplanin displayed defective migratory function toward C. trachomatis-infected HeLa 229 cells. Lastly, using immunoprecipitation-mass spectrometry method, we identified two potential podoplanin interacting proteins, namely, Cofilin 1 and Talin 1 actin-binding proteins. The present study reports a role of podoplanin in directing macrophage migration to the chlamydial infection site. Our results suggest a potential for reducing inflammation in individuals with chronic chlamydial infections by targeting podoplanin.

Regulation of chlamydial colonization by IFNγ delivered via distinct cells.

The mouse-adapted pathogen Chlamydia muridarum (CM) induces pathology in the mouse genital tract but fails to do so in the gastrointestinal tract. CM is cleared from both the genital tract and small intestine by IFNγ delivered by antigen-specific CD4+ T cells but persists for a long period in the large intestine. The long-lasting colonization of CM in the large intestine is regulated by IFNγ delivered by group 3 innate lymphoid cells (ILC3s). Interestingly, the ILC3-delivered IFNγ can inhibit the human pathogen Chlamydia trachomatis (CT) in the mouse endometrium. Thus, IFNγ produced/delivered by different cells may selectively restrict chlamydial colonization in different tissues. Revealing the underlying mechanisms of chlamydial interactions with IFNγ produced by different cells may yield new insights into both chlamydial pathogenicity and mucosal immunity.

Chlamydia transmitting from the genital to gastrointestinal tract and inducing tubal disease: Double attack pattern. / ç”Ÿæ®–é“è¡£åŽŸä½“ä¼ æ’­è‡³èƒƒè‚ é“å¹¶é¶å‘è¯±å¯¼è¾“åµç®¡ç— å˜­­åŒé‡æ”»å‡»æ¨¡å¼.

Chlamydia trachomatis ( CT ) genital tract infection is insidious, and patients often have no conscious symptoms.Delayed treatment after infection can lead to serious complications. Chlamydia muridarum ( CM ) genital tract infection in female mice can simulate CT genital tract infection in women, which is an ideal model to investigate the pathogenesis of CT . CM plasmid protein pGP3, chromosomal protein TC0237/TC0668, CM -specific CD8 + T cells, TNF-α, and IL-13 can induce genital tract inflammation, CD4 + T cells are responsible for CM clearance. However, tubal inflammation persists after genital tract CM is removed. Genital tract CM can spread spontaneously in vivo and colonize the gastrointestinal (GI) tract, but the GI tract CM cannot reverse spread to the genital tract. The survival time and number of CM transmitted from genital tract to GI tract are positively correlated with the long-term lesion of oviduct, while the CM inoculated directly into the GI tract has no pathogenicity in both the genital and GI tract. The double attack pattern of Chlamydia -induced genital tract inflammatory lesions is as follows: CM infection of oviduct epithelial cells initiates the process of oviduct repair as the first attack. After genital CM spreads to the GI tract, activated chlamydia-specific CD8 + T cells are recruited to the genital tract and secreted pro-fibrotic cytokines such as TNF-α and IL-13. This process is called the second attack which transform tubal repair initiated by the first attack into long-term tubal fibrosis/hydrosalpinx. Elucidating the pathogenic mechanism of Chlamydia infection can provide new ideas for the development of Chlamydia vaccine, which is expected to solve the problems of infertility caused by repeated CT infection in women.

IFNγ and Antibody Synergize To Enhance Protective Immunity against Chlamydia Dissemination and Female Reproductive Tract Reinfections.

CD4 T cell-dependent IFNγ production and antibody are the two best known effectors for protective immunity against Chlamydia female reproductive tract (FRT) infection. Nevertheless, mice lacking either IFNγ or B cells can clear the vast majority of Chlamydia from the FRT, while suffering from varying degrees of disseminated infection. In this study, we investigated whether IFNγ and B cells play complementary roles in host defense against Chlamydia and evaluated their relative contributions in systemic and mucosal tissues. Using mice deficient in both IFNγ and B cells (IFNγ-/- x µMT), we showed that mice lacking both effectors were highly susceptible to lethal systemic bacterial dissemination following Chlamydia muridarum intravaginal infection. Passive transfer of immune convalescent serum, but not recombinant IFNγ, reduced bacterial burden in both systemic and mucosal tissues in IFNγ-/- x µMT mice. Notably, over the course of primary infection, we observed a reduction of bacterial shedding of more than 2 orders of magnitude in IFNγ-/- x µMT mice following both C. muridarum and C. trachomatis FRT infections. In contrast, no protective immunity against C. muridarum reinfection was detected in the absence of IFNγ and B cells. Together, our results suggest that IFNγ and B cells synergize to combat systemic Chlamydia dissemination, while additional IFNγ and B cell-independent mechanisms exist for host resistance to Chlamydia in the lower FRT.

A Minimal Replicon Enables Efficacious, Species-Specific Gene Deletion in Chlamydia and Extension of Gene Knockout Studies to the Animal Model of Infection Using Chlamydia muridarum.

The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause specific diseases, it is imperative to develop a mutagenesis approach that would be applicable to every chlamydial species. We present functional evidence that the region between Chlamydia trachomatis and Chlamydia muridarum pgp6 and pgp7, containing four 22-bp tandem repeats that are present in all chlamydial endogenous plasmids, represents the plasmid origin of replication. Furthermore, by introducing species-specific ori regions into an engineered 5.45-kb pUC19-based plasmid, we generated vectors that can be successfully transformed into and propagated under selective pressure by C. trachomatis serovars L2 and D, as well as C. muridarum. Conversely, these vectors were rapidly lost upon removal of the selective antibiotic. This conditionally replicating system was used to generate a tarP deletion mutant by fluorescence-reported allelic exchange mutagenesis in both C. trachomatis serovar D and C. muridarum. The strains were analyzed using in vitro invasion and fitness assays. The virulence of the C. muridarum strains was then assessed in a murine infection model. Our approach represents a novel and efficient strategy for targeted genetic manipulation in Chlamydia beyond C. trachomatis L2. This advance will support comparative studies of species-specific infection biology and enable studies in a well-established murine model of chlamydial pathogenesis.