Circulating immunity protects the female reproductive tract from Chlamydia infection.

Anatomical positioning of memory lymphocytes within barrier tissues accelerates secondary immune responses and is thought to be essential for protection at mucosal surfaces. However, it remains unclear whether resident memory in the female reproductive tract (FRT) is required for Chlamydial immunity. Here, we describe efficient generation of tissue-resident memory CD4 T cells and memory lymphocyte clusters within the FRT after vaginal infection with Chlamydia Despite robust establishment of localized memory lymphocytes within the FRT, naïve mice surgically joined to immune mice, or mice with only circulating immunity following intranasal immunization, were fully capable of resisting Chlamydia infection via the vaginal route. Blocking the rapid mobilization of circulating memory CD4 T cells to the FRT inhibited this protective response. These data demonstrate that secondary protection in the FRT can occur in the complete absence of tissue-resident immune cells. The ability to confer robust protection to barrier tissues via circulating immune memory provides an unexpected opportunity for vaccine development against infections of the FRT.

T cell responses to Chlamydia.

Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection.

Immunopathogenesis of genital Chlamydia infection: insights from mouse models.

Chlamydiae are pathogenic intracellular bacteria that cause a wide variety of diseases throughout the globe, affecting the eye, lung, coronary arteries and female genital tract. Rather than by direct cellular toxicity, Chlamydia infection generally causes pathology by inducing fibrosis and scarring that is largely mediated by host inflammation. While a robust immune response is required for clearance of the infection, certain elements of that immune response may also damage infected tissue, leading to, in the case of female genital infection, disease sequelae such as pelvic inflammatory disease, infertility and ectopic pregnancy. It has become increasingly clear that the components of the immune system that destroy bacteria and those that cause pathology only partially overlap. In the ongoing quest for a vaccine that prevents Chlamydia-induced disease, it is important to target mechanisms that can achieve protective immunity while preventing mechanisms that damage tissue. This review focuses on mouse models of genital Chlamydia infection and synthesizes recent studies to generate a comprehensive model for immunity in the murine female genital tract, clarifying the respective contributions of various branches of innate and adaptive immunity to both host protection and pathogenic genital scarring.

Comparison of In Vitro Chlamydia muridarum Infection Under Aerobic and Anaerobic Conditions.

Although Chlamydia infects host body regions that are hypoxic to anoxic, standard Chlamydiae culture conditions are in CO2 enriched (5%) atmospheric oxygen (21%). Because of its success in causing disease in principally anaerobic body sites, e.g., vaginal tract, we hypothesize that Chlamydia has an anaerobic life cycle that plays a role in its maintenance in the host. Using a model system developed for the anaerobic culture of mammalian cells, we assessed the anoxic infectious cycle of C. muridarum in anaerobically cultured HeLa 229 cells. In the absence of oxygen, C. muridarum is capable of going through their life cycle, although its cycle is slowed (2 days post-infection anaerobic vs. 1 day aerobic). Interestingly, in addition to a slower rate of replication, there is a reduction in Chlamydia inclusion number and size as compared to aerobic controls. Anaerobic infected host cell physiology also changed with IL-6 and IL-8 production significantly lower (p ≤ 0.05) compared to aerobic infected host cells (day 4 post-infection). These findings demonstrate that Chlamydia are capable of replicating in the absence of oxygen.

Optimal cultivation of Chlamydia requires testing of serum on individual species.

OBJECTIVE: This report is a side product of experiments aimed at identifying serum for culturing obligate intracellular bacteria Chlamydia trachomatis and C. muridarum in mouse fibroblast L929 cells. RESULTS: Of five commercial serum samples tested, two showed optimal efficiencies at supporting growth of the human pathogen Chlamydia trachomatis as control fetal bovine serum, whereas two showed modest ~ 40% inhibitions in progeny production, and the remaining one showed a 20% inhibition. Three of the six sera poorly supported growth of the murine pathogen Chlamydia muridarum, resulting in 73-90% reduction in progeny formation. Most significantly, the one with the strongest (90%) C. muridarum inhibition activity showed optimal C. trachomatis-supporting efficiency. These findings indicate that in laboratories that study multiple Chlamydia species, serum samples should be prescreened on a species basis. Considering Chlamydial biology and epidemiology, it may even be necessary to perform serum tests on a serovar- or strain-basis for studying some animal chlamydiae.

The Cryptic Plasmid Improves Chlamydia Fitness in Different Regions of the Gastrointestinal Tract.

The cryptic plasmid is important for chlamydial colonization in the gastrointestinal tract. We used a combination of intragastric, intrajejunal, and intracolon inoculations to reveal the impact of the plasmid on chlamydial colonization in distinct regions of gastrointestinal tract. Following an intragastric inoculation, the plasmid significantly improved chlamydial colonization. At the tissue level, plasmid-positive Chlamydia produced infectious progenies throughout gastrointestinal tract. However, to our surprise, plasmid-deficient Chlamydia failed to produce infectious progenies in small intestine, although infectious progenies were eventually detected in large intestine, indicating a critical role of the plasmid in chlamydial differentiation into infectious particles in small intestine. The noninfectious status may represent persistent infection, since Chlamydia genomes proliferated in the same tissues. Following an intrajejunal inoculation that bypasses the gastric barrier, plasmid-deficient Chlamydia produced infectious progenies in small intestine but was 530-fold less infectious than plasmid-positive Chlamydia, suggesting that (i) the noninfectious status developed after intragastric inoculation might be induced by a combination of gastric and intestinal effectors and (ii) chlamydial colonization in small intestine was highly dependent on plasmid. Finally, following an intracolon inoculation, the dependence of chlamydial colonization on plasmid increased over time. Thus, we have demonstrated that the plasmid may be able to improve chlamydial fitness in different gut regions via different mechanisms, which has laid a foundation to further reveal the specific mechanisms.

A primary study on genes with selected mutations by in vitro passage of Chlamydia muridarum strains.

OBJECTIVE: This study is to investigate the functions of newly discovered genes in Chlamydia muridarum (C. muridarum) strains with single gene differences. METHODS: Using whole genome sequencing and plaque formation assays, C. muridarum parental and passaging strains were established, and the isogenic clones expressing certain genotypes were isolated. Strains with single gene differences were obtained. Based on prediction, the valuable strains with single gene differences of tc0412, tc0668 or tc0237 were subjected to the in vitro and in vivo experiments for biological characterization and virulence analysis. RESULTS: Insertional -472840T mutation of the tc0412 gene (T28T/B3 type) matching with the nonmutant tc0668 gene and tc0237 gene with point mutations G797659T (Q117E) might slow the growth of Chlamydia due to the lack of a plasmid. The nonmutant tc0668 in the strain might induce a high incidence of hydrosalpinx in mice, while tc0668 with a G797659T point mutation was significantly attenuated. Compared with the nonmutant tc0237, the strains containing mutant tc0237 were characterized by reduced centrifugation dependence during infection. CONCLUSION: The identification and characterization of these genes might contribute to the comprehensive understanding of the pathogenic mechanism of Chlamydia.

Chlamydia muridarum with Mutations in Chromosomal Genes tc0237 and/or tc0668 Is Deficient in Colonizing the Mouse Gastrointestinal Tract.

Chlamydiae colonize the gastrointestinal tracts of both animals and humans. However, their medical significance remains unknown. We have previously shown that wild-type Chlamydia muridarum spreads to and establishes stable colonization of the gastrointestinal tract following intravaginal inoculation. In the present study, we found that C. muridarum with mutations in chromosomal genes tc0237 and/or tc0668 was defective in spreading to the mouse gastrointestinal tract, which correlated with its attenuated pathogenicity in the upper genital tract. This correlation was more consistent than that of chlamydial pathogenicity with ascending infection in the genital tract, since attenuated C. muridarum spread significantly less to the gastrointestinal tract but maintained robust ascending infection of the upper genital tract. Transcervical inoculation further confirmed the correlation between C. muridarum spreading to the gastrointestinal tract and its pathogenicity in the upper genital tract. Finally, defective spreading of C. muridarum mutants was due to their inability to colonize the gastrointestinal tract since intragastric inoculation did not rescue the mutants' colonization. Thus, promoting C. muridarum colonization of the gastrointestinal tract may represent a primary function of the TC0237 and TC0668 proteins. Correlation of chlamydial colonization of the gastrointestinal tract with chlamydial pathogenicity in the upper genital tract suggests a potential role for gastrointestinal chlamydiae in genital tract pathogenicity.

Infection of Hysterectomized Mice with Chlamydia muridarum and Chlamydia trachomatis.

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.

Effect of cold water-induced stress on immune response, pathology and fertility in mice during Chlamydia muridarum genital infection.

Genital infection by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. It causes serious reproductive health complications, including pelvic inflammatory disease and infertility. Stress is implicated as a risk factor for various infections; however, its effect on chlamydia genital infection is unknown. We previously showed that repeated exposure of mice to cold water results in increased severity of chlamydia genital infection. In this study, cold water-induced stress resulted in (i) elevated levels of norepinephrine (NE) and epinephrine in the spleen and genital tract of stressed mice; (ii) elevated IL-1ß, TNF-α, IL-6 and nitric oxide production in macrophage-rich peritoneal cells of mice; (iii) supplement of NE in vitro exerts an immunosuppressive effect on splenic T-cell production of cytokines; (iv) decreased C. muridarum shedding in the genital tract of ß1Adr/ß2Adr receptor KO mice; and (v) a higher rate of infertility in infected mice. These results suggest that cold water stress induces the production of catecholamines, which may play a critical role in the modulation of the immune system leading to increased intensity of C. muridarum genital infection.

Host Nectin-1 Promotes Chlamydial Infection in the Female Mouse Genital Tract, but Is Not Required for Infection in a Novel Male Murine Rectal Infection Model.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection.

The Chromosome-Encoded Hypothetical Protein TC0668 Is an Upper Genital Tract Pathogenicity Factor of Chlamydia muridarum.

We previously associated a missense mutation of the tc0668 gene of serial in vitro-passaged Chlamydia muridarum, a murine model of human urogenital C. trachomatis, with severely attenuated disease development in the upper genital tract of female mice. Since these mutants also contained a TC0237 Q117E missense mutation that enhances their in vitro infectivity, an effort was made here to isolate and characterize a tc0668 single mutant to determine its individual contribution to urogenital pathogenicity. Detailed genetic analysis of C. muridarum passages revealed a truncated variant with a G216* nonsense mutation of the 408-amino-acid TC0668 protein that does not produce a detectable product. Intracellular growth and infectivity of C. muridarum in vitro remain unaffected in the absence of TC0668. Intravaginal inoculation of the TC0668 null mutant into C3H/HeJ mice results in a typical course of lower genital tract infection but, unlike a pathogenic isogenic control, is unable to elicit significant chronic inflammation of the oviduct and fails to induce hydrosalpinx. Thus, TC0668 is demonstrated as an important chromosome-encoded urogenital pathogenicity factor of C. muridarum and the first with these characteristics to be discovered for a Chlamydia pathogen.

Modeling the transcriptome of genital tract epithelial cells and macrophages in healthy mucosa versus mucosa inflamed by Chlamydia muridarum infection.

Chlamydia trachomatis urogenital serovars are intracellular bacteria that parasitize human reproductive tract epithelium. As the principal cell type supporting bacterial replication, epithelial cells are central to Chlamydia immunobiology initially as sentries and innate defenders, and subsequently as collaborators in adaptive immunity-mediated bacterial clearance. In asymptomatic individuals who do not seek medical care a decisive struggle between C. trachomatis and host defenses occurs at the epithelial interface. For this study, we modeled the immunobiology of epithelial cells and macrophages lining healthy genital mucosa and inflamed/infected mucosa during the transition from innate to adaptive immunity. Upper reproductive tract epithelial cell line responses were compared to bone marrow-derived macrophages utilizing gene expression microarray technology. Those comparisons showed minor differences in the intrinsic innate defenses of macrophages and epithelial cells. Major lineage-specific differences in immunobiology relate to epithelial collaboration with adaptive immunity including an epithelial requirement for inflammatory cytokines to express MHC class II molecules, and a paucity and imbalance between costimulatory and coinhibitory ligands on epithelial cells that potentially limits sterilizing immunity (replication termination) to Chlamydia-specific T cells activated with limited or unconventional second signals.

Genomic variant representation in a Chlamydia population is dynamic and adaptive with dependence on in vitro and in vivo passage.

We have previously shown that Chlamydia muridarum has multiple genomic variants that concomitantly vary in their in vitro and in vivo phenotype. Herein, we used real-time polymerase chain reaction-based genotyping assays to query plaque-cloned isolates of C. muridarum for the frequency of eight selected polymorphisms. These strains had no history of passage in vivo since their original isolation from laboratory mice. There was significant variance in the frequency of two of the eight polymorphisms assessed with the remaining exhibiting a low rate of variance. To determine if any of these polymorphisms were more favorable for in vivo conditions, we blindly passaged non-clonal C. muridarum three times at 7-day intervals through the urogenital tract of mice. Seven of the eight polymorphisms varied in frequency following in vivo passage and four of these varied between C. muridarum strains. Selected isolates displayed variable growth rates and cytopathic effect in vitro. We conclude that multiple genotypic variants are present within the existing known C. muridarum strains and that the frequency of these variants changes upon introduction into the mouse host. These findings lend support to the concept that genotypic proportional representation in a chlamydial population is dynamic and adaptive.

Benzylidene acylhydrazides inhibit chlamydial growth in a type III secretion- and iron chelation-independent manner.

Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value.

In vivo whole animal body imaging reveals colonization of Chlamydia muridarum to the lower genital tract at early stages of infection.

PURPOSE: The leading cause of sexually transmitted bacterial infection is Chlamydia trachomatis. The aim of this study is to investigate the early events in colonization of this bacterium within the murine genital tract. PROCEDURES: An in vivo animal body imaging technology was used to track fluorophore labeled C. muridarum elementary bodies (EBs) inoculated intravaginally in C57BL/6 mice during the first 24 h of infection. RESULTS: Ascension of viable EBs was observed (1) to be localized to the lower regions of the murine genital tract within the first 24 h post challenge and (2) was dose independent during this early exposure period. Molecular detection revealed enhanced bacterial load in lower regions of the genital tract with increasing bacterial load in the upper region beginning 12 h post inoculation. CONCLUSION: This study provides additional insight into chlamydial colonization in the murine genital tract during the first 12-24 h following inoculation.

Intracellular survival and persistence of Chlamydia muridarum is determined by macrophage polarization.

Macrophages can display a number of distinct phenotypes, known collectively as polarized macrophages. The best defined of these phenotypes are the classically-activated, interferon gamma (IFNγ)/LPS induced (M1) and alternatively-activated, IL-4 induced (M2) macrophages. The goal of this study is to characterize macrophage-Chlamydia interactions in the context of macrophage polarization. Here we use Chlamydia muridarum and murine bone-marrow derived macrophages to show Chlamydia does not induce M2 polarization in macrophages as a survival strategy. Unexpectedly, the infection of macrophages was silent with no upregulation of M1 macrophage-associated genes. We further demonstrate that macrophages polarized prior to infection have a differential capacity to control Chlamydia. M1 macrophages harbor up to 40-fold lower inclusion forming units (IFU) than non-polarized or M2 polarized macrophages. Gene expression analysis showed an increase in 16sRNA in M2 macrophages with no change in M1 macrophages. Suppressed Chlamydia growth in M1 macrophages correlated with the induction of a bacterial gene expression profile typical of persistence as evident by increased Euo expression and decreased Omp1 and Tal expression. Observations of permissive Chlamydia growth in non-polarized and M2 macrophages and persistence in M1 macrophages were supported through electron microscopy. This work supports the importance of IFNγ in the innate immune response to Chlamydia. However, demonstration that the M1 macrophages, despite an antimicrobial signature, fail to eliminate intracellular Chlamydia supports the notion that host-pathogen co-evolution has yielded a pathogen that can evade cellular defenses against this pathogen, and persist for prolonged periods of time in the host.

Endosulfatases SULF1 and SULF2 limit Chlamydia muridarum infection.

The first step in attachment of Chlamydia to host cells is thought to involve reversible binding to host heparan sulfate proteoglycans (HSPGs), polymers of variably sulfated repeating disaccharide units coupled to diverse protein backbones. However, the key determinants of HSPG structure that are involved in Chlamydia binding are incompletely defined. A previous genome-wide Drosophila RNAi screen suggested that the level of HSPG 6-O sulfation rather than the identity of the proteoglycan backbone maybe a critical determinant for binding. Here, we tested in mammalian cells whether SULF1 or SULF2, human endosulfatases, which remove 6-O sulfates from HSPGs, modulate Chlamydia infection. Ectopic expression of SULF1 or SULF2 in HeLa cells, which decreases cell surface HSPG sulfation, diminished C. muridarum binding and decreased vacuole formation. ShRNA depletion of endogenous SULF2 in a cell line that primarily expresses SULF2 augmented binding and increased vacuole formation. C. muridarum infection of diverse cell lines resulted indownregulation of SULF2 mRNA. In a murine model of acute pneumonia, mice genetically deficient in both endosulfatases or in SULF2 alone demonstrated increased susceptibility to C. muridarum lung infection. Collectively, these studies demonstrate that the level of HSPG 6-O sulfation is a critical determinant of C. muridarum infection in vivo and that 6-O endosulfatases are previously unappreciated modulators of microbial pathogenesis.