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.

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.

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.

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.

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.

Chlamydia causes fragmentation of the Golgi compartment to ensure reproduction.

The obligate intracellular bacterium Chlamydia trachomatis survives and replicates within a membrane-bound vacuole, termed the inclusion, which intercepts host exocytic pathways to obtain nutrients. Like many other intracellular pathogens, C. trachomatis has a marked requirement for host cell lipids, such as sphingolipids and cholesterol, produced in the endoplasmic reticulum and the Golgi apparatus. However, the mechanisms by which intracellular pathogens acquire host cell lipids are not well understood. In particular, no host cell protein responsible for transporting Golgi-derived lipids to the chlamydial inclusions has yet been identified. Here we show that Chlamydia infection in human epithelial cells induces Golgi fragmentation to generate Golgi ministacks surrounding the bacterial inclusion. Ministack formation is triggered by the proteolytic cleavage of the Golgi matrix protein golgin-84. Inhibition of golgin-84 truncation prevents Golgi fragmentation, causing a block in lipid acquisition and maturation of C. trachomatis. Golgi fragmentation by means of RNA-interference-mediated knockdown of distinct Golgi matrix proteins before infection enhances bacterial maturation. Our data functionally connect bacteria-induced golgin-84 cleavage, Golgi ministack formation, lipid acquisition and intracellular pathogen growth. We show that C. trachomatis subverts the structure and function of an entire host cell organelle for its own advantage.

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.

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.

Immunization with live and dead Chlamydia muridarum induces different levels of protective immunity in a murine genital tract model: correlation with MHC class II peptide presentation and multifunctional Th1 cells.

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ(+)-producing CD4(+) T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4(+) T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.

Chlamydia-specific CD4 T cell clones control Chlamydia muridarum replication in epithelial cells by nitric oxide-dependent and -independent mechanisms.

Chlamydia trachomatis serovars D-K are sexually transmitted intracellular bacterial pathogens that replicate in epithelial cells lining the human reproductive tract. It is clear from knockout mice and T cell depletion studies using Chlamydia muridarum that MHC class II and CD4 T cells are critical for clearing bacteria from the murine genital tract. It is not clear how CD4 T cells interact with infected epithelial cells to mediate bacterial clearance in vivo. Previous work using an epithelial tumor cell line showed that a Chlamydia-specific CD4 T cell clone was able to inhibit C. muridarum replication in vitro via induction of epithelial NO production. We have previously shown that Chlamydia-specific CD4 T cell clones can recognize and be activated by infected reproductive tract epithelial cells and block Chlamydia replication in them. We extend those observations by investigating the mechanism used by a panel of CD4 T cell clones to control Chlamydia replication in epithelial cells. We found that Chlamydia-specific CD4 T cell clones were cytolytic, but that cytolysis was not likely critical for controlling C. muridarum replication. For one, CD4 T cell clone-induced epithelial NO production was critical for controlling replication; however, the most potent CD4 T cell clones were dependent on T cell degranulation for replication control with only a minor additional contribution from NO production. We discuss our data as they relate to existing knockout mouse studies addressing mechanisms of T cell-mediated control of Chlamydia replication and their implications for intracellular epithelial pathogens in mouse models.

Regulation of chlamydial infection by host autophagy and vacuolar ATPase-bearing organelles.

As arguably the most successful parasite, Chlamydia is an obligate intracellular bacterium replicating inside a vacuole of eukaryotic host cells. The chlamydial vacuole does not fuse with the defense cell organelle lysosome. We previously showed that chlamydial infection increases markers of autophagy, an innate antimicrobial activity requiring lysosomal function. However, the work presented here demonstrates that p62, an autophagy protein that is degraded in lysosomes, either remained unchanged or increased in chlamydia-infected human epithelial, mouse fibroblast, and mouse macrophage cell lines. In addition, the activities of three lysosomal enzymes analyzed were diminished in chlamydia-infected macrophages. Bafilomycin A1 (BafA), a specific inhibitor of vacuolar ATPase (vATPase) required for lysosomal function, increased the growth of the human pathogen Chlamydia trachomatis (L2) in wild-type murine fibroblasts and macrophages but inhibited growth in the autophagy-deficient ATG5(-/-) fibroblasts. BafA exhibited only slight inhibition or no effect on L2 growth in multiple human genital epithelial cell lines. In contrast to L2, the mouse pathogen Chlamydia muridarum (MoPn) was consistently inhibited by BafA in all cell lines examined, regardless of species origin and autophagy status. Finally, L2 but not MoPn grew more efficiently in the ATG5(-/-) cells than in wild-type cells. These results suggest that there are two types of vATPase-bearing organelles that regulate chlamydial infection: one supports chlamydial infection, while the other plays a defensive role through autophagy when cells are artificially infected with certain chlamydiae that have not been adapted to the host species.

A MyD88-dependent early IL-17 production protects mice against airway infection with the obligate intracellular pathogen Chlamydia muridarum.

We found that IL-17, a signature cytokine of Th17, was produced early in the innate immunity phase after an intranasal infection with the obligate intracellular pathogen Chlamydia muridarum. The airway IL-17, which peaked at 48 h after infection, was dependent on live chlamydial organism replication and MyD88-mediated signaling pathways. Treatment with antibiotics or knockout of the MyD88 gene, but not Toll/IL receptor domain-containing adapter-inducing IFN-beta, can block the early IL-17 production. Treatment of mice with an anti-IL-17-neutralizing mAb enhanced growth of chlamydial organisms in the lung, dissemination to other organs, and decreased mouse survival, whereas treatment with an isotype-matched control IgG had no effect. Although IL-17 did not directly affect chlamydial growth in cell culture, it enhanced the production of other inflammatory cytokines and chemokines by Chlamydia-infected cells and promoted neutrophil infiltration in mouse airways during chlamydial infection, which may contribute to the antichlamydial effect of IL-17. These observations suggest that an early IL-17 response as an innate immunity component plays an important role in initiating host defense against infection with intracellular bacterial pathogens in the airway.

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.

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.

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.

Regulation of surfactant protein D in the mouse female reproductive tract in vivo.

Surfactant protein D (SP-D) plays a role in innate immunity in the lung and is expressed at many other mucosal surfaces throughout the human body. In this study, we show that SP-D mRNA and protein are present in the murine female reproductive tract; i.e. in the vagina, cervix, uterus and oviduct. SP-D protein is primarily localized to epithelial cells lining the genital tract and is also present in secretory material within the lumen of the uterus and cervix. The levels of SP-D mRNA in the uterus vary by a factor of 10 during the estrous cycle with peak levels present at estrus and the lowest levels at diestrus. In contrast, SP-D mRNA levels in the lung do not change during the estrous cycle. Since SP-D is an innate host defense protein present in the mouse reproductive tract, we studied the influence of infection on SP-D levels in vivo. We found that Chlamydia muridarum infection caused an increase in the SP-D protein content of reproductive tract epithelial cells. These data are suggestive that SP-D may play a role in innate immunity in the female reproductive tract in vivo.

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.

COX-2 inhibition affects growth rate of Chlamydia muridarum within epithelial cells.

Chlamydiae alter apoptosis of host target cells, which regulates their growth. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme for prostaglandin E2 (PGE2) production, modulates epithelial cell survival. We addressed whether endogenous PGE2 alters chlamydial growth or apoptosis of epithelial cells infected with Chlamydia muridarum. PGE2 is secreted by infected host cells in the genital tract (GT). Using immunohistochemical techniques, we found that COX-2 enzyme was localized to epithelial cells in the GT in vivo. Pellets of the COX-2 enzyme inhibitor, NS-398, and placebo were implanted in mice subcutaneously and released a constant amount of these chemicals throughout the infection. NS-398-treated mice were found to exhibit 10-fold lower bacterial load than the placebo group on day 3 post infection, suggesting disruption of the chlamydial developmental cycle. To prove this, the human lung adenocarcinoma cell line A549 was then infected with different MOIs of C. muridarum in the presence of multiple concentrations of NS-398 in vitro. There was no difference in inclusion forming units (IFUs) between NS-389-treated and untreated cells. We also found no alterations in C. muridarum IFUs in A549 cells transfected with a 2.0 kb cDNA fragment of human COX-2 cloned in the sense (S) or anti-sense (AS) orientation. However, the inclusion size was reduced and the number of EB was significantly diminished during reinfection in AS-transfected cells. In addition, the absence of COX-2 did not significantly modify apoptosis in infected cells. In total, COX-2 deficiency reduces the infectious burden in vivo and may modulate transmission of the organism.