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Tail-specific proteases (Tsp) are members of a widely distributed family of serine proteases that commonly target and process periplasmic proteins in Gram-negative bacteria. The obligately intracellular, Gram-negative Chlamydia encode a highly conserved Tsp homolog whose target and function are unclear. We identified a Chlamydia muridarum mutant with a nonsense mutation in tsp . Differentiation of the tsp mutant elementary bodies into vegetative reticulate bodies was delayed at 37°C and completely blocked at 40°C. Tsp localized to C. muridarum cells but was not detected outside the inclusion, suggesting that it targets chlamydial rather than host proteins. The abundance of key chlamydia outer membrane complex and virulence-related proteins differed in wild-type and tsp mutant elementary bodies, consistent with the possibility that Tsp regulates developmental cycle progression. The altered abundances of chlamydial structural and virulence factors could explain why the mutant, but not an isogenic recombinant with wild-type tsp , was highly attenuated in a mouse intravaginal infection model. Thus, chlamydial Tsp is required for timely differentiation of elementary bodies into reticulate bodies in vitro and is an essential virulence factor in vivo ., Competing Interests: The authors declare no conflict of interest.

Background: Chlamydia trachomatis (CT) infection remains highly prevalent, and young women are disproportionately affected. Most CT-infected women are asymptomatic, and their infection often goes unrecognized and untreated. We hypothesized that testing for active CT infection with molecular diagnostics and obtaining a reported history of CT infection underestimate the prevalence of current and past CT infection, and incorporating serum CT antibody testing in addition to these other prevalence measures would generate more accurate estimates of the prevalence of CT infection in asymptomatic young women., Methods: We enrolled 362 asymptomatic women aged 16 to 29 years at 4 different clinical settings in Birmingham, AL, between August 2016 and January 2020 and determined the prevalence of CT infection based on having 1 or more of the following prevalence measures: an active urogenital CT infection based on molecular testing, reported prior CT infection, and/or being CT seropositive. Multivariable regression analysis was used to determine predictors of the prevalence of CT infection after adjustment for participant characteristics., Results: The prevalence of CT infection was 67.7% (95% confidence interval, 62.6%-72.5%). Addition of CT antibody testing to the other individual prevalence measures more than doubled the CT infection prevalence. Non-Hispanic Black race, reported prior gonorrhea, and reported prior trichomoniasis predicted a higher prevalence of CT infection., Conclusions: More than half of women were unaware of ever having CT infection, suggesting many were at risk for CT-associated reproductive complications. These data reinforce the need to adhere to chlamydia screening guidelines and to increase screening coverage in those at risk., Competing Interests: Conflict of Interest and Sources of Funding: W.M.G. and B.V.D.P. report receiving research funding from Hologic, Inc, outside the range of the current work. The other authors report no potential conflicts of interest., (Copyright © 2021 American Sexually Transmitted Diseases Association. All rights reserved.)

Chlamydia spp. productively infect mucosal epithelial cells of multiple anatomical sites, including the conjunctiva, lungs, gastrointestinal (GI) tract, and urogenital tract. We, and others, previously established that chlamydial GI tropism is mediated by distinct chromosomal and plasmid factors. In this study, we describe a genital infection-attenuated Chlamydia muridarum mutant (GIAM-1) that is profoundly and specifically attenuated in the murine genital tract. GIAM-1 infected the murine GI tract similarly to wild-type (WT) Chlamydia muridarum but did not productively infect the lower genital tract of female mice, ascend to infect the upper genital tract, or cause hydrosalpinx. However, GI infection of mice with GIAM-1 elicited a transmucosal immune response that protected against subsequent genital challenge with WT Chlamydia muridarum Collectively, our results demonstrate that chlamydia mutants that are profoundly attenuated for specific organ tissues can be derived and demonstrate that live-attenuated vaccine strains that infect the GI tract, but do not elicit genital tract disease, could be used to protect against chlamydia genital tract infection and disease. IMPORTANCE Chlamydia is the most common sexually transmitted bacterial infection in the United States. Most chlamydia genital infections resolve without serious consequences; however, untreated infection in women can cause pelvic inflammatory disease and infertility. Antibiotics are very effective in treating chlamydia, but most genital infections in both men and women are asymptomatic and go undiagnosed. Therefore, there is a critical need for an effective vaccine. In this work, we show that a mutant chlamydia strain, having substantially reduced virulence for genital infection, colonizes the gastrointestinal tract and produces robust immunity to genital challenge with fully virulent wild-type chlamydia. These results are an important advance in understanding chlamydial virulence and provide compelling evidence that safe and effective live-attenuated chlamydia vaccines may be feasible., (Copyright © 2020 Morrison et al.)

Some members of the genus Chlamydia , including the human pathogen Chlamydia trachomatis , infect multiple tissues, including the genital and gastrointestinal (GI) tracts. However, it is unknown if bacterial targeting to these sites is mediated by multifunctional or distinct chlamydial factors. We previously showed that disruption of individual large clostridial toxin homologs encoded within the Chlamydia muridarum plasticity zone were not critical for murine genital tract infection. Here, we assessed whether cytotoxin genes contribute to C. muridarum GI tropism. Infectivity and shedding of wild-type (WT) C. muridarum and three mutants containing nonsense mutations in different cytotoxin genes, tc0437 , tc0438 , and tc0439 , were compared in mouse genital and GI infection models. One mutant, which had a nonsense mutation in tc0439 , was highly attenuated for GI infection and had a GI 50% infectious dose (ID 50 ) that was 1,000 times greater than that of the WT. GI inoculation with this mutant failed to elicit anti-chlamydial antibodies or to protect against subsequent genital tract infection. Genome sequencing of the tc0439 mutant revealed additional chromosomal mutations, and phenotyping of additional mutants suggested that the GI attenuation might be linked to a nonsense mutation in tc0600 The molecular mechanism underlying this dramatic difference in tissue-tropic virulence is not fully understood. However, isolation of these mutants demonstrates that distinct chlamydial chromosomal factors mediate chlamydial tissue tropism and provides a basis for vaccine initiatives to isolate chlamydia strains that are attenuated for genital infection but retain the ability to colonize the GI tract and elicit protective immune responses., (Copyright © 2018 Morrison et al.)

Determining the effector populations involved in humoral protection against genital chlamydia infection is crucial to development of an effective chlamydial vaccine. Antibody has been implicated in protection studies in multiple animal models, and we previously showed that the passive transfer of immune serum alone does not confer immunity in the mouse. Using the Chlamydia muridarum model of genital infection, we demonstrate a protective role for both Chlamydia -specific immunoglobulin G (IgG) and polymorphonuclear neutrophils and show the importance of an antibody/effector cell interaction in mediating humoral immunity. While neutrophils were found to contribute significantly to antibody-mediated protection in vivo , natural killer (NK) cells were dispensable for protective immunity. Furthermore, gamma interferon (IFN-γ)-stimulated primary peritoneal neutrophils (PPNs) killed chlamydiae in vitro in an antibody-dependent manner. The results from this study support the view that an IFN-γ-activated effector cell population cooperates with antibody to protect against genital chlamydia and establish neutrophils as a key effector cell in this response., (Copyright © 2017 Naglak et al.)

Chlamydia trachomatis elementary body enzyme-linked immunosorbent assay (ELISA) was used to investigate serum anti-CT immunoglobulin G1 (IgG1; long-lived response) and immunoglobulin G3 (IgG3; short-lived response indicating more recent infection) from treatment (enrollment) and 6-month follow-up visits in 77 women previously classified as having spontaneous resolution of chlamydia. Of these women, 71.4% were IgG1+IgG3+, consistent with more recent chlamydia resolution. 15.6% were IgG3- at both visits, suggesting absence of recent chlamydia. Using elementary body ELISA, we demonstrated approximately 1 in 6 women classified as having spontaneous resolution of chlamydia might have been exposed to C. trachomatis but not infected. Further, we classified their possible infection stage., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. Chlamydia trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed--and some may produce functional proteins--but are dispensable for infection of the murine genital tract., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Renewed interest in chlamydia vaccination has revealed the need for a greater understanding of the seroprevalence of chlamydial infection in US populations. We used a Chlamydia trachomatis elementary body (EB)-based enzyme-linked immunosorbent assay to define the characteristics of the humoral immune response and to determine seroprevalence. Two groups were analyzed: one consisting of patients with current, laboratory confirmed, genital chlamydial infection (n = 98) and one group of individuals whose chlamydia infection history was unknown (n = 367). C. trachomatis seropositivity was detected in 90% of the infected group and in 31% of the chlamydia-unknown group. IgG1 and IgG3 comprised the predominant anti-Chlamydia serum antibody responses. Serum IgA1 responses were variably positive, and individuals were rarely positive for anti-chlamydia IgG2, IgG4 or IgA2. The magnitude of the IgG1 and IgG3 responses was greatest in female and African American individuals and was sustained for at least 6 months. Antibody responses were not serovar restricted or confounded by Chlamydia pneumoniae cross-reactivity.

Chlamydia muridarum and Chlamydia trachomatis mouse models of genital infection have been used to study chlamydial immunity and vaccine development. To assess the protective role of CD4(+) T cells in resolving C. trachomatis and C. muridarum genital tract infections, we used the female mouse model and evaluated infection in the presence and absence of CD4(+) T cells. In contrast to C. muridarum infection, C. trachomatis infection was unaltered in the absence of CD4(+) T cells. Mice infected with C. trachomatis developed protective immunity to re-challenge, but unlike C. muridarum infection, optimum resistance required multiple infectious challenges, despite the generation of adaptive serum and local chlamydial specific immune responses. Thus, understanding the chlamydial pathogenic and host immunologic factors that result in a diminished protective role for CD4(+) T cells in C. trachomatis murine infection might lead to new insights important to human immunity and vaccine development., (© The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.)

Chlamydia trachomatis genital infection is a worldwide public health problem, and considerable effort has been expended on developing an efficacious vaccine. The murine model of C. muridarum genital infection has been extremely useful for identification of protective immune responses and in vaccine development. Although a number of immunogenic antigens have been assessed for their ability to induce protection, the majority of studies have utilized the whole organism, the major outer membrane protein (MOMP), or the chlamydial protease-like activity factor (CPAF). These antigens, alone and in combination with a variety of immunostimulatory adjuvants, have induced various levels of protection against infectious challenge, ranging from minimal to nearly sterilizing immunity. Understanding of the mechanisms of natural infection-based immunity and advances in adjuvant biology have resulted in studies that are increasingly successful, but a vaccine licensed for use in humans has not yet been brought to fruition. Here we review immunity to chlamydial genital infection and vaccine development using the C. muridarum model.

Despite effective antimicrobial chemotherapy, control of Chlamydia trachomatis urogenital infection will likely require a vaccine. We have assessed the protective effect of an outer membrane protein-based vaccine by using a murine model of chlamydial genital infection. Female mice were first vaccinated with Chlamydia muridarum major outer membrane protein (MOMP) plus the adjuvants CpG-1826 and Montanide ISA 720; then they were challenged with C. muridarum. Vaccinated mice shed 2 log(10) to 3 log(10) fewer inclusion-forming units (IFU) than ovalbumin-vaccinated or naïve animals, resolved infection sooner, and had a lower incidence of hydrosalpinx. To determine the relative contribution of T cells to vaccine-induced protection, mice were vaccinated, depleted of CD4(+) or CD8(+) T cells, and then challenged vaginally with C. muridarum. Depletion of CD4(+) T cells, but not depletion of CD8(+) T cells, diminished vaccine-induced protection, with CD4-depleted mice shedding 2 log(10) to 4 log(10) more IFU than CD8-depleted or nondepleted mice. The contribution of antibodies to vaccine-induced protection was demonstrated by the absence of protective immunity in vaccinated B-cell-deficient mice and by a 2 log(10) to 3 log(10) decrease in bacterial shedding by mice passively administered an anti-MOMP serum. Thus, optimal protective immunity in this model of vaccine-induced protection depends on contributions from both CD4(+) T cells and antibody.

Acquired immunity to murine Chlamydia trachomatis genital tract reinfection has long been assumed to be solely dependent on cell-mediated immunity. However, in this study, we identify a previously unrecognized protective role for Ab. Immunity develops in Ab-deficient mice following the resolution of primary chlamydial genital infection. Subsequent depletion of CD4+ T cells, but not CD8+ T cells, in those immune Ab-deficient mice before secondary infectious challenge, resulted in an infection that did not resolve. Passive immunization with immune (convalescent) serum conferred a marked level of protective immunity to reinfection, which was characterized by a striking decrease in bacterial shedding, from >100,000 inclusion forming units to fewer than 10 inclusion forming units, and a shortened duration of infection. Furthermore, mAbs to the chlamydial major outer membrane protein and LPS conferred significant levels of immunity to reinfection and reduced chlamydial shedding by >100-fold. Anti-heat shock protein 60 mAb had no protective effect. In contrast to the marked protective efficacy of immune serum on reinfection, the course of primary infection was essentially unaltered by the passive transfer of immune serum. Our results convincingly demonstrate that Abs contribute importantly to immunity to chlamydial genital tract reinfection, and that Ab-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection. These results impact our understanding of immunity to chlamydial genital infection and may provide important insight into vaccine development.

The resolution of primary and secondary chlamydial genital infection in immunoglobulin A (IgA)-deficient (IgA(-/-)) mice was not different from that in IgA(+/+) mice. Furthermore, depletion of either CD4(+) or CD8(+) T cells prior to reinfection of IgA(+/+) or (-/-) mice had limited impact on immunity to reinfection. Thus, although antibody contributes importantly to immunity to chlamydial genital tract reinfection, IgA antibodies are not an absolute requirement of that protective response.

Tissue tropism of clinical ocular and genital Chlamydia trachomatis strains is shown to be linked to the tryptophan synthase genotype. It is suggested that, in the presence of IFN-gamma, which depletes available tryptophan, there exist unique host-parasite interactions that may contribute to persistent chlamydial infection.

Examines the correlation of model systems of chlamydial persistence to features of chlamydial disease. Association of chronic inflammation with detrimental immune system-mediated pathologic changes; Alterations in the chlamydia-host cell relationship; Recognition of abnormal chlamydiae under a variety of cell culture systems.