Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

The role of immunoglobulins and their transporters in urogenital Chlamydial infections

Chlamydia trachomatis infections of the male and female reproductive tracts are the world's leading sexually transmitted bacterial disease, and can lead to damaging pathology, scarring and infertility. The resolution of chlamydial infection requires the development of adaptive immune responses to infection, and includes cell-mediated and humoral immunity. Whilst cluster of differentiation (CD)4+ T cells are known to be essential in clearance of infection [1], they are also associated with immune cell infiltration, autoimmunity and infertility in the testes [2-3]. Conversely, antibodies are less associated with inflammation, are readily transported into the reproductive tracts, and can offer lumenal neutralization of chlamydiae prior to infection. Antibodies, or immunoglobulins (Ig), play a supportive role in the resolution of chlamydial infections, and this thesis sought to define the function of IgA and IgG, against a variety of chlamydial antigens expressed during the intracellular and extracellular stages of the chlamydial developmental cycle. Transport of IgA and IgG into the mucosal lumen is facilitated by receptor-mediated transcytosis yet the expression profile (under normal conditions and during urogenital chlamydial infection) of the polymeric immunoglobulin receptor (pIgR) and the neonatal Fc receptor (FcRn) remains unknown. The expression profile of pIgR and FcRn in the murine male reproductive tract was found to be polarized to the lower and upper reproductive tract tissues respectively. This demonstrates that the two receptors have a tissue tropism, which must be considered when targeting pathogens that colonize different sites. In contrast, the expression of pIgR and FcRn in the female mouse was found to be distributed in both the upper and lower reproductive tracts. When urogenitally infected with Chlamydia muridarum, both male and female reproductive tracts up-regulated expression of pIgR and down-regulated expression of FcRn. Unsurprisingly, the up-regulation of pIgR increased the concentration of IgA in the lumen. However, down-regulation of FcRn, prevented IgG uptake and led to an increase or pooling of IgG in lumenal secretions. As previous studies have identified the importance of pIgR-mediated delivery of IgA, as well as the potential of IgA to bind and neutralize intracellular pathogens, IgA against a variety of chlamydial antigens was investigated. The protection afforded by IgA against the extracellular antigen major outer membrane protein (MOMP), was found to be dependent on pIgR expression in vitro and in vivo. It was also found that in the absence of pIgR, no protection was afforded to mice previously immunized with MOMP. The protection afforded from polyclonal IgA against the intracellular chlamydial antigens; inclusion membrane protein A (IncA), inclusion membrane proteins (IncMem) and secreted chlamydial protease-like activity factor (CPAF) were produced and investigated in vitro. Antigen-specific intracellular IgA was found to bind to the respective antigen within the infected cell, but did not significantly reduce inclusion formation (p > 0.05). This suggests that whilst IgA specific for the selected antigens was transported by pIgR to the chlamydial inclusion, it was unable to prevent growth. Similarly, immunization of male mice with intracellular chlamydial antigens (IncA or IncMem), followed by depletion CD4+ T cells, and subsequent urogenital C. muridarum challenge, provided minimal pIgR-mediated protection. Wild type male mice immunized with IncA showed a 57 % reduction (p < 0.05), and mice deficient in pIgR showed a 35 % reduction (p < 0.05) in reproductive tract chlamydial burden compared to control antigen, and in the absence of CD4+ T cells. This suggests that pIgR and secretory IgA (SIgA) were playing a protective role (21 % pIgR-mediated) in unison with another antigen-specific immune mechanism (36 %). Interestingly, IgA generated during a primary respiratory C. muridarum infection did not provide a significant amount of protection to secondary urogenital C. muridarum challenge. Together, these data suggest that IgA specific for an extracellular antigen (MOMP) can play a strong protective role in chlamydial infections, and that IgA targeting intracellular antigens is also effective but dependent on pIgR expression in tissues. However, whilst not investigated here, IgA targeting and blocking other intracellular chlamydial antigens, that are more essential for replication or type III secretion, may be more efficacious in subunit vaccines. Recently, studies have demonstrated that IgG can neutralize influenza virus by trafficking IgG-bound virus to lysosomes [4]. We sought to determine if this process could also traffic chlamydial antigens for degradation by lysosomes, despite Chlamydia spp. actively inhibiting fusion with the host endocytic pathway. As observed in pIgR-mediated delivery of anti-IncA IgA, FcRn similarly transported IgG specific for IncA which bound the inclusion membrane. Interestingly, FcRn-mediated delivery of anti-IncA IgG significantly decreased inclusion formation by 36 % (p < 0.01), and induced aberrant inclusion morphology. This suggests that unlike IgA, IgG can facilitate additional host cellular responses which affect the intracellular niche of chlamydial growth. Fluorescence microscopy revealed that IgG also bound the inclusion, but unlike influenza studies, did not induce the recruitment of lysosomes. Notably, anti-IncA IgG recruited sequestosomes to the inclusion membrane, markers of the ubiquitin/proteasome pathway and major histocompatibility complex (MHC) class I loading. To determine if the protection against C. muridarum infection afforded by IncA IgG in vitro translated in vivo, wild type mice and mice deficient in functional FcRn and MHC-I, were immunized, depleted of CD4+, and urogenitally infected with C. muridarum. Unlike in pIgR-deficient mice, the protection afforded from IncA immunization was completely abrogated in mice lacking functional FcRn and MHC-I/CD8+. Thus, both anti-IncA IgA and IgG can bind the inclusion in a pIgR and FcRn-mediated manner, respectively. However, only IgG mediates a higher reduction in chlamydial infection in vitro and in vivo suggesting more than steric blocking of IncA had occurred. Unlike anti-MOMP IgA, which reduced chlamydial infection of epithelial cells and male mouse tissues, IgG was found to enhance infectivity in vitro, and in vivo. Opsonization of EBs with MOMP-IgG enhanced inclusion formation of epithelial cells in a MOMP-IgG dose-dependent and FcRn-dependent manner. When MOMP-IgG opsonized EBs were inoculated into the vagina of female mice, a small but non-significant (p > 0.05) enhancement of cervicovaginal C. muridarum shedding was observed three days post infection in mice with functional FcRn. Interestingly, infection with opsonized EBs reduced the intensity of the peak of infection (day six) but protracted the duration of infection by 60 % in wild type mice only. Infection with EBs opsonized in IgG also significantly increased (p < 0.05) hydrosalpinx formation in the oviducts and induced lymphocyte infiltration uterine horns. As MOMP is an immunodominant antigen, and is widely used in vaccines, the ability of IgG specific to extracellular chlamydial antigens to enhance infection and induce pathology needs to be considered. Together, these data suggest that immunoglobulins play a dichotomous role in chlamydial infections, and are dependent on antigen specificity, FcRn and pIgR expression. FcRn was found to be highly expressed in upper male reproductive tract, whilst pIgR was dominantly expressed in the lower reproductive tract. Conversely, female mice expressed FcRn and pIgR in both the lower and upper reproductive tracts. In response to a normal chlamydial infection, pIgR is up-regulated increasing secretory IgA release, but FcRn is down-regulated preventing IgG uptake. Similarly to other studies [5-6], we demonstrate that IgA and IgG generated during primary chlamydial infections plays a minor role in recall immunity, and that antigen-specific subunit vaccines can offer more protection. We also show that both IgA and IgG can be used to target intracellular chlamydial antigens, but that IgG is more effective. Finally, IgA against the extracellular antigen MOMP can afford protection, whist IgG plays a deleterious role by increasing infectivity and inducing damaging immunopathology. Further investigations with additional antigens or combination subunit vaccines will enhance our understanding the protection afforded by antibodies against intracellular and extracellular pathogenic antigens, and help improve the development of an efficacious chlamydial vaccine.

Estrogen receptor α antagonists mediate changes in CCL20 and CXCL1 secretions in the murine female reproductive tract

PROBLEM Estradiol regulates chemokine secretion from uterine epithelial cells, but little is known about estradiol regulation in vivo or the role of estrogen receptors (ERs). METHOD CCL20 and CXCL1 present in reproductive washes following treatment with selective estrogen receptor modulators (SERMs) were compared with that during estrous and following estradiol-treated ovariectomized BALB/c mice. Cellular regulation was determined using isolated vaginal and uterine epithelial/stromal cells in vitro. RESULTS Uterine and vaginal chemokine secretion is cyclically regulated with CCL20 at low levels but CXCL1 at high levels during high estradiol, generally mimicking estradiol effect in vivo. ERα but not ERβ regulated CCL20/CXCL1 secretion by uterine epithelial cells in vitro and vaginal CCL20 in vivo. Estradiol/SERMs failed to alter uterine CCL20 secretion in ovariectomized mice. Diminished uterine epithelial ERα staining following ovariectomy corresponded with estradiol unresponsiveness of uterine tissue. CONCLUSION Estrogen receptors α regulates CCL20/CXCL1 secretion in the female reproductive tract, and ERα antagonists directly oppose the regulation by estradiol. Understanding ER-mediated antimicrobial chemokine expression is important to elucidate cyclic susceptibility to sexually transmitted pathogens.

The mouse model of Chlamydia genital tract infection: a review of infection, disease, immunity and vaccine development

Chlamydia trachomatis is the most common sexually transmitted bacterial infection worldwide. The impact of this pathogen on human reproduction has intensified research efforts to better understand chlamydial infection and pathogenesis. Whilst there are animal models available that mimic the many aspects of human chlamydial infection, the mouse is regarded as the most practical and widely used of the models. Studies in mice have greatly contributed to our understanding of the host-pathogen interaction and provided an excellent medium for evaluating vaccines. Here we explore the advantages and disadvantages of all animal models of chlamydial genital tract infection, with a focus on the murine model and what we have learnt from it so far.

Changes to commercial service provision among sex workers from Queensland, Australia

Introduction: The role of commercial sex in facilitating infection transmission is a subject of ongoing empirical enquiry, with little attention to the variety and extent of ‘non-traditional’ commercial services that pose a lesser risk of infection. This study sought to examine the supply and demand of a wide range of traditional and non-traditional commercial sexual services among sex workers and their clients from Queensland, Australia. Methods: Cross-sectional convenience sampling was used to compare female sex workers in 1991 (n=200, aged 16-46 years) and 2003 (n=247, aged 18-57 years) and from male clients in 2003. The client sample comprised 160 male clients aged between 19 and 72 years. Results: Over the comparison period there was a significant increase in the provision of ‘exotic’ or non-traditional sexual services. In 2003, the availability of bondage and discipline, submission, fantasy, use of sex toys, golden showers, fisting and lesbian double acts had increased dramatically, while ‘traditional’ services had mostly remained at similar levels. Moreover, the proportion of sex workers in some industry sectors providing ‘exotic’ commercial services seem to have risen over time. Conclusion: Undoubtedly, the sex industry has professionalised and now includes more sophisticated and specialized suppliers. As with any commercial business, the diversification of services is largely driven with client demand, with the ‘menu’ being generally broader than the majority of client preferences. However, although clients demands for particular commercial sexual services seems to have been met, with regard to anal sex and anal play, supply has failed to meet client demand. Disclosure of Interest Statement: Funding for the 2003 study was provided by the Prostitution Licensing Authority. Acknowledgement and sincere thanks to the men and women who participated in this study.

‘It's all scientific to me’ : focus group insights into why young people do not apply safe-sex knowledge

Despite rising levels of safe-sex knowledge in Australia, sexually transmitted infection notifications continue to increase. A culture-centred approach suggests it is useful in attempting to reach a target population first to understand their perspective on the issues. Twenty focus groups were conducted with 89 young people between the ages of 14 and 16 years. Key findings suggest that scientific information does not articulate closely with everyday practice, that young people get the message that sex is bad and they should not be preparing for it and that it is not appropriate to talk about sex. Understanding how young people think about these issues is particularly important because the focus groups also found that young people disengage from sources of information that do not match their own experiences.

Sex workers can be screened too often : a cost-effectiveness analysis in Victoria, Australia

Objectives Commercial sex is licensed in Victoria, Australia such that sex workers are required to have regular tests for sexually transmitted infections (STIs). However, the incidence and prevalence of STIs in sex workers are very low, especially since there is almost universal condom use at work. We aimed to conduct a cost-effectiveness analysis of the financial cost of the testing policy versus the health benefits of averting the transmission of HIV, syphilis, chlamydia and gonorrhoea to clients. Methods We developed a simple mathematical transmission model, informed by conservative parameter estimates from all available data, linked to a cost-effectiveness analysis. Results We estimated that under current testing rates, it costs over $A90 000 in screening costs for every chlamydia infection averted (and $A600 000 in screening costs for each quality-adjusted life year (QALY) saved) and over $A4 000 000 for every HIV infection averted ($A10 000 000 in screening costs for each QALY saved). At an assumed willingness to pay of $A50 000 per QALY gained, HIV testing should not be conducted less than approximately every 40 weeks and chlamydia testing approximately once per year; in comparison, current requirements are testing every 12 weeks for HIV and every 4 weeks for chlamydia. Conclusions Mandatory screening of female sex workers at current testing frequencies is not cost-effective for the prevention of disease in their male clients. The current testing rate required of sex workers in Victoria is excessive. Screening intervals for sex workers should be based on local STI epidemiology and not locked by legislation.

Bang and bust: almost everything you wanted to know about sex and the mining boom (but were afraid to ask)

Recent growth and expansion of the fly-in/fly-out (FIFO) model of mining in remote rural Australia has led to concerns about the health and well-being of those employed by the mines and those in the small rural communities where they are based. A particular concern has been the potential disruption to sexual norms in mining towns and increases in sexually transmitted infections (STIs) and HIV.

Mining, sex work and STIs: why force a connection?

Can the mining boom be blamed for the rising rates of sexually transmitted infections (STIs) in some states? The Australian Medical Association thinks so, with its Queensland president Dr Richard Kidd attributing rising rates of gonorrhoea, syphilis and chlamydia in Queensland and Western Australia to bored and cashed-up miners.

A new approach to estimating trends in chlamydia incidence

Objectives Directly measuring disease incidence in a population is difficult and not feasible to do routinely. We describe the development and application of a new method of estimating at a population level the number of incident genital chlamydia infections, and the corresponding incidence rates, by age and sex using routine surveillance data. Methods A Bayesian statistical approach was developed to calibrate the parameters of a decision-pathway tree against national data on numbers of notifications and tests conducted (2001-2013). Independent beta probability density functions were adopted for priors on the time-independent parameters; the shape parameters of these beta distributions were chosen to match prior estimates sourced from peer-reviewed literature or expert opinion. To best facilitate the calibration, multivariate Gaussian priors on (the logistic transforms of) the time-dependent parameters were adopted, using the Matérn covariance function to favour changes over consecutive years and across adjacent age cohorts. The model outcomes were validated by comparing them with other independent empirical epidemiological measures i.e. prevalence and incidence as reported by other studies. Results Model-based estimates suggest that the total number of people acquiring chlamydia per year in Australia has increased by ~120% over 12 years. Nationally, an estimated 356,000 people acquired chlamydia in 2013, which is 4.3 times the number of reported diagnoses. This corresponded to a chlamydia annual incidence estimate of 1.54% in 2013, increased from 0.81% in 2001 (~90% increase). Conclusions We developed a statistical method which uses routine surveillance (notifications and testing) data to produce estimates of the extent and trends in chlamydia incidence.

Pathogenesis of Fallopian tube damage caused by Chlamydia trachomatis infections

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease worldwide resulting in 4–5 million new cases of Chlamydia annually and an estimated 100 million cases per annum. Infections of the lower female genital tract (FGT) frequently are asymptomatic so they often remain undiagnosed or untreated. If infections are either not resolved, or are left untreated, chlamydia can ascend to the upper FGT and infect the fallopian tubes (FTs) causing salpingitis that may lead to functional damage of the FTs and tubal factor infertility (TFI). Clinical observations and experimental data have indicated a role for antibodies against C. trachomatis proteins such as the 60 kDa heat-shock protein 60 (cHSP60) in the immunopathogenesis of TFI. When released from infected cells cHSP60 can induce pro-inflammatory immune responses that may functionally impair the FTs leading to fibrosis and luminal occlusion. Chlamydial pathogenesis of irreversible and permanent tubal damage is a consequence of innate and adaptive host immune responses to ongoing or repeated infections. The extracellular matrix (ECM) that is regulated by metalloproteinases (MMPs) may also be modified by chlamydial infections of the FGT. This review will highlight protective and pathogenic immune responses to ongoing and repeated chlamydial infections of the FGT. It will also present two recent hypotheses to explain mechanisms that may contribute to FT damage during a C. trachomatis infection. If Chlamydia immunopathology can be controlled it might yield a method of inducing fibrosis and thus provide a means of non-surgical permanent contraception for women.

Awareness and knowledge of Human Papillomavirus (HPV) among ethnically diverse women varying in generation status

Although Human papillomavirus (HPV) is a common sexually transmitted infection, there is limited knowledge of HPV with ethnic/racial minorities experiencing the greatest disparities. This cross-sectional study used the most recent available data from the California Health Interview Survey to assess disparities in awareness and knowledge of HPV among ethnically/racially diverse women varying in generation status (N = 19,928). Generation status emerged as a significant predictor of HPV awareness across ethnic/racial groups, with 1st generation Asian-Americans and 1st and 2nd generation Latinas reporting the least awareness when compared to same-generation White counterparts. Also, generation status was a significant predictor of HPV knowledge, but only for Asian-Americans. Regardless of ethnicity/race, 1st generation women reported lowest HPV knowledge when compared to 2nd and 3rd generation women. These findings underscore the importance of looking at differences within and across ethnic/racial groups to identify subgroups at greatest risk for poor health outcomes. In particular, we found generation status to be an important yet often overlooked factor in the identification of health disparities.

A Chlamydia trachomatis strain with a chemically generated amino acid substitution (P370L) in the cthtrA gene shows reduced elementary body production

Background Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism’s intracellular developmental cycle. HtrA is a critical stress response serine protease and chaperone for many bacteria and in C. trachomatis has been previously shown to be important for heat stress and the replicative phase of development using a chemical inhibitor of the CtHtrA activity. In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized. Methods SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis. The C. trachomatis strains containing the SNVs with marked reductions in proteolysis were investigated in cell culture to identify phenotypes that could be linked to CtHtrA function. Results The strain harboring the SNV with the most marked impact on proteolysis (cthtrAP370L) was detected to have a significant reduction in the production of infectious elementary bodies. Conclusions This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.

Development of a vaccine for Chlamydia trachomatis: Challenges and current progress

Chlamydia trachomatis remains an enigmatic bacterial pathogen with no vaccine yet available to treat human ocular and genital tract infections caused by tissue-tropic serovars of the organism. Globally, it is the leading cause of preventable blindness as well as the leading cause of bacterial sexually transmitted infections. The pathogen has a range of virulence factors that enable it to successfully evade both the innate and adaptive immune system of the host. The host immune system, although protective, paradoxically is also associated closely with the pathologies of trachoma and pelvic inflammatory disease – disease sequelae of some chlamydial infections and reinfections in some genetically susceptible hosts. In this review, we focus on what is known currently about the pathogenesis of ocular and genital infections caused by this mucosal pathogen. We also discuss novel insights into the pathogenesis of infections caused by the genital and ocular serovars of C. trachomatis, including a discussion of both pathogen and host factors, such as the human microbiota at these mucosal sites as well as the current immunological challenges facing vaccine development. Finally, we discuss the current progress toward development of a vaccine against C. trachomatis. A wide range of recombinant protein antigens are being identified and, hence, are available for vaccine trials. A plasmid-free live strain has recently been produced and evaluated in the mouse (Chlamydia muridarum) and monkey (C. trachomatis) models. The data for ocular infections in the monkey model was particularly encouraging, although the path to regulatory approval of a live vaccine is still uncertain. While still a major challenge, vaccines for ocular and genital C. trachomatis infections are looking more promising.