Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

Development of novel vaccine strategies to prevent genital tract chlamydial infections

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection in the developed world and the leading cause of preventable blindness worldwide. As reported by the World Health Organization in 2001, there are approximately 92 million new infections detected annually, costing health systems billions of dollars to treat not only the acute infection, but also to treat infection-associated sequelae. The majority of genital infections are asymptomatic, with 50-70% going undetected. Genital tract infections can be easily treated with antibiotics when detected. Lack of treatment can lead to the development of pelvic inflammatory disease, ectopic pregnancies and tubal factor infertility in women and epididymitis and prostatitis in men. With infection rates on the continual rise and the large number of infections going undetected, there is a need to develop an efficacious vaccine which prevents not only infection, but also the development of infection-associated pathology. Before a vaccine can be developed and administered, the pathogenesis of chlamydial infections needs to be fully understood. This includes the kinetics of ascending infection and the effects of inoculating dose on ascension and development of pathology. The first aim in this study was to examine these factors in a murine model. Female BALB/c mice were infected intravaginally with varying doses of C. muridarum, the mouse variant of human C. trachomatis, and the ascension of infection along the reproductive tract and the time-course of infection-associated pathology development, including inflammatory cell infiltration, pyosalpinx and hydrosalpinx, were determined. It was found that while the inoculating dose did affect the rate and degree of infection, it did not affect any of the pathological parameters examined. This highlighted that the sexual transmission dose may have minimal effect on the development of reproductive sequelae. The results of the first section enabled further studies presented here to use an optimal inoculating dose that would ascend the reproductive tract and cause pathology development, so that vaccine efficacy could be determined. There has been a large amount of research into the development of an efficacious vaccine against genital tract chlamydial infections, with little success. However, there have been no studies examining the effects of the timing of vaccination, including the effects of vaccination during an active genital infection, or after clearance of a previous infection. These are important factors that need to be examined, as it is not yet known whether immunization will enhance not only the individual's immune response, but also pathology development. It is also unknown whether any enhancement of the immune responses will cause the Chlamydia to enter a dormant, persistent state, and possibly further enhance any pathology development. The second section of this study aimed to determine if vaccination during an active genital tract infection, or after clearance of a primary infection, enhanced the murine immune responses and whether any enhanced or reduced pathology occurred. Naïve, actively infected, or previously infected animals were immunized intranasally or transcutaneously with the adjuvants cholera toxin and CpG-ODN in combination with either the major outer membrane protein (MOMP) of C. muridarum, or MOMP and ribonucleotide reductase small chain protein (NrdB) of C. muridarum. It was found that the systemic immune responses in actively or previously infected mice were altered in comparison to animals immunized naïve with the same combinations, however mucosal antibodies were not enhanced. It was also found that there was no difference in pathology development between any of the groups. This suggests that immunization of individuals who may have an asymptomatic infection, or may have been previously exposed to a genital infection, may not benefit from vaccination in terms of enhanced immune responses against re-exposure. The final section of this study aimed to determine if the vaccination regimes mentioned above caused in vivo persistence of C. muridarum in the upper reproductive tracts of mice. As there has been no characterization of C. muridarum persistence in vitro, either ultrastructurally or via transcriptome analysis, this was the first aim of this section. Once it had been shown that C. muridarum could be induced into a persistent state, the gene transcriptional profiles of the selected persistent marker genes were used to determine if persistent infections were indeed present in the upper reproductive tracts of the mice. We found that intranasal immunization during an active infection induced persistent infections in the oviducts, but not the uterine horns, and that intranasal immunization after clearance of infection, caused persistent infections in both the uterine horns and the oviducts of the mice. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. It is possible that the induction of persistent infections in the reproductive tract might enhance the development of pathology and thereby enhance the risk of infertility, factors that need to be prevented by vaccination, not enhanced. Overall, this study has shown that the inoculating dose does not affect pathology development in the female reproductive tract of infected mice, but does alter the degree and rate of ascending infection. It has also been shown that intranasal immunization during an active genital infection, or after clearance of one, induces persistent infections in the uterine horns and oviducts of mice. This suggests that potential vaccine candidates will need to have these factors closely examined before progressing to clinical trials. This is significant, because if the same situation occurs in humans, a vaccine administered to an asymptomatic, or previously exposed individual may not afford any extra protection and may in fact enhance the risk of development of infection-associated sequelae. This suggests that a vaccine may serve the community better if administered before the commencement of sexual activity.

Vaccination to protect against infection of the female reproductive tract

Infection of the female genital tract can result in serious morbidities and mortalities from reproductive disability, pelvic inflammatory disease and cancer, to impacts on the fetus, such as infant blindness. While therapeutic agents are available, frequent testing and treatment is required to prevent the occurrence of the severe disease sequelae. Hence, sexually transmitted infections remain a major public health burden with ongoing social and economic barriers to prevention and treatment. Unfortunately, while there are two success stories in the development of vaccines to protect against HPV infection of the female reproductive tract, many serious infectious agents impacting on the female reproductive tract still have no vaccines available. Vaccination to prevent infection of the female reproductive tract is an inherently difficult target, with many impacting factors, such as appropriate vaccination strategies/mechanisms to induce a suitable protective response locally in the genital tract, variation in the local immune responses due to the hormonal cycle, selection of vaccine antigen(s) that confers effective protection against multiple variants of a single pathogen (e.g., the different serovars of Chlamydia trachomatis) and timing of the vaccine administration prior to infection exposure. Despite these difficulties, there are numerous ongoing efforts to develop effective vaccines against these infectious agents and it is likely that this important human health field will see further major developments in the next 5 years.

Insights into the role and function of L2, the minor capsid protein of papillomaviruses

Human papillomaviruses (HPV) are responsible for the most common human sexually transmitted viral infections, and high-risk types are responsible for causing cervical and other cancers. The minor capsid protein L2 of HPV plays important roles in virus entry into cells, localisation of viral components to the nucleus, in DNA binding, capsid formation and stability. It also elicits antibodies that are more cross-reactive between HPV types than does the major capsid protein L1, making it an attractive potential target for new-generation, more broadly protective subunit vaccines against HPV infections. However, its low abundance in natural capsids-12-72 molecules per 360 copies of L1-limits its immunogenicity. This review will explore the biological roles of the protein, and prospects for its use in new vaccines. © 2009 Springer-Verlag.

Development status and future prospects for a vaccine against Chlamydia trachomatis infection

Chlamydia trachomatis continues to be the most commonly reported sexually transmitted bacterial infection in many countries with more than 100 million new cases estimated annually. These acute infections translate into significant downstream health care costs, particularly for women, where complications can include pelvic inflammatory disease and other disease sequelae such as tubal factor infertility. Despite years of research, the immunological mechanisms responsible for protective immunity versus immunopathology are still not well understood, although it is widely accepted that T cell driven IFN-g and Th17 responses are critical for clearing infection. While antibodies are able to neutralize infections in vitro, alone they are not protective, indicating that any successful vaccine will need to elicit both arms of the immune response. In recent years, there has been an expansion in the number and types of antigens that have been evaluated as vaccines, and combined with the new array of mucosal adjuvants, this aspect of chlamydial vaccinology is showing promise. Most recently, the opportunities to develop successful vaccines have been given a significant boost with the development of a genetic transformation system for Chlamydia, as well as the identification of the key role of the chlamydial plasmid in virulence. While still remaining a major challenge, the development of a successful C.trachomatis vaccine is starting to look more likely.

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.