Effect of female sex hormones on Chlamydia trachomatis growth and gene expression

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

Modeling the impact of potential vaccines on epidemics of sexually transmitted chlamydia trachomatis infection

Background. We investigated the likely impact of vaccines on the prevalence of and morbidity due to Chlamydia trachomatis (chlamydia) infections in heterosexual populations. Methods.An individual‐based mathematical model of chlamydia transmission was developed and linked to the infection course in chlamydia‐infected individuals. The model describes the impact of a vaccine through its effect on the chlamydial load required to infect susceptible individuals (the “critical load”), the load in infected individuals, and their subsequent infectiousness. The model was calibrated using behavioral, biological, and clinical data. Results.A fully protective chlamydia vaccine administered before sexual debut can theoretically eliminate chlamydia epidemics within 20 years. Partially effective vaccines can still greatly reduce the incidence of chlamydia infection. Vaccines should aim primarily to increase the critical load in susceptible individuals and secondarily to decrease the peak load and/or the duration of infection in vaccinated individuals who become infected. Vaccinating both sexes has a beneficial impact on chlamydia‐related morbidity, but targeting women is more effective than targeting men. Conclusions.Our findings can be used in laboratory settings to evaluate vaccine candidates in animal models, by regulatory bodies in the promotion of candidates for clinical trials, and by public health authorities in deciding on optimal intervention strategies.

HtrA, RseP, and Tsp do not elicit a pathology related serum IgG response during sexually transmitted infection with Chlamydia trachomatis

Chlamydia trachomatis sexually transmitted infection can cause serious reproductive morbidities. This study determined the prevalence of serum IgG response to C. trachomatis putative stress response proteins in females to test for an association with genital tract pathology. There was no significant association of serum IgG to HtrA, Tsp, or RseP with infection or pathology. cHSP60 serum IgG prevalence was significantly associated with infection compared to negative (infertile) controls (p = 0.002), but not with upper genital tract pathology. Serum IgG1-4 antibody subclasses reactive with the antigens was not significantly different between cohorts, although different responses to each antigen were detected.

Identification of novel markers for uncomplicated lower genital tract infections and upper genital tract pathology due to Chlamydia trachomatis.

Background: Untreated Chlamydia trachomatis infections in women can result in disease sequelae such as salpingitis and pelvic inflammatory disease (PID), ultimately culminating in tubal occlusion and infertility. Whilst nucleic acid amplification tests can effectively diagnose uncomplicated lower genital tract (LGT) infections, they are not suitable for diagnosing upper genital tract (UGT) pathological sequelae. As a consequence, this study aimed to identify serological markers that can, with a high degree of sensitivity and specificity, discriminate between LGT infections and UGT pathology. Methods: Plasma was collected from 73 women with a history of LGT infection, UGT pathology due to C. trachomatis or no serological evidence of C. trachomatis infection. Western blotting was used to analyse antibody reactivity against extracted chlamydial proteins. Sensitivity and specificity of differential markers were also calculated. Results: Four antigens (CT157, CT423, CT727 and CT396) were identified and found to be capable of discriminating between the infection and disease sequelae state. Sensitivity and specificity calculations showed that our assay for diagnosing LGT infection had a sensitivity and specificity of 75% and 76% respectively, whilst the assay for identifying UGT pathology demonstrated 80% sensitivity and 86% specificity. Conclusions: The use of these assays could potentially facilitate earlier diagnoses in women suffering UGT pathology due to C. trachomatis.

Apoptosis is induced in Chlamydia trachomatis infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Problem: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. ----- ----- Method of study: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. ----- ----- Results: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. ----- ----- Conclusion: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone.

Tubal damage, infertility and tubal ectopic pregnancy : chlamydia trachomatis and other microbial aetiologies

Infertility is a worldwide health problem with one in six couples suffering from this condition and with a major economic burden on the global healthcare industry. Estimates of the current global infertility rate suggest that 15% of couples are infertile (Zegers-Hochschild et al 2009) defined as: (1) failure to conceive after 12 months of unprotected sexual intercourse (i.e. infertility); (2) repeated implantation failure following ART cycles; or (3) recurrent miscarriage without difficulty conceiving (natural conceptions). Tubal factor infertility is among the leading causes of female factor infertility accounting for 7-9.8% of all female factor infertilities. Tubal disease directly causes from 36% to 85% of all cases of female factor infertility in developed and developing nations respectively and is associated with polymicrobial aetiologies. One of the leading global causes of tubal factor infertility is thought to be symptomatic (and asymptomatic in up to 70% cases) infection of the female reproductive tract with the sexually transmitted pathogen, Chlamydia trachomatis. Infection-related damage to the Fallopian tubes caused by Chlamydia accounts for more than 70% of cases of infertility in women from developing nations such as sub-Saharan Africa (Sharma et al 2009). Bacterial vaginosis, a condition associated with increased transmission of sexually transmitted infections including those caused by Neisseria gonorrhoeae and Mycoplasma genitalium is present in two thirds of women with pelvic inflammatory disease (PID). This review will focus on (1) the polymicrobial aetiologies of tubal factor infertility and (2) studies involved in screening for, and treatment and control of, Chlamydial infection to prevent PID and the associated sequelae of Fallopian tube inflammation that may lead to infertility and ectopic pregnancy.

Ovarian steroid hormones: effects on immune responses and Chlamydia trachomatis infections of the female genital tract

Female sex hormones are known to regulate the adaptive and innate immune functions of the female reproductive tract. This review aims to update our current knowledge of the effects of the sex hormones estradiol and progesterone in the female reproductive tract on innate immunity, antigen presentation, specific immune responses, antibody secretion, genital tract infections caused by Chlamydia trachomatis, and vaccine-induced immunity.

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.

Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

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.