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The Chlamydiaceae are a bacterial family that contains a single genus: Chlamydia. The genus Chlamydia consists of 9 species that are obligate, intracellular pathogens. Untreated C. trachomatis infections can lead to serious health ramifications, such as ectopic pregnancy, tubal factor infertility, pelvic inflammatory disease, and long-term pelvic pain. In this study, it was found that a primary antibody dilution of 1:400 using methanol fixed HeLA cells, as derived from Carrasco, et al. protocol, was only optimal for PMP-C staining. Pmp-A, Pmp-B, and Pmp-F were found to stain brighter with formaldehyde fixed, infected HeLa cells and using different primary antibody dilutions. The manuscript by Carrasco, et al., demonstrated that chlamydial persistence caused by penicillin-stressed conditions showed a decrease in Pmp-B and Pmp-C protein expression between 24-48 hpi, while Pmp-A and Pmp-F expression stayed the same under the stressful conditions. We hypothesized that under HSV- induced persistence the same results would occur. However, our data indicates that the chlamydial response to stressful conditions is not the same among persistence-inducers and implies that various inducers of persistence may affect PMP expression differently. Initially, we also hypothesized that PMP expression could be utilized as an indicator to determine whether an infected individual has a productive or persistent chlamydial infection. Due to the experiments’ results, PMP expression is most likely not a good marker to identify the type of chlamydial infection (ie. productive or persistent) in the host.

Chlamydia trachomatis is a human pathogen of the genital tract and ocular epithelium. It is an obligate intracellular parasite with a unique biphasic development cycle. C.trachomatis infection is the most common bacterial sexually transmitted disease in industrialized nations. Its ability to cause chronic disease makes it a serious economic burden and health threat to developed and developing countries. Although treatable, approximately 70% of C.trachomatis infections are asymptomatic, potentially leading to the development of chronic sequelae. Furthermore, chlamydial genital tract infection has been associated with an increased risk of cervical cancer and human immunodeficiency virus infection. Consequently, the development of an efficacious vaccine is the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Anti-chlamydial protective immunity is essentially mediated by a T helper, type 1 (Th1), response that is dependent upon the presentation of antigen via major histocompatibility (MHC) class II molecules. While antibody secreting cells are not critical components of the primary effector response, they have been shown to be important for clearance of re-infection. Thus an ideal vaccine would be one capable of inducing both a strong Th1 T cell response and a strong mucosal antibody response. Currently there are very few efficacious vaccine candidates that have been identified and characterized. More specifically, there is only a limited number of known T cell antigens processed and presented by the human leukocyte antigen (HLA) class II molecules. This type of antigen is going to be essential to the development of an efficacious chlamydial vaccine. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. In an attempt to overcome HLA polymorphism the whole chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. predicted ‘promiscuous’ T cell epitopes). A wide range of HLA class II molecules were used in this screen to identify vaccine antigens that could potentially offer broad and ethnically balanced population coverage. This analysis identified a number of novel targets and was validated by the identification of a known chlamydial T cell epitope. A selection of these target proteins was cloned, expressed and purified. Recombinant protein was screened against serum samples from patients with both acute and chronic chlamydial infections. Two novel targets, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. The in vivo protective efficacy of NrdB was analyzed using a mouse model. CD4+ T cells were harvested from NrdB immunized mice and adoptively transferred to naïve mice, which were subsequently infected at the genital site. NrdB immunization was found to confer a CD4+ T cell driven degree of protection similar to that seen with CD4+ T cells primed from a live challenge. The adjuvants and route of immunization used ensured immunological responses were initiated at both the systemic and local sites of infection. Immunization elicited a predominant Th1 response with primed T cells producing high levels of interferon gamma, an essential requirement for the development of an efficacious chlamydial vaccine. Furthermore, high titres of antigen specific IgG and IgA were produced following immunization, with sera derived antibodies demonstrating neutralization properties. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could play a central role in a multi-subunit vaccine against chlamydial genital tract infections.

Chlamydiae may exist at the site of infection in an alternative replicative form, called the aberrant body (AB). ABs are produced during a viable but non-infectious developmental state termed "persistence" or "chlamydial stress." As persistent/stressed chlamydiae: (i) may contribute to chronic inflammation observed in diseases like trachoma; and (ii) are more resistant to current anti-chlamydial drugs of choice, it is critical to better understand this developmental stage. We previously demonstrated that porcine epidemic diarrhea virus (PEDV) co-infection induced Chlamydia pecorum persistence/stress in culture. One critical characteristic of persistence/stress is that the chlamydiae remain viable and can reenter the normal developmental cycle when the stressor is removed. Thus, we hypothesized that PEDV-induced persistence would be reversible if viral replication was inhibited. Therefore, we performed time course experiments in which Vero cells were C. pecorum/PEDV infected in the presence of cycloheximide (CHX), which inhibits viral but not chlamydial protein synthesis. CHX-exposure inhibited PEDV replication, but did not inhibit induction of C. pecorum persistence at 24 h post-PEDV infection, as indicated by AB formation and reduced production of infectious EBs. Interestingly, production of infectious EBs resumed when CHX-exposed, co-infected cells were incubated 48-72 h post-PEDV co-infection. These data demonstrate that PEDV co-infection-induced chlamydial persistence/stress is reversible and suggest that this induction (i) does not require viral replication in host cells; and (ii) does not require de novo host or viral protein synthesis. These data also suggest that viral binding and/or entry may be required for this effect. Because the PEDV host cell receptor (CD13 or aminopeptidase N) stimulates cellular signaling pathways in the absence of PEDV infection, we suspect that PEDV co-infection might alter CD13 function and induce the chlamydiae to enter the persistent state.

Abstract Chlamydiae are obligatory intracellular gram-negative bacteria with a unique growth cycle. They are very successful pathogens and responsible for a wide variety infections in humans and different animal species. In addition, they have a tendency to cause recurrent, persistent or chronic infections with potentially severe sequelae years or decades later. The general purpose of this work was to study the possible serological associations between chronic chlamydial infection, systemic inflammation and reproductive health in a general population. The chlamydial heat shock proteins 60 and 10 (Hs10 and Hsp60) have been suggested to contribute to the pathogenesis of chronic chlamydial infections. Thus, the antibodies to chlamydial Hsp10 and Hsp60 were also investigated in complications of pregnancy. The present study was a longitudinal population-based birth cohort study, and all of the original papers of this dissertation are based on a nested case - control design. Our results confirmed the serological association between C. trachomatis infections and subfertility and the rather high incidence of undiagnosed C. trachomatis infections in the male partners of subfertile couples. We further demonstrated a serological association between previous C. trachomatis infections, immunity to chlamydial Hsps and female subfertility. We also showed that serological markers of chronic chlamydial infection present as early as the first trimester are associated with preterm delivery among nulliparous women. When elevated levels of C. trachomatis IgG and hsCRP were present, the estimated risk for preterm delivery was over 4-fold. According to our study, nulliparous women who subsequently developed preeclampsia leading to preterm delivery, which was used as a marker of more serious illness, had significantly more often serum IgG antibodies to C. pneumoniae during the first trimester of pregnancy compared to the preeclamptic women who delivered at term. In conclusion, chronic C. trachomatis infection was found to associate with subfertility both in men and in women. In addition, a subclinical chronic inflammatory process associated at least partly with chronic C. trachomatis infection and present in the first trimester already may be important in the development of preterm delivery. Chronic C. pneumoniae infection and systemic low-grade inflammation were found to associate with pregnancies that lead to preeclampsia and preterm delivery.

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other ß-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.

Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.

In culture, exposure to penicillin and other stressors induce chlamydiae to enter a non-infectious but viable state termed persistence. Chlamydiae may reenter their normal developmental cycle after stressor removal. Though aberrant RB similar to those present in culture models of persistence have been observed within infected tissues, the existence of persistent chlamydiae has not been definitively demonstrated in vivo. As a result, the role of persistent organisms in pathogenesis is undefined. In order to establish an experimentally tractable model of in vivo persistence, Chlamydia muridarum vaginally-infected mice were gavaged with either water or amoxicillin (amox). Vaginal swabs were collected for chlamydial titration and RNA isolated for quantification of pre-16s rRNA. Uterine tissue was analyzed by transmission electron microscopy (TEM). Although amox-treatment reduced vaginal shedding by >99%, C. muridarum pre-16s rRNA accumulation was unchanged by treatment. These data indicate that the amox-exposed organisms were viable but not infectious. Furthermore, TEM analyses demonstrated that inclusions in amox-treated animals contained primarily large, aberrant RB, but those observed in untreated control animals were normal. Collectively, these data suggest that amoxicillin treatment induces C. muridarum to enter the persistent state in vivo. This model also represents the first experimentally tractable animal model of chlamydial persistence.

Under stress, chlamydiae can enter a non-infectious but viable state termed persistence. In the absence of a tractable genetic system, persistence induction provides an important experimental tool with which to study these fascinating organisms. This review will discuss examples of: i) persistence studies that have illuminated critical chlamydiae/host interactions; and ii) novel persistence models that will do so in the future.

Objective: The clinical significance of the potential for persistent human chlamydial infections in vivo is being actively reassessed because of the increased frequency of recurrent infection with the same serovar despite compliance with an effective antibiotic regimen. The ability to extend the length of time of in vitro cultivation of polarized human endometrial epithelial cells (HEC-1B) provided the opportunity to establish a model system to determine if a persistent form of Chlamydia trachomatis had the same susceptibility as the actively growing form to a cidal concentration of azithromycin. Methods: Polarized HEC-1B cells cultivated on extracellular matrix were infected with C. trachomatis serovar E and exposed to penicillin at 24 h post-infection (hpi) to induce a persistent infection characterized by slowly metabolizing but non-dividing, ultrastructurally aberrant reticulate bodies within the chlamydial inclusion; at 48hpi, infected cultures were exposed to a bactericidal concentration of azithromycin for 72 h. Results: Persistent chlamydiae were phenotypically resistant to azithromycin; the number of chlamydial inclusions on subpassage of progeny from persistent chlamydiae following removal of penicillin and recovery was essentially the same as from progeny from persistent chlamydiae following removal of penicillin and azithromycin and recovery. Neutrophils were attracted in vitro to persistently infected HEC-1B cells that had been exposed to penicillin and azithromycin. Conclusions: Thus, this study provides evidence at the cellular microbiology level in vitro for mechanisms that could exist in vivo to create sustained, but perhaps clinically inapparent inflammation, which might eventually lead to conditions such as silent pelvic inflammatory disease.