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Key Points Chlamydia spp. are obligate intracellular pathogens that are important causes of human and animal diseases. Chlamydiae share a common developmental cycle in which they alternate between the extracellular, infectious elementary body and the intracellular, non-infectious reticulate body. Chlamydiae use several redundant mechanisms to enter host cells and to establish their intracellular membrane bound niche — the inclusion. Chlamydiae deliver effector proteins into the inclusion membrane and into host cells to promote replication and survival. Chlamydiae encode a unique set of T3SS effectors, the inclusion membrane proteins (Incs), which are inserted into the inclusion membrane where they may function as structural determinants of the membrane or as scaffolds to interface with various cell pathways in the host. Recent studies have solved the 'chlamydial anomaly' and reveal that Chlamydia spp. do synthesize peptidoglycan and use an atypical mechanism of cell division. The recent major advances in chlamydial genetics open the door for the development of tools and avenues of research that were not previously accessible to this historically intractable pathogen. Chlamydia spp. are intracellular bacteria that depend on the host for their metabolic requirements, while hiding from host immune defences. In this Review, Elwell, Mirrashidi and Engel detail the molecular mechanisms that enable these pathogens to shape and thrive in their niche in host cells. Chlamydia spp. are important causes of human disease for which no effective vaccine exists. These obligate intracellular pathogens replicate in a specialized membrane compartment and use a large arsenal of secreted effectors to survive in the hostile intracellular environment of the host. In this Review, we summarize the progress in decoding the interactions between Chlamydia spp. and their hosts that has been made possible by recent technological advances in chlamydial proteomics and genetics. The field is now poised to decipher the molecular mechanisms that underlie the intimate interactions between Chlamydia spp. and their hosts, which will open up many exciting avenues of research for these medically important pathogens.

Chlamydia , a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53–MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen’s anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection. Protein p53 plays key roles in cellular stress responses and is frequently deregulated in cancer. Here the authors show that infection with chlamydiae activates the ubiquitin ligase MDM2 in infected cells, leading to proteasomal degradation of p53 and thus promoting apoptosis resistance.

ABSTRACT Chlamydiae are pathogenic intracellular bacteria that cause a wide variety of diseases throughout the globe, affecting the eye, lung, coronary arteries and female genital tract. Rather than by direct cellular toxicity, Chlamydia infection generally causes pathology by inducing fibrosis and scarring that is largely mediated by host inflammation. While a robust immune response is required for clearance of the infection, certain elements of that immune response may also damage infected tissue, leading to, in the case of female genital infection, disease sequelae such as pelvic inflammatory disease, infertility and ectopic pregnancy. It has become increasingly clear that the components of the immune system that destroy bacteria and those that cause pathology only partially overlap. In the ongoing quest for a vaccine that prevents Chlamydia-induced disease, it is important to target mechanisms that can achieve protective immunity while preventing mechanisms that damage tissue. This review focuses on mouse models of genital Chlamydia infection and synthesizes recent studies to generate a comprehensive model for immunity in the murine female genital tract, clarifying the respective contributions of various branches of innate and adaptive immunity to both host protection and pathogenic genital scarring. This review summarizes mouse model studies that significantly contributed to our current understanding of the protective and pathogenic immune responses taking place during genital Chlamydia infections.

The ability to introduce targeted genetic modifications in microbial genomes has revolutionized our ability to study the role and mode of action of individual bacterial virulence factors. Although the fastidious lifestyle of obligate intracellular bacterial pathogens poses a technical challenge to such manipulations, the last decade has produced significant advances in our ability to conduct molecular genetic analysis in Chlamydia trachomatis, a major bacterial agent of infertility and blindness. Similar approaches have not been established for the closely related veterinary Chlamydia spp., which cause significant economic damage, as well as rare but potentially life-threatening infections in humans. Here we demonstrate the feasibility of conducting site-specific mutagenesis for disrupting virulence genes in C. caviae, an agent of guinea pig inclusion conjunctivitis that was recently identified as a zoonotic agent in cases of severe community-acquired pneumonia. Using this approach, we generated C. caviae mutants deficient for the secreted effector proteins IncA and SinC. We demonstrate that C. caviae IncA plays a role in mediating fusion of the bacteria-containing vacuoles inhabited by C. caviae. Moreover, using a chicken embryo infection model, we provide first evidence for a role of SinC in C. caviae virulence in vivo.

ABSTRACT Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection. Here, the authors review the functions by which T cells limit Chlamydia trachomatis infections as well as the ways this pathogen inhibits T cell immunity.

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

Chlamydia trachomatis infections are the leading cause of sexually transmitted infections of bacterial origin. Lower genital tract infections are often asymptomatic, and therefore left untreated, leading to ascending infections that have long-term consequences on female reproductive health. Human pathology can be recapitulated in mice with the mouse adapted strain C. muridarum. Eight years into the post-genetic era, significant advances to expand the Chlamydia genetic toolbox have been made to facilitate the study of this important human pathogen. However, the need for additional tools remains, especially for C. muridarum. Here, we describe a new set of spectinomycin resistant E. coli-Chlamydia shuttle vectors, for C. trachomatis and C. muridarum. These versatile vectors allow for expression and localization studies of Chlamydia effectors, such as Inc proteins, and will be instrumental for mutant complementation studies. In addition, we have exploited the differential expression of specific Chlamydia genes during the developmental cycle to engineer an omcA::gfp fluorescent transcriptional reporter. This novel tool allows for monitoring RB to EB conversion at the bacterial level. Spatiotemporal tracking of GFP expression within individual inclusions revealed that RB to EB conversion initiates in bacteria located at the edge of the inclusion and correlates with the time post initiation of bacterial replication and inclusion size. Comparison between primary and secondary inclusions potentially suggests that the environment in which the inclusions develop influences the timing of conversion. Altogether, the Chlamydia genetic tools described here will benefit the field, as we continue to investigate the molecular mechanisms underlying Chlamydia-host interaction and pathogenesis.

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 . infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host protein synthesis. and showed less capacity for NFκB activation compared to , suggesting a species-specific variation in NFκB activation. At 24 hpi, induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. -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 and that stressed chlamydiae can promote inflammation.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitr o exposure of spermatozoa to Chlamydia trachomatis , and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum , respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum , respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm ( p  > 0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility.

Trachoma is the leading infectious cause of blindness. Repeated or persistent ocular infection with Chlamydia trachomatis in childhood leads to conjunctival scarring, usually in adulthood but often earlier in areas with greater disease burden. There are limited longitudinal data examining change in scarring in children, especially where trachoma rates are low. A cohort of children, ages 1-9 years, were randomly selected at baseline from 38 communities in Kongwa, Tanzania and followed for 2 years. Rates of trachomatous inflammation-follicular (TF) were <5% over the survey period. At baseline, 1,496 children were recruited and 1,266 (85%) were followed-up. Photographs were obtained at baseline and follow-up and graded for the presence and severity of scarring using a four-point scale ranging between S1-S4. In children without scarring at baseline, 1.6% (20/1,246) were found to have incident scarring, and incident scarring was more common among girls compared to boys. Among children with scarring at baseline, 21% (4/19) demonstrated progression. In this formerly hyper-endemic district, the incidence of new scarring in children ages 1-9 years is low, although 21% of those who had scarring at baseline progressed in severity over the 2-year follow-up period. These data provide support for the thesis that while incident scarring more closely reflects ongoing exposure, progression may involve factors independent of ongoing transmission of trachoma.