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 β-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.

Circulating lactate levels are a critical biomarker for sepsis and are positively correlated with sepsis-associated mortality. We investigated whether lactate plays a biological role in causing endothelial barrier dysfunction in sepsis. We showed that lactate causes vascular permeability and worsens organ dysfunction in CLP sepsis. Mechanistically, lactate induces ERK-dependent activation of calpain1/2 for VE-cadherin proteolytic cleavage, leading to the enhanced endocytosis of VE-cadherin in endothelial cells. In addition, we found that ERK2 interacts with VE-cadherin and stabilizes VE-cadherin complex in resting endothelial cells. Lactate-induced ERK2 phosphorylation promotes ERK2 disassociation from VE-cadherin. In vivo suppression of lactate production or genetic depletion of lactate receptor GPR81 mitigates vascular permeability and multiple organ injury and improves survival outcome in polymicrobial sepsis. Our study reveals that metabolic cross-talk between glycolysis-derived lactate and the endothelium plays a critical role in the pathophysiology of sepsis.

The oestrogen receptor (ER) α-β+ HEC-1B and the ERα+β+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERβ alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERβ antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.

Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singly-infected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans.

Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis.