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A vaginal microbiota dominated by certain Lactobacillus species may have a protective effect against Chlamydia trachomatis infection. One of the key antimicrobial compounds produced is lactic acid, which is believed to play a central role in host defense. Lactobacillus strains producing the D(−)-lactic acid isomer are known to exert stronger protection. However, the molecular mechanisms underlying this antimicrobial action are not well understood. The aim of this study was to investigate the role of D(−)-lactic acid isomer in the prevention of C. trachomatis infection in an in vitro HeLa cell model. We selected two strains of lactobacilli belonging to different species: a vaginal isolate of Lactobacillus crispatus that releases both D(−) and L(+) isomers and a strain of Lactobacillus reuteri that produces only the L(+) isomer. Initially, we demonstrated that L. crispatus was significantly more effective than L. reuteri in reducing C. trachomatis infectivity. A different pattern of histone acetylation and lactylation was observed when HeLa cells were pretreated for 24 h with supernatants of Lactobacillus crispatus or L. reuteri, resulting in different transcription of genes such as CCND1, CDKN1A, ITAG5 and HER-1. Similarly, distinct transcription patterns were found in HeLa cells treated with 10 mM D(−)- or L(+)-lactic acid isomers. Our findings suggest that D(−) lactic acid significantly affects two non-exclusive mechanisms involved in C. trachomatis infection: regulation of the cell cycle and expression of EGFR and α5β1-integrin.

The oropharynx represents a crucial site for the emergence of multi-drug resistance in Neisseria gonorrhoeae. The mosaic penA alleles, associated with decreased susceptibility to cephalosporins, have emerged by DNA recombination with partial penA genes, particularly those from commensal pharyngeal Neisseria species. Here, we investigated the prevalence of the mosaic structure of the penA gene in the oropharynx of men who have sex with men testing negative for pharyngeal gonorrhoea. From January 2016 to June 2018, 351 gonorrhoea-negative men who have sex with men attending a sexually transmitted infection clinic in Italy were enrolled. Pharyngeal swabs underwent a real-time polymerase chain reaction (PCR) for the detection of the mosaic penA gene. In case of positivity, PCR products were sequenced and searched against several sequences of Neisseria strains. Overall, 31 patients (8.8%) were found positive for the presence of the mosaic penA gene. The positivity was significantly associated with previous cases of pharyngeal gonorrhoea (relative risk [RR]: 3.56, 95% confidence interval 1.44–8.80) and with recent exposure to beta-lactams (RR: 4.29, 95% confidence interval 2.20–8.38). All penA-positive samples showed a high relatedness (90–99%) with mosaic-positive Neisseria strains. Our data underline that commensal Neisseria species of the oropharynx may be a significant reservoir for genetic material conferring antimicrobial resistance in N. gonorrhoeae.

Background. Previous works suggest that sugars can have a beneficial effect on C. trachomatis (CT) survival and virulence. In this study, we investigated the effect of different sugars on CT infectivity, elucidating some of the molecular mechanisms behind CT-sugar interaction. Methods. CT infectivity was investigated on HeLa cells after 2 hour-incubation of elementary bodies (EBs) with glucose, sucrose, or mannitol solutions (0.5, 2.5, 5.0 mM). The effect of sugars on EB membrane fluidity was investigated by fluorescence anisotropy measurement, whereas the changes in lipopolysaccharide (LPS) exposure were examined by cytofluorimetric analysis. By means of a Western blot, we explored the phosphorylation state of Focal Adhesion Kinase (FAK) in HeLa cells infected with EBs pre-incubated with sugars. Results. All sugar solutions significantly increased CT infectivity on epithelial cells, acting directly on the EB structure. Sugars induced a significant increase of EB membrane fluidity, leading to changes in LPS membrane exposure. Especially after incubation with sucrose and mannitol, EBs led to a higher FAK phosphorylation, enhancing the activation of anti-apoptotic and proliferative signals in the host cells. Conclusions. Sugars can increase CT infectivity and virulence, by modulating the expression/exposure of chlamydial membrane ligands. Further in-depth studies are needed to better understand the molecular mechanisms involved.

The sexually transmitted pathogen Chlamydia trachomatis (CT) is able to replicate and survive in human intestinal epithelial cells, being the gastro-intestinal tract a suitable site of residence for this microorganism. In this context, no detailed information about the mechanisms of cell death in intestinal cell lines after a chlamydial infection is available. The aim of this study was to compare the effect of two different CT serovars (D and L2) on the survival/death of different intestinal cell lines (Caco-2 and COLO-205), using endocervical cells (HeLa) as a reference model of genital infection. Seventy two hours after chlamydial infection at different multiplicity of infection (MOI) levels, the viability of HeLa, Caco-2 and COLO 205 cells was evaluated through dose-response experiments by means of a MTS-based assay. To get deeper insights in the mechanisms of cell death induced by CT, cell viability was assessed in presence of different inhibitors (i.e. pan-caspase inhibitor Z-VAD, necroptosis inhibitor Necrostatin-1, hydrogen peroxide scavenger catalase, caspase-1 inhibitor Ac-YVAD-cmk). Moreover, the activation of effector caspases and the presence of cellular apoptotic/necrotic changes were evaluated at different time points after CT infection. Our results demonstrated that, for both chlamydial serovars, intestinal cell lines are more resistant to CT-induced cell death compared to HeLa, thus representing a suitable 'niche' for chlamydial residence and replication. In literature, apoptosis has been widely described to be the main cell death mechanism elicited by chlamydia infection. However, our data demonstrate that necroptosis plays a relevant role, proceeding in parallel with apoptosis. The protective effect of catalase suggests the involvement of oxidative stress in triggering both cell death pathways. Moreover, we demonstrated that caspase-1 is involved in CT-induced cell death, potentially contributing to host inflammatory response and tissue damage. Cells infected by L2 serovar displayed a higher activation of effector caspases compared to cells infected with serovar D, suggesting a serovar-specific activation of apoptotic pathways and potentially explaining the greater virulence of L serovars. Finally, we found that Chlamydia elicits the early externalization of phosphatidylserine on the external leaflet of plasma membrane independently of caspase activation.

Chlamydia persistence is a viable but non-replicative stage, induced by several sub-lethal stressor agents, including beta-lactam antibiotics. So far, no data about the connection between doxycycline and chlamydial persistence has been described in literature. We investigated the ability of doxycycline to induce C. trachomatis (CT) persistence in an in vitro model of epithelial cell infection (HeLa cells), comparing the results with the well-established model of penicillin-induced persistence. The effect of doxycycline was explored on 10 different CT strains by analysing (i) the presence of aberrant inclusions, (ii) chlamydial recovery, (iii) the expression of different chlamydial genes (omcB, euo, Ct110, Ct604, Ct755, HtrA) and (iv) the effects on epithelial cell viability. For each strain, the presence of foreign genomic islands responsible of tetracycline resistance was excluded. We found that low doses of doxycycline can induce a condition of CT persistence. For concentrations of doxycycline equal to 0.03–0.015 mg/L, CT inclusions are smaller and aberrant and CT cycle is characterized by the presence of viable but non-dividing RBs with the complete abolishment of chlamydial cytotoxic effect. Infectious EBs can be recovered after removal of the drug. During doxycycline-induced persistence, the expression of the late gene omcB is decreased, indicating the blocking of RB-to-EB conversion. Conversely, as for penicillin G, a significant up-regulation of the stress response HtrA gene is found in doxycycline-treated cells. This study provides a novel in vitro cell model to examine the characteristics of doxycycline-induced persistent CT infection.