Lactic acid from vaginal microbiota enhances cervicovaginal epithelial barrier integrity by promoting tight junction protein expression.

BACKGROUND: Women with a cervicovaginal microbiota dominated by Lactobacillus spp. are at reduced risk of acquiring sexually transmitted infections including HIV, but the biological mechanisms involved remain poorly defined. Here, we performed metaproteomics on vaginal swab samples from young South African women (n = 113) and transcriptomics analysis of cervicovaginal epithelial cell cultures to examine the ability of lactic acid, a metabolite produced by cervicovaginal lactobacilli, to modulate genital epithelial barrier function. RESULTS: Compared to women with Lactobacillus-depleted microbiota, women dominated by vaginal lactobacilli exhibit higher abundance of bacterial lactate dehydrogenase, a key enzyme responsible for lactic acid production, which is independently associated with an increased abundance of epithelial barrier proteins. Physiological concentrations of lactic acid enhance epithelial cell culture barrier integrity and increase intercellular junctional molecule expression. CONCLUSIONS: These findings reveal a novel ability of vaginal lactic acid to enhance genital epithelial barrier integrity that may help prevent invasion by sexually transmitted pathogens. Video abstract.

Three-dimensional models of the cervicovaginal epithelia to study host-microbiome interactions and sexually transmitted infections.

2D cell culture systems have historically provided controlled, reproducible means to analyze host-pathogen interactions observed in the human reproductive tract. Although inexpensive, straightforward, and requiring a very short time commitment, these models recapitulate neither the functionality of multilayered cell types nor the associated microbiome that occurs in a human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications of animal models are required to recreate interactions that resemble those in the human reproductive tract. 3D cell culture models have emerged as alternative means of reproducing vital elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments. Here, we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extracellular matrix (ECM)-like layer. The model produced tissues with morphologic and physiological resemblance to human cervical and vaginal epithelia, including mucus levels produced by cervical cells. Infection by Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated, as well as the growth of bacterial species observed in the human vaginal microbiota. This enabled controlled mechanistic analyses of the interactions between host cells, the vaginal microbiota, and STI pathogens. Affordable and semi high-throughput 3D models of the cervicovaginal epithelia that are physiologically relevant by sustaining vaginal bacterial colonization, and facilitate studies of chlamydial and gonococcal infections.

The Cervicovaginal Microbiota-Host Interaction Modulates Chlamydia trachomatis Infection.

The mechanism(s) by which Lactobacillus-dominated cervicovaginal microbiota provide a barrier to Chlamydia trachomatis infection remain(s) unknown. Here we evaluate the impact of different Lactobacillus spp. identified via culture-independent metataxonomic analysis of C. trachomatis-infected women on C. trachomatis infection in a three-dimensional (3D) cervical epithelium model. Lactobacillus spp. that specifically produce d(-) lactic acid were associated with long-term protection against C. trachomatis infection, consistent with reduced protection associated with Lactobacillus iners, which does not produce this isoform, and with decreased epithelial cell proliferation, consistent with the observed prolonged protective effect. Transcriptomic analysis revealed that epigenetic modifications involving histone deacetylase-controlled pathways are integral to the cross talk between host and microbiota. These results highlight a fundamental mechanism whereby the cervicovaginal microbiota modulates host functions to protect against C. trachomatis infection.IMPORTANCE The vaginal microbiota is believed to protect women against Chlamydia trachomatis, the etiologic agent of the most prevalent sexually transmitted infection (STI) in developed countries. The mechanism underlying this protection has remained elusive. Here, we reveal the comprehensive strategy by which the cervicovaginal microbiota modulates host functions to protect against chlamydial infection, thereby providing a novel conceptual mechanistic understanding. Major implications of this work are that (i) the impact of the vaginal microbiota on the epithelium should be considered in future studies of chlamydial infection and other STIs and (ii) a fundamental understanding of the cervicovaginal microbiota's role in protection against STIs may enable the development of novel microbiome-based therapeutic strategies to protect women from infection and improve vaginal and cervical health.

Inhibition of EGFR Signaling Protects from Mucormycosis.

Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. Rhizopus species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled Rhizopus spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We performed an unbiased survey of the host transcriptional response during early stages of Rhizopus arrhizus var. delemar (R. delemar) infection in a murine model of pulmonary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis revealed activation of the host's epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon in vitro infection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with R. delemar spores. Inhibition of EGFR signaling with cetuximab or gefitinib, specific FDA-approved inhibitors of EGFR, significantly reduced the ability of R. delemar to invade and damage airway epithelial cells. Furthermore, gefitinib treatment significantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epithelial cells by R. delemar, and inhibition of EGFR signaling provides a novel approach for treating mucormycosis by repurposing an FDA-approved drug.IMPORTANCE Mucormycosis is an increasingly common, highly lethal fungal infection with very limited treatment options. Using a combination of in vivo animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs significantly increased survival following R. arrhizus var. delemar infection in mice. This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of-concept for the repurposing of FDA-approved drugs that target EGFR function.

Neisseria gonorrhoeae infects the human endocervix by activating non-muscle myosin II-mediated epithelial exfoliation.

Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation.

Lactobacillus crispatus inhibits growth of Gardnerella vaginalis and Neisseria gonorrhoeae on a porcine vaginal mucosa model.

BACKGROUND: The vaginal microbiota can impact the susceptibility of women to bacterial vaginosis (BV) and sexually transmitted infections (STIs). BV is characterized by depletion of Lactobacillus spp., an overgrowth of anaerobes (often dominated by Gardnerella vaginalis) and a pH > 4.5. BV is associated with an increased risk of acquiring STIs such as chlamydia and gonorrhea. While these associations have been identified, the molecular mechanism(s) driving the risk of infections are unknown. An ex vivo porcine vaginal mucosal model (PVM) was developed to explore the mechanistic role of Lactobacillus spp. in affecting colonization by G. vaginalis and Neisseria gonorrhoeae. RESULTS: The data presented here demonstrate that all organisms tested can colonize and grow on PVM to clinically relevant densities. Additionally, G. vaginalis and N. gonorrhoeae form biofilms on PVM. It was observed that lactic acid, acetic acid, and hydrochloric acid inhibit the growth of G. vaginalis on PVM in a pH-dependent manner. N. gonorrhoeae grows best in the presence of lactic acid at pH 5.5, but did not grow well at this pH in the presence of acetic acid. Finally, a clinical Lactobacillus crispatus isolate (24-9-7) produces lactic acid and inhibits growth of both G. vaginalis and N. gonorrhoeae on PVM. CONCLUSIONS: These data reveal differences in the effects of pH, various acids and L. crispatus on the growth of G. vaginalis and N. gonorrhoeae on a live vaginal mucosal surface. The PVM is a useful model for studying the interactions of commensal vaginal microbes with pathogens and the mechanisms of biofilm formation on the vaginal mucosa.

Neisseria gonorrhoeae breaches the apical junction of polarized epithelial cells for transmigration by activating EGFR.

Neisseria gonorrhoeae initiates infection at the apical surface of columnar endocervical epithelial cells in the female reproductive tract. These cells provide a physical barrier against pathogens by forming continuous apical junctional complexes between neighbouring cells. This study examines the interaction of gonococci (GC) with polarized epithelial cells. We show that viable GC preferentially localize at the apical side of the cell-cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84. In GC-infected cells, continuous apical junctional complexes are disrupted, and the junction-associated protein ß-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites; however, overall cellular levels remain unchanged. This redistribution of junctional proteins is associated with a decrease in the 'fence' function of the apical junction but not its 'gate' function. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and ß-catenin, while inhibition of EGFR kinase activity significantly reduces both GC-induced ß-catenin redistribution and GC transmigration. Therefore, the gonococcus is capable of weakening the apical junction and polarity of epithelial cells by activating EGFR, which facilitates GC transmigration across the epithelium.

Construction and characterization of a derivative of Neisseria gonorrhoeae strain MS11 devoid of all opa genes.

To better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11Δopa) that had the coding sequence for all 11 Opa proteins deleted. The MS11Δopa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11Δopa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11Δopa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11Δopa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ∼85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11Δopa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11Δopa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.

Neisseria gonorrhoeae-induced transactivation of EGFR enhances gonococcal invasion.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, adheres to and invades into genital epithelial cells. Here, we investigate host components that are used by the bacteria for their entry into epithelial cells. We found that gonococcal microcolony formation on the surface of HEC-1-B cells disrupted the polarized, basolateral distribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member, and induced their accumulation under the microcolonies at the apical membrane. Gonococcal infection increased EGFR and ErbB2 phosphorylation. The EGFR kinase inhibitor, AG1478, reduced gonococcal invasion by 80%, but had no effect on adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the mRNA levels of several ligands of EGFR. Prevention of EGFR ligand shedding by blocking matrix metalloproteinase activation reduced gonococcal invasion without altering their adherence, while the addition of the EGFR ligand, HB-EGF, was able to restore invasion to 66% of control levels. These data indicate that N. gonorrhoeae modulates the activity and cellular distribution of host EGFR, facilitating their invasion. EGFR activation does not appear to be due to direct gonococcal binding to EGFR, but instead by its transactivation by gonococcal induced increases in EGFR ligands.

Human squamous cell carcinomas evade the immune response by down-regulation of vascular E-selectin and recruitment of regulatory T cells.

Squamous cell carcinomas (SCCs) of the skin are sun-induced skin cancers that are particularly numerous in patients on T cell immunosuppression. We found that blood vessels in SCCs did not express E-selectin, and tumors contained few cutaneous lymphocyte antigen (CLA)(+) T cells, the cell type thought to provide cutaneous immunosurveillance. Tumors treated with the Toll-like receptor (TLR)7 agonist imiquimod before excision showed induction of E-selectin on tumor vessels, recruitment of CLA(+) CD8(+) T cells, and histological evidence of tumor regression. SCCs treated in vitro with imiquimod also expressed vascular E-selectin. Approximately 50% of the T cells infiltrating untreated SCCs were FOXP3(+) regulatory T (T reg) cells. Imiquimod-treated tumors contained a decreased percentage of T reg cells, and these cells produced less FOXP3, interleukin (IL)-10, and transforming growth factor (TGF)-beta. Treatment of T reg cells in vitro with imiquimod inhibited their suppressive activity and reduced FOXP3, CD39, CD73, IL-10, and TGF-beta by indirect mechanisms. In vivo and in vitro treatment with imiquimod also induced IL-6 production by effector T cells. In summary, we find that SCCs evade the immune response at least in part by down-regulating vascular E-selectin and recruiting T reg cells. TLR7 agonists neutralized both of these strategies, supporting their use in SCCs and other tumors with similar immune defects.