Inerolysin and vaginolysin, the cytolysins implicated in vaginal dysbiosis, differently impair molecular integrity of phospholipid membranes

Abstract

The pore-forming toxins, inerolysin (INY) and vaginolysin (VLY), produced by vaginal bacteria Lactobacillus iners and Gardnerella vaginalis were studied using the artificial cholesterol-rich tethered bilayer membranes (tBLMs) by electrochemical techniques. The electrochemical impedance spectroscopy (EIS) of tBLMs attested for the toxin-induced impairment of the integrity of phospholipid membranes. This observation was in line with the atomic force microscopy data demonstrating formation of oligomeric protein assemblies in tBLMs. These assemblies exhibited different morphologies: VLY mostly formed complete rings, whereas INY produced arciform structures. We found that both EIS (membrane damage) and the surface plasmon resonance (protein binding) data obtained on tBLMs are in-line with the data obtained in human cell lysis experiments. EIS, however, is capable of capturing effects inaccessible for biological activity assays. Specifically, we found that the INY-induced damage of tBLMs is nearly a linear function of membrane cholesterol content, whereas VLY triggered significant damage only at high (50 mol%) cholesterol concentrations. The observed differences of INY and VLY activities on phospholipid membranes might have clinical importance: both toxin-producing bacteria have been found in healthy vagina and dysbiosis, suggesting the need for adaptation at different vaginal conditions. Our results broaden the possibilities of application of tBLMs in medical diagnostics.

Cholesterol-Dependent Cytolysins Produced by Vaginal Bacteria: Certainties and Controversies

“…Superimposition of available CDC structures indicates that amino acids in the UPD loop of non-hCD59-dependent cytolysins interact with the L1 loop via hydrogen bonds, which leads to the proper conformation of L1 for initial binding with membrane FIGURE 3 | (A) Atomic force microscopy three-dimensional topography image of the oligomeric structure obtained after reconstitution of VLY into a cholesterol-rich artificial bilayer. Calculated protrusion of the ring-shaped defect above the membrane is consistent with the height of inserted pore (reproduced with permission from Ragaliauskas et al, 2019). (B) Crystal structure of VLY in complex with hCD59 (PDB ID: 5IMY; Lawrence et al, 2016).…”

Colonization at birth with human CST IV cervicovaginal microbiota alters development and increases neonatal mortality in mice

“…G. vaginalis strain 11E4 was isolated from a women with BV (Nugent Score 7), a vaginal pH of 4.7, and a CST IV vaginal microbiota (subject 15 in (Ravel et al, 2013). The strain carries all known pathogenic traits associated with G. vaginalis proper, including the expression of vaginolysin, a cholesterol-dependent pore-forming cytolysins implicated in vaginal dysbiosis (Gelber et al, 2008; Ragaliauskas et al, 2019). This strain was selected based on pilot testing conducted by the Ravel lab in which 14 strains of human G. vaginalis were evaluated for biofilm formation, a feature associated with symptomatic bacterial vaginosis (Gelber et al, 2008; Ragaliauskas et al, 2019).…”

“…The strain carries all known pathogenic traits associated with G. vaginalis proper, including the expression of vaginolysin, a cholesterol-dependent pore-forming cytolysins implicated in vaginal dysbiosis (Gelber et al, 2008; Ragaliauskas et al, 2019). This strain was selected based on pilot testing conducted by the Ravel lab in which 14 strains of human G. vaginalis were evaluated for biofilm formation, a feature associated with symptomatic bacterial vaginosis (Gelber et al, 2008; Ragaliauskas et al, 2019). Based on this, we tested two biofilm forming strains (11E4 and 16B1) and confirmed the presence of G. vaginalis 48-hours post-inoculation in mouse vaginal fluid by qPCR for rpoB .…”

“…vaginalis were evaluated for biofilm formation, a feature associated with symptomatic bacterial vaginosis (Gelber et al, 2008;Ragaliauskas et al, 2019) were split by sample using the QIIME-dependent script split_sequence_file_on_sample_ids.py, and primer sequences were removed using TagCleaner (version 0.16). Further processing followed the DADA2 workflow for Big Data and DADA2 (v.1.5.2) (https://benjjneb.github.io/dada2/bigdata.html).…”

Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

“…CDCs such as inerolysin and vaginolysin (G. vaginalis speci c) have been associated to membrane adhesion and subsequent pathogenicity. Pore formation in the host cell membranes, a characteristic of CDCs, has been speculated to allow the microbes to gain access to nutrients and potentially kill immune cells, allowing L. iners to survive in a dysbiotic state [66,69,70]. A repertoire of enzymes involved in the biosynthesis and degradation of inosine monophosphate (IMP) were observed in samples containing G. vaginalis.…”