Purification of a Bacteriocin-like Inhibitor Produced by Enterococcus faecium strain 62-6 Antagonistic to the Growth of Vaginal Lactobacilli: Potential Significance for the Pathogenesis of Bacterial Vaginosis

Abstract

Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a shift in the ecology of the vaginal tract, and reflected by an alteration in the micro flora. During BV, Lactobacillus populations that are generally dominant in the vaginal tract of healthy women are replaced by a consortium of other organisms including Gardnerella vaginalis and anaerobes. Currently, the mechanism(s) which could account for the shift in the ecology and for the decrease in the prevalence and concentration of lactobacilli are not well understood. One approach being used is to investigate inhibitory substances produced by vaginal bacteria, which are antagonistic to the growth of vaginal lactobacilli. Previous studies have characterized a heat stable, proteinaceous inhibitor (a bacteriocin) produced by Enterococcus faecium strain 62-6. The aim of this study was to purify the bacteriocin, and ultimately determine its molecular mass and amino acid sequence. The bacteriocin was grown to one-liter volumes in brain heart infusion (BHI), isolated using 60% w/v ammonium sulfate precipitation, and dialyzed. The resulting concentrated inhibitor-containing preparation was subjected to Sepharose cation exchange chromatography and the inhibitor was eluted using a continuous salt gradient. All stages of the isolation and purification procedure were tested for the presence of the bacteriocin using the well diffusion assay. Sepharose column fractions positive for inhibitory activity against sensitive lactobacilli were analyzed by SDS-PAGE to isolate the protein. Agar overlayers of the resulting PAGE gels were used to identify the inhibitor and suggested that it was approximately 6kDa in mass. A protein band associated with inhibitory activity was transferred to a PVDF membrane and subsequently analyzed by Edman degradation to determine its amino acid sequence. Due to N-terminally blockage, no, amino acid sequence has been obtained. Also, the determination of molecular mass by HPLC has been unsuccessful, but further MALDI analysis is in progress.