Identifcation and Characterization of Inerolysin, the Cholesterol Dependent Cytolysin Produced by Lactobacillus Iners

Lactobacillus iners, is a gram positive organism recently identified through the use of culture independent techniques. Identified as a major constituent of the vaginal microbiota, epidemiological studies have suggested that this organism may not provide the protective effects ascribed to other vaginal lactobacilli. Our work here has identified and characterized a pore forming toxin, Inerolysin, produced by this organism. This pore forming toxin was present in all strains of L. iners tested and possessed characteristics which firmly categorize it as a member of the Cholesterol Dependent Cytolsin superfamily. Additionally, we identified pH as a regulatory factor for the activity of Inerolysin as well as other CDCs. Inerolysin was shown to had optimal activity at acidic pH, while other toxins such as pneumolysin and arcanolysin had optimal activity at basic pH. We demonstrate that pH induced changes in activity were reversible, suggesting that a reversible conformational change takes place in the protein. Furthermore, our results show that it is the last step in pore formation, the transition from pre-pore to pore, which is impaired. Our attempts to localize this to particular residues were unsuccessful. Finally, we sought to understand what the vaginal environment of a Lactobacillus dominated flora would look like. We demonstrate that L. iners induced unique signaling in the vaginal epithelium, leading to the production of a unique profile of proinflammatory cytokines as well as antimicrobial peptides. We further demonstrate that some of these responses are mediated by the activity of the pore forming toxin of L. iners, Inerolysin.