Structure-Activity Relationships of the Enterococcal Cytolysin

Enterococcal cytolysin is a hemolytic virulence factor linked to human disease and increased patient mortality. Produced by pathogenic strains of Enterococcus faecalis, cytolysin is made up of two small, post-translationally modified peptides called CylL(L)” and CylL(S)”. They exhibit a unique toxicity profile where lytic activity is observed for both mammalian cells and gram-positive bacteria that is dependent on the presence of both peptides. In this study, we performed alanine substitution of all residues in CylL(L)” and CylL(S)” and determined the effect on both activities. We identified key residues involved in overall activity and residues that dictate cell type specificity. All (methyl)lanthionines as well as a Gly-rich hinge region were critical for both activities. In addition, we investigated the binding of the two subunits to bacterial cells suggesting that the large subunit CylL(L)” has stronger affinity for the membrane or a target molecule therein. Genome mining identified other potential two-component lanthipeptides and provides insights into potential evolutionary origins.