Biosynthesis and mechanism of action of enterococcal cytolysin

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large, structurally diverse group of natural products that are united by a common biosynthetic logic. During RiPP biosynthesis, a genetically encoded precursor peptide is post-translationally modified by enzymes. The precursor peptide is typically composed of an N-terminal leader peptide and a C-terminal core peptide. The leader peptide serves as a recognition motif for the modification enzymes that subsequently catalyze transformations on the core peptide. The separation between substrate recognition on the leader peptide from catalysis on the core peptide allows RiPP modification enzymes to be inherently substrate tolerant. Changes to the core peptide are generally well tolerated, as long as the recognition motif on the leader peptide is not perturbed. Detailed understanding of the mechanism of substrate recognition in RiPP biosynthesis can enable RiPP enzymes to be utilized in a variety of biotechnological platforms.

In chapter 2, the mode of substrate recognition between the class II lanthipeptide synthetase, HalM2, and the leader peptide of its cognate substrate, HalA2 is described. Critical residues on the leader peptide required for substrate recognition and processing were identified. Furthermore, the capping helices on HalM2 were determined to be involved in leader peptide binding. In chapter 3, the interactions identified were applied to strategically position cysteine residues on both the substrate and enzyme to generate covalent substrate-enzyme complexes. The complexes were found to be catalytically competent, thus providing a platform to investigate the positional requirements of the leader peptide during catalysis.

Chapter 4 focuses on the structure-activity relationships of an unusual class II lanthipeptide known as enterococcal cytolysin. Cytolysin is composed of two subunits, a large subunit, CylLL” and a small subunit, CylLS”. Cytolysin is a virulence factor produced by pathogenic strains of Enterococci and has been directly linked to human disease and mortality. Amongst lanthipeptides, cytolysin displays a unique bioactivity profile with potent lytic activity against both gram-positive bacteria and mammalian erythrocytes. Alanine scanning mutagenesis of every residue in both subunits of cytolysin was performed and bioactivity was determined for every mutant. Specific residues appear to dictate cell-type specificity, suggesting that the molecular target of cytolysin may be a structurally related molecule between the two cell types. Additionally, I provide evidence that CylLL” binds to bacterial cell membranes first, potentiating the lytic activity of CylLS”. In chapter 5, I provide insights into the molecular target of cytolysin in both bacterial and mammalian systems. These insights set the stage to understanding the molecular recognition of cytolysin and target cells.