Roles of novel cationic lipids in bacterial pathogenesis

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Abstract Lipids are major components of bacterial membranes and play vital roles in bacterial survival, host-pathogen interactions and pathogenesis. An important mechanism used by bacteria to interact with hosts and cope with antimicrobial stresses is the modulation of surface charge via alteration of membrane lipid composition. However, membrane lipids remain uncharacterized for many significant bacterial pathogens, and our understanding of how the bacterial cell surface contributes to pathogenesis is still very limited. We aim to address these important knowledge gaps in our research on Gram-positive pathogens. We recently discovered novel, highly positively charged glycolipids in a range of Gram-positive pathogens, including the neonatal meningitis pathogen Streptococcus agalactiae (Group B Streptococcus; GBS). The overarching hypothesis of the project is that cationic glycolipids impact Gram-positive bacterial cell surface biochemical properties and play crucial roles in pathogenesis. Using GBS as a model organism, in Aim 1 we propose to investigate mechanisms for cationic glycolipid-dependent blood-brain barrier disruption by GBS. We will examine key mechanisms involved in barrier disruption, including loss of tight junctions, direct bacterial invasion and transcytosis, and inflammatory damage. We will also characterize the role of GBS membrane vesicles during the pathogenesis of meningitis. In Aim 2, we will investigate regulatory interactions between cationic glycolipids and lipoteichoic acid (LTA) biosynthesis. Our genetic and lipidomic analysis show that these pathways have an inverse relationship because they utilize the same substrates. This is a novel mechanism for regulating the cell surface structure-function of a Gram- positive pathogen, and has relevance beyond GBS, as we have also identified novel cationic glycolipids in multiple pathogenic enterococci. We will investigate this proposed mechanism further and quantify how this co- regulation impacts the structure and level of GBS LTA using a combination of chemical, biochemical and genetic approaches. In Aim 3, we will investigate the molecular mechanism for cationic glycolipid synthesis. The enzyme that synthesizes novel cationic glycolipids is the Multiple Peptide Resistance Factor (MprF), a broadly distributed virulence factor in bacteria. The molecular mechanism for lipid substrate selection by MprF enzymes is unknown. Using molecular evolution, global statistical models, and machine learning approaches that analyze the entire MprF family, we propose a rigorous dissection of MprF lipid substrate specificity in GBS, which will inform studies of MprF structure-function in GBS and other pathogenic bacteria, and guide the discovery of other pathogenic species producing cationic glycolipids. Overall, this project will elucidate the biosynthesis, distribution and physiological functions of the novel cationic lipids in GBS and other Gram-positive bacteria and uncover their roles in bacterial pathogenesis.