Understanding the Dynamics of Protein Lipoylation in Staphylococcus Aureus

Staphylococcus aureus is a commensal bacterium that also acts as an opportunistic pathogen. The pathogenicity of S. aureus has often been attributed to the wide range of virulence factors that the bacterium produces. While virulence factors do contribute a great deal, there is a growing field of research that aims to investigate the role of metabolism in bacterial virulence. My project focuses on the necessity of a metabolic cofactor, lipoic acid. To ensure sufficient amounts of lipoic acid are available for enzyme activity, S. aureus has evolved two pathways to obtain the important nutrient. The lipoic acid salvage pathway is composed of two lipoic acid ligases, LplA1 and LplA2, that sequester environmental lipoic acid. The de novo biosynthesis pathway allows for the transfer of octanoic acid from an acyl carrier protein to GcvH. The octanoic acid is converted to lipoic acid, and LipL facilitates the transfer to the bacterial metabolic protein. While a significant amount is known about these pathways from recent studies in our lab, some questions remain. So far, the Alonzo lab studied the function of LplA1 and LplA2 in the presence of free lipoic acid. While this is valuable in understanding ligase activity, it does not necessarily model the host environment in which there is limited free lipoic acid. A more prevalent substrate is lipoylpeptides. This led me to ask if LplA1 and LplA2 ligases can use lipoamide as a source of lipoic acid for protein lipoylation. Through biochemical lipoylation assays, we discovered that these enzymes are unable to directly use lipoylated peptides; instead, S. aureus harbors lipoamidase functions that allow for generation of the free acid from its peptide-bound form. My thesis also interrogates the activity of LipL. Previously attempts to purify LipL and study it in a lipoylation assay led to no observable function of the protein, and complementation of a ˆ†lipL strain with lipL did not restore of growth defects. I evaluated the lipL gene sequence and determined that the LipL coding sequence in NCBI yields a truncated non-functional gene. Use of an extended LipL in biochemical assays showed that LipL can utilize free lipoic acid alone or in conjunction with LplA1 to lipoylate a limited range of substrates