Peptidoglycan hydrolases, their protein partners, and related membrane proteins in Staphylococcus aureus

Abstract

In almost all bacteria, the peptidoglycan (PG) cell wall is required to maintain cell shape and protect the cell from the environment. PG is made up of sugar polymers connected by crosslinked peptides that form a single meshwork encasing the cell. Some of the enzymes that synthesize PG have received a lot of attention because they are the targets of some of the most successful clinical antibiotics. However, in addition to the polymerases and transpeptidases that make PG, there is a host of other enzymes involved in PG biogenesis, including hydrolases that cleave bonds in PG. Two hydrolases that act in early stages of PG synthesis in Staphylococcus aureus have recently been identified in our lab. First, LytH is a membrane-bound amidase that is important for regulating cell growth and division. It also requires another membrane protein, ActH, for activity. Here, I show that the extracellular portion of ActH activates LytH by stabilizing metals in the active site. This work suggests both that ActH has multiple functions and that metal cofactor stabilization by a partner protein is a widespread mechanism used to activate amidases. Next, SagB, a glucosaminidase, and its regulator SpdC control glycan strand length by releasing newly synthesized PG strands from the membrane. Here, I develop a new assay for monitoring glycan strand length and discuss the coordination between PG polymerization and release by SagB-SpdC. Finally, I describe preliminary studies of another membrane protein, SAOUHSC_01908, that is important for cell envelope integrity. Our studies suggest that this protein plays a role in cell division. This thesis advances our understanding of hydrolases and creates opportunities for future study to elucidate the function of an important membrane protein. All of the proteins studied are potential targets for beta-lactam sensitizing agents; better understanding their mechanisms may aid in future antibiotic development.