Investigating genetic networks of cell envelope targets in Staphylococcus aureus

Abstract

Antibiotic resistance in bacterial pathogens is a significant concern for human health. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of difficult-to-treat infections, and novel antibiotics are needed. In this thesis, I describe studies investigating targets in the S. aureus cell envelope, which is a critical protective barrier surrounding the bacterial cell and the site of many successful antibiotic targets. Here, we used a genome-wide sequencing platform called transposon sequencing (Tn-Seq) to study the genetic networks of S. aureus cell envelope pathways. First, we profiled an optimized novel antibiotic belonging to the arylomycin class using Tn-Seq. The arylomycins target the type I signal peptidase, which is an essential and conserved enzyme that is responsible for the release of secreted proteins at the cell surface. This Tn-Seq study revealed that targeting the pathway responsible for lipoprotein maturation synergizes with the arylomycins and can be exploited to reverse arylomycin resistance in S. aureus. We also used Tn-Seq to identify a novel regulator of teichoic acid biosynthesis. An uncharacterized gene encoding a membrane protein was a hit in Tn-Seq profiles of inhibitors of both wall teichoic acid synthesis and lipoteichoic acid synthesis. Here, I describe studies of this membrane protein that show that it regulates lipoteichoic acid (LTA) length and abundance. This protein regulates LTA synthesis independently of a known LTA length regulatory mechanism dependent on membrane glycolipid levels, and its expression also affects processing of LtaS, the lipoteichoic acid synthase. Together, these studies demonstrate how studying the genetic interactions of antibiotic targets can be used to both investigate antibiotic resistance mechanisms as well as explore the biology of important pathways.