| The Staphylococcus aureus cell envelope is a large, complex structure essential for cell shape and protection from the environment. It consists of membrane lipids, glycan polymers, and proteins, many of which have not been characterized despite decades of research. One method for elucidating biological function is to identify synthetic lethal interactions. Specific chemical inhibitors of known proteins are powerful tools in these studies. Here I describe a systematic approach to identify small molecule inhibitors useful for synthetic lethal interaction mapping. I first probe a transposon mutant library with an inhibitor of wall teichoic acid (WTA) biosynthesis and perform transposon insertion sequencing (Tn-seq) to refine a growing network of genetic interactions centered on WTAs. I next carried out a whole-cell pathway-directed high-throughput chemical screen for inhibitors of proteins within the WTA interaction network based on differential growth inhibition of WTA-deficient cells versus wild-type. To identify hits, I developed a flexible method of analysis that ranks hit compounds by likelihood of being a true positive. Through this screen I found a direct relationship between teichoic acid D-alanylation and permeability to positively charged antibiotics such as aminoglycosides. I also identified amsacrine as an inhibitor of WTA-deficient strains and established its target as the D-alanyl aceyltransferase DltB. I showed that amsacrine phenocopies a D-alanylation-deficient strain in sensitizing S. aureus to aminoglycosides and preventing biofilm formation. My research represents the first iteration of a "discovery cycle" for using a specific small molecule probe to identify genetic interactions, then exploiting those genetic interactions to perform a chemical screen that identifies additional probes. This work describes a rapid and adaptable method for exploring the complex interactions within cell wall biosynthesis. |
