Investigating Staphylococcus aureus Cell Envelope Enzymes for Antibiotic Discovery

Abstract

The rise of antibiotic-resistant Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA), has created an urgent need for new antibacterial strategies. The bacterial cell envelope—comprising the peptidoglycan cell wall, membrane-bound enzymes, and teichoic acids—remains a validated but underexploited target space for antibiotic discovery. This thesis describes three complementary approaches to address antibiotic resistance by investigating S. aureus cell envelope enzymes. The first approach focuses on discovering small-molecule inhibitors of FtsW, a highly conserved and essential peptidoglycan polymerase involved in septal cell wall synthesis. To identify FtsW inhibitors, we developed a time-resolved FRET (TR-FRET) assay capable of detecting polymerase activity in vitro. Screening a curated library of compounds lethal to S. aureus revealed a set of candidate inhibitors, including compounds that act through substrate competition. Biochemical validation and substrate displacement assays confirmed their activity and provided insights into the mechanism of inhibition. The second approach centers on the identification and characterization of SpbR, a previously uncharacterized membrane protein discovered through transposon sequencing under teichoic acid inhibition conditions. SpbR modulates lipoteichoic acid (LTA) length and physically interacts with SpsB, the essential type I signal peptidase in S. aureus. Genetic and biochemical evidence shows that SpbR inhibits SpsB-mediated cleavage of LtaS, the LTA polymerase, resulting in extended LTA polymers. SpbR also partially inhibits cleavage of other canonical SpsB substrates, functioning as a general modulator of SpsB activity. To our knowledge, SpbR is the first described modulator of a bacterial type I signal peptidase. The third approach involves structure-guided discovery of inhibitors targeting DltB, a membrane protein involved in D-alanylation of teichoic acids. While D-alanylation is not essential under normal conditions, it plays a key role in virulence and antimicrobial resistance. Virtual screening against the cryo-EM structure of DltB identified compounds that inhibit growth only in strains missing a membrane protein that is synthetically lethal with the Dlt pathway, but not in wild-type S. aureus. Genetic suppressor analysis and saturation mutagenesis revealed a previously uncharacterized binding site and provided insight into the mechanism of inhibition. Together, these studies advance our understanding of S. aureus cell envelope biology and highlight diverse strategies for antibiotic development—through inhibition of essential enzymes, identification of modulators of envelope biogenesis, and targeting of non-essential enzymes to potentiate existing antibiotics.