Enzymatic Synthesis of Peptidoglycan in Methicillin-Resistant Staphylococcus Aureus and Its Inhibition by Beta-lactams

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for more than half of the deaths due to antibiotic resistant bacterial infection in the United States, killing over ten thousand people a year. Beta-lactams, our first-line treatment against Staph infection, target the transpeptidase domain of penicillin-binding proteins (PBPs), the enzymes involved in the final steps of peptidoglycan biosynthesis, rendering the bacteria unable to synthesize the cell wall. These clinically important drugs however are ineffective against MRSA because MRSA acquired PBP2a, a non-native PBP, that can still crosslink peptidoglycan when S. aureus native PBPs are inhibited. Despite the importance of PBPs as antibiotic targets, their transpeptidase activities had not been reconstituted in vitro and there was no method to directly evaluate beta-lactam inhibition of the PBP activity. One major hurdle to study the PBPs was the inaccessibility of their substrate, Lipid II. In this thesis, I will describe a simple and versatile strategy for isolating native Lipid II from S. aureus and other bacteria. A chemical probe triggers Lipid II accumulation and a two-step extraction yields a practical amount of clean Lipid II. I used the isolated Lipid II to reconstitute peptidoglycan synthesis by S. aureus PBP2 and PBP2a. I also reported a direct assay of transpeptidase inhibition by beta-lactams. Comparing the substrate preference of S. aureus PBPs, I found that PBP2a can unexpectedly crosslink peptidoglycan strands bearing a triglycine branch but not a monoglycine one. This work offers biochemical tools to gain a better understanding of cell wall biosynthesis and especially transpeptidases which are important drug targets. The methods reported here will be useful for developing the next generation of antibiotics and also potentiator compounds that restore sensitivity of MRSA to the currently available beta-lactams.