In vitro and in vivo studies of two essential MRSA cell wall synthesis enzymes

Abstract

Methicillin resistant Staphylococcus aureus (MRSA) is a growing healthcare threat worldwide. The mechanism of antibiotic resistance in MRSA relies on the acquired cell wall synthesis enzyme PBP2a, which rescues transpeptidase (TP) activity upon inhibition by β-lactam drugs. Previous evidence reveals a functional coordination between PBP2a and PBP2, an essential S. aureus enzyme with glycosyltransferase (GT) and TP activities. Excitingly, recent data have demonstrated that PBP2 and PBP2a form a physical complex, which can be isolated via tandem affinity purification and size exclusion chromatography. In Chapter 2 of this thesis work, we replicate the PBP2/PBP2a complex purification in an effort to further characterize the heterodimer. However, binding assays and structural studies present challenges that suggest a weak binding interaction in vitro. To investigate the coordination between PBP2 and PBP2a within the S. aureus cell, we express PBP2a in a methicillin sensitive Staphylococcus aureus (MSSA) background and study its impact on PBP2 localization. In Chapter 3, we explore PBP2’s recruitment to the division site and the relevance of PBP2a for this recruitment under antibiotic treatment. Here, we find that PBP2a can rescue PBP2’s septal localization when it is disrupted by β-lactams. We further demonstrate that antibiotic inhibition of either GT or TP activity displaces PBP2 from the division site, but a catalytically inactive copy of PBP2 is still able to localize properly in the presence of other active PBP2 enzymes. Ultimately, our data suggest a unique model of PBP2 septal recruitment, in which nascent septal peptidoglycan recruits PBP2 to the division site in a positive feedback loop, and other divisome components stabilize the enzyme’s localization once it arrives. These discoveries add important insights into how S. aureus is positioned for its essential role in cell division and how PBP2a acts to rescue MRSA strains from β-lactam inhibition.