Reconstitution of the PBP2-PBP2a Complex from Methicillin-Resistant Staphylococcus aureus

Abstract

β-lactams are commonly-used antibiotics that target penicillin-binding proteins (PBPs), the enzymes which build the bacterial cell wall. In methicillin-resistant Staphylococcus aureus (MRSA), resistance to these drugs is mediated by the β-lactam-insensitive PBP, PBP2a. PBP2a replaces the transpeptidase (TP) activity of the essential bifunctional enzyme PBP2 when it is inhibited by β-lactam, although its other activity as a glycosyltransferase (GT) remains intact. Previous work has demonstrated that PBP2 and PBP2a must coordinate their functions to assemble peptidoglycan (PG), although the mechanism of this cooperative behavior is unknown. In this work, we demonstrate that PBP2 and PBP2a form a stable heterodimeric complex, which can be purified by heterologous production in E. coli. The isolated complex has in vitro PG assembly activity on native S. aureus substrates. We identified the transmembrane helices as contributing to the stability of PBP2-PBP2a interaction. The co-purification suggests that PBP2 and PBP2a interact directly between their extracellular domains, which may facilitate their cooperative function in vivo. Many PBPs function in complexes, including the recently-identified SEDS-bPBPs, which require cognate binding for GT activity. Admixing of purified PBP2 and PBP2a revealed that PBP2a stimulates the GT activity of PBP2, causing an increase in PG strand length and substrate depletion. PBP2a is able to restore processivity to a moenomycin-resistant mutant of PBP2 which generates short polymer products. We demonstrate that this stimulatory effect is dependent on the transmembrane helix of PBP2a, but not its TP domain. These results provide evidence that addition of the resistant PBP2a enzyme causes changes in the existing methicillin-sensitive cell wall machinery, allowing cells to overcome the growth disadvantage posed by β-lactam treatment. The isolation of the PBP2-PBP2a complex provides an opportunity to better understand the cell wall synthesis of MRSA to help address the mounting problem of acquired antibiotic resistance. The work described here also suggests an ancestral function of PBP2a homologues that is not related to their role in antibiotic resistance. Indeed, the isolation and reconstitution of this complex may be the first example of a new type of widely-distributed cell wall synthesis machinery.