Divergent LD-transpeptidase-independent effects of peptidoglycan carboxypeptidases on intrinsic ss-lactam and vancomycin resistance

Vancomycin and {beta}-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is D-alanine-D-alanine (D-Ala-D-Ala), whereas that of {beta}-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidases on vancomycin and {beta}-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of PG carboxypeptidases induced sensitivity to most {beta}-lactams, whereas it induced strong resistance toward vancomycin. Notably, both of two phenotypes did not have strong association with LD-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by increased amount of decoy D-Ala-D-Ala residues within PG, whereas {beta}-lactam sensitivity was associated with physical interactions between PG carboxypeptidase and PBPs. The presence of OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened {beta}-lactam sensitivity. Collectively, our results revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases, which involved inverse modulation of bacterial resistance to clinically important antibiotics, {beta}-lactams and vancomycin, and presented evidence for a link between PG carboxypeptidase and PBPs. IMPORTANCEBacterial peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. To date, most studies on PG carboxypeptidases are focused on LD-transpeptidase-related roles. We have revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases with respect to antibiotic resistance. The deletion of PG carboxypeptidases led to sensitivity to most {beta}-lactams, while it caused strong resistance to vancomycin. The underlying molecular mechanisms of two phenotypes were not associated with LD-transpeptidases. Therefore, our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of {beta}-lactam antibiotics. One sentence summaryEffect of peptidoglycan carboxypeptidase on antibiotic