A D-alanine aminotransferase S180F substitution confers resistance to β-chloro-D-alanine in Staphylococcus aureus via antibiotic inactivation

Alanine transport and metabolism impact MRSA pathophysiology by dictating the availability of D-alanine for cell wall synthesis, the target of {beta}-lactam antibiotics. Furthermore cycA-dependent alanine transport controls MRSA {beta}-lactam susceptibility in chemically defined medium (CDM) in a glucose-dependent manner. Here we report that S. aureus was auxotrophic for L-alanine in CDM, and that this growth defect was rescued by glucose (or compensatory mutations), but only when the alanine racemase (alr1) and D-alanine aminotransferase (dat) genes were functional. No role was observed for the alanine dehydrogenase 1 (ald1) and ald2 genes. As previously reported, alr1 and, to a lesser extent, cycA mutations increased susceptibility to D-cycloserine (DCS). In contrast, only alr1 mutation increased susceptibility to {beta}-chloro-D-alanine (BCDA), suggesting distinct targets for these alanine analogue antibiotics, which act synergistically against MRSA. Genome sequencing of a BCDA-resistant mutant identified a C539T mutation in dat, predicted to result in a S180F substitution. Expression of the datC539T operon in wild-type increased BCDA resistance. alr1/dat::Em and alr1/datC539T double mutants were auxotrophic for D-alanine, indicating that Dat-S180F transaminase activity is impaired, a conclusion supported by in vitro enzyme assays. Structural modeling revealed an active-site loop shift in Dat-S180F that altered PLP co-factor binding. Molecular docking showed that the S180F substitution promotes BCDA-PLP adduct dissociation by releasing inactivated BCDA, thereby conferring resistance. These data reveal essential roles for Alr1 and Dat during growth under nutrient-limiting conditions and the potential of combination therapy separately targeting both enzymes with DCS and BCDA to extend the treatment options for MRSA infections.