Abstract
Alanine transport and metabolism impact MRSA pathophysiology by dictating the availability of d-alanine for cell wall synthesis, the target of β-lactam antibiotics. Furthermore cycA-dependent alanine transport controls MRSA β-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 β-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 C(539)T mutation in dat, predicted to result in a S(180)F substitution. Expression of the dat (C539T) operon in wild-type increased BCDA resistance. alr1/dat::Em and alr1/dat (C539T) double mutants were auxotrophic for d-alanine, indicating that Dat-S(180)F transaminase activity is impaired, a conclusion supported by in vitro enzyme assays. Structural modeling revealed an active-site loop shift in Dat-S(180)F that altered PLP co-factor binding. Molecular docking showed that the S(180)F 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.