Impaired Alanine Transport or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to ß-lactam Antibiotics.

Prolonging the clinical effectiveness of ß-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between ß-lactams and DCS. DCS resensitized MRSA to ß-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to ß-lactam antibiotics.

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics.

Penicillin binding protein 2a (PBP2a)-dependent resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact ß-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to ß-lactam antibiotics. IMPORTANCEmecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to ß-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of ß-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome ß-lactam resistance.