D-alanine synthesis and exogenous alanine affect the antimicrobial susceptibility of Staphylococcus aureus

D-alanine is an important amino acid for peptidoglycan biosynthesis in Staphylococcus aureus. In addition, D-alanine is used for the modification of teichoic acids to weaken the net surface negative charge, leading to decreased susceptibility to cationic antimicrobial agents. D-alanine synthesis is dependent on only two enzymes. One is alanine racemase, encoded by the alr1 gene, which reversibly converts L-alanine and D-alanine. The other is D-amino acid transaminase, encoded by the dat gene, which synthesizes D-amino acids from α-keto acids and other D-amino acids. In addition, the uptake of L- and D-alanine is dependent on the alanine transporter CycA. To reveal the relationship between D-alanine supply and antimicrobial susceptibility, we evaluated antimicrobial susceptibility in alr1, dat, and cycA inactivation mutants. These mutants, especially the Δalr1 and ΔcycA mutants, presented increased susceptibility to β-lactams, D-cycloserine, bacitracin, lysostaphin, and cationic antimicrobial agents such as aminoglycosides, nisin A, and daptomycin. The net negative charge of the cell surface increased in the Δalr1 and ΔcycA mutants. The changes in susceptibility to antimicrobial agents and cell surface charge were restored in their gene-complemented mutants. Furthermore, in an alanine-depleted medium, the MIC for oxacillin decreased significantly, and the MIC for gentamicin also decreased slightly. Clinical MRSA strains also showed significantly increased susceptibility to oxacillin in the alanine-depleted medium. These results indicate that D-alanine deficiency leads to impaired peptidoglycan and increased net surface negative charge, resulting in increased antimicrobial susceptibility.