Genetic basis of glycine and L-serine toxicity in Staphylococcus aureus and the case for glycine as an antibiotic adjuvant

The toxicity of the amino acids glycine and L-serine at high concentrations in bacteria was discovered decades ago. In this work, we used deep transposon insertion sequencing (Tn-seq) experiments to determine the genes necessary to tolerate excess L-serine, diglycine or glycine in the human pathogen Staphylococcus aureus. Our results indicate that intracellular accumulation of specific counterbalancing amino acids--such as alanine in excess glycine--is the primary mechanism of resistance to amino acid toxicity. Consistent with this model, specific amino acid and peptide uptake transporters were required for fitness in each treatment; the peptide transporter DtpT was crucial for fitness in excess L-serine or glycine, and the alanine transporter AapA was essential in diglycine. Tn-seq results also identified the cystine/cysteine uptake transporter TcyABC as necessary in excess L-serine, suggesting that both peptide and cysteine uptake contribute to L-serine tolerance. In addition to uptake mechanisms, glycine and diglycine toxicity is neutralized by D-alanine aminotransferase (Dat), which is required for D-alanine synthesis. The requirement for Dat and DtpT function--but not AapA--in excess glycine is explained by excess glycine inhibiting alanine uptake. Building on this finding, we found that combined treatment with glycine and the alanine analog antibiotic D-cycloserine was strongly synergistic in inhibiting S. aureus growth. Overall, our findings identify targetable mechanisms underlying excess amino acid tolerance in S. aureus, with implications for developing novel combination treatments using the accessible and biocompatible amino acids glycine and L-serine. IMPORTANCEGrowing evidence supports the beneficial effects of glycine and L-serine supplementation in combating bacterial infections. Previous researchers have found that combining antibiotic treatment with high glycine concentrations has additive effects with many antibiotics, even reversing resistance to antibiotics in some bacteria. In vivo, activating glycine and L-serine metabolism heightens the sensitivity of bacterial pathogens to the host complement system, and studies of glycine or L-serine treatments show low toxicity and reduced inflammation in mouse and human subjects. This study reveals that glycine may be an effective antibiotic adjuvant with D-cycloserine, and treatment with glycine or L-serine could potentiate other drugs that target alanine or cysteine metabolism, respectively.