Distinct roles for theStaphylococcus aureusPOT transporter DtpT in di/tripeptide uptake and glutathione utilisation inside human macrophages

Abstract

Peptides available in biological niches inhabited by the human pathogenStaphylococcus aureusserve as a rich source of amino acids required for growth and successful host colonisation. Uptake of peptides byS. aureusinvolves at least two transport systems: the POT family di/tri-peptide permease DtpT and the oligopeptide ABC transporter Opp3. Here we study the individual and combined functions of DtpT and Opp3 in enabling utilisation of 282 di-/tri-peptides via a high-throughput phenotypic screen. We reveal that many peptides can be utilised via either transporter, though DtpT appears to be the primary route of uptake for dipeptides. Intriguingly, we demonstrate a preference for Asp/Glu-containing peptides among putative DtpT substrates. To measure DtpT-mediated peptide transport directly, the protein was purified and reconstituted into proteoliposomes and active transport of diverse di- and tri-peptides was demonstrated, supporting the conclusions of the phenotypic screen. During thisin vitroanalysis, we also found that DtpT could transport the biologically prevalent tripeptide reduced glutathione (GSH). In the absence of the known glutathione transporter Gis,dtpTis essential for growth under conditions where GSH is supplied as the sole sulphur source, identifying DtpT as the predicted second GSH uptake system ofS. aureus. Site-directed mutagenesis of the predicted ligand binding site of DtpT suggests GSH binds the protein vertically in a manner dependent on interactions with Gln310. Finally, we demonstrate that GSH transport is required byS. aureusfor complete fitness duringin vitromacrophage infection experiments, and our data suggest that GSH uptake plays a role in modulating host-pathogen interactions in these infections. Together, these data reveal important new functions for DtpT, both in the utilisation of diverse peptides for growth but also providing the first clues toward a distinct role of DtpT during intracellular infection.