Defining the routes of di/tri-peptide uptake in Staphylococcus aureus

Peptides available in biological niches inhabited by the human pathogen Staphylococcus aureus serve as a rich source of amino acids required for growth and successful host colonisation. Uptake of peptides by S. aureus involves at least two transport systems: the POT family di/tripeptide permease DtpT and the oligopeptide ABC transporter Opp3. Previously, genetic screening approaches revealed that dtpT contributes toward bacterial fitness in vivo, but further functional characterisation of the DtpT transporter protein (i.e. in regard to substrate range and physiological roles) remains limited.

In this thesis, the individual and combined functions of DtpT and Opp3 in enabling utilisation of 282 di/tripeptides are studied using a high-throughput phenotypic screen. This approach reveals that many peptides can be utilised via either transporter, though DtpT appears to be the primary route of uptake for dipeptides. Intriguingly, a clear preference is observed for Asp/Glu-containing peptides among putative DtpT substrates. To measure DtpT-mediated peptide transport directly, the protein was purified and reconstituted into proteoliposomes. Active transport of diverse diand tripeptides was demonstrated using this approach, supporting the conclusions of the phenotypic screen. During this in vitro analysis, DtpT was also found to transport the biologically prevalent tripeptide metabolite reduced glutathione (GSH). In the absence of the known glutathione transporter – Gis – the dtpT gene is essential for growth when GSH is supplied as the sole sulphur source, identifying DtpT as the predicted second GSH uptake system of S. aureus. Site-directed mutagenesis of the predicted ligand binding site of DtpT suggests GSH binds the protein vertically, in a similar conformation to that observed for S-Cys-Gly-3M3SH binding in the homologous POT protein PepTSh. Finally, GSH transport is revealed to be required by S. aureus for complete fitness during in vitro macrophage infection experiments, revealing a potential new role for GSH uptake in orchestrating 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 glutathione transporters during intracellular infection.