Inactivation of branched-chain amino acid uptake halts Staphylococcus aureus growth and induces bacterial quiescence within macrophages

Staphylococcus aureus is a notorious human pathogen that thrives in macrophages. It resides in mature phagolysosomes, where a subset of the bacteria eventually begin to proliferate. How S. aureus acquires essential nutrients, such as amino acids, for growth in this niche is poorly understood. Using a long-term primary human macrophage infection model, we show that branched-chain amino acid (BCAA) uptake mediated by the major transporter BrnQ1 is required by S. aureus for intracellular replication in macrophages and we provide mechanistic insight into the role of BCAAs in the success of intracellular S. aureus. Loss of BrnQ1 function renders intracellular S. aureus non-replicative and non-cytotoxic. The defective intracellular growth of S. aureus brnQ1 mutants can be rescued by supplementation with BCAAs or by overexpression of the BCAA transporters BrnQ1 or BcaP. Inactivation of the CodY repressor rescues the ability of S. aureus brnQ1 mutants to proliferate intracellularly independent of endogenous BCAA synthesis but dependent on BcaP expression. Non-replicating brnQ1 mutants in primary human macrophages become metabolically quiescent and display aberrant gene expression marked by failure to respond to intraphagosomal iron starvation. The bacteria remain, however, viable for an inordinate length of time. This dormant, yet viable bacterial state is distinct from classical persisters and small colony variants. S. aureus JE2 wild-type or its isogenic brnQ1 mutant were either used to infect human macrophages or were grown in the infection medium (RPMI1640+10% [v/v] fetal bovine serum [heat-inactivated at 56 degrees C for 30 minutes]). RNA of both, pathogen and host cells were isolated together (dual RNA-Seq of infected macrophages) or was isolated from planktonic bacteria (grown in infection medium and were labelled "RPMI").