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. Overall design: 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").

金黄色葡萄球菌（Staphylococcus aureus）是一种臭名昭著的人类致病菌，可在巨噬细胞内定植。该菌寄生于成熟吞噬溶酶体中，其中一部分细菌最终会开始增殖。目前人们对金黄色葡萄球菌在该微环境中获取氨基酸等必需营养物质以支持生长的机制仍知之甚少。
本研究采用长期原代人巨噬细胞感染模型，证实由主要转运蛋白BrnQ1介导的支链氨基酸（branched-chain amino acid, BCAA）摄取，是金黄色葡萄球菌在巨噬细胞内完成胞内复制的必要条件，并阐明了支链氨基酸在胞内金黄色葡萄球菌存活过程中的作用机制。
BrnQ1功能缺失会使胞内金黄色葡萄球菌丧失复制能力与细胞毒性。brnQ1突变株的胞内生长缺陷，可通过添加支链氨基酸，或过表达支链氨基酸转运蛋白BrnQ1与BcaP得以挽救。
CodY阻遏蛋白（CodY repressor）的失活可挽救brnQ1突变株的胞内增殖能力，该挽救过程不依赖于内源性支链氨基酸合成，但依赖于BcaP的表达。
原代人巨噬细胞内的非复制型brnQ1突变株会进入代谢静息状态，并表现出异常的基因表达特征：无法响应吞噬溶酶体内的铁饥饿胁迫。尽管如此，该细菌仍可在极长的时间内保持存活。这种处于休眠状态但仍具活性的细菌状态，有别于经典持留菌（persisters）与小菌落变异株（small colony variants）。
总体实验设计：本研究使用金黄色葡萄球菌JE2野生型菌株及其同基因brnQ1突变株，分别感染人巨噬细胞，或在感染培养基（RPMI1640+10%[体积分数]胎牛血清，经56℃热灭活30分钟）中进行浮游培养。分别收集两类样本的总RNA：一类为感染巨噬细胞的病原体与宿主细胞总RNA（感染巨噬细胞双转录组测序，dual RNA-Seq），另一类为浮游培养细菌的RNA（培养于上述感染培养基中，标注为"RPMI"组）。