The Staphylococcus aureus α-Acetolactate Synthase ALS Confers Resistance to Nitrosative Stress. Staphylococcus aureus subsp. aureus USA300_FPR3757

Staphylococcus aureus is a worldwide pathogen that colonizes the human nasal cavity and is a major cause of respiratory and cutaneous infections. In the nasal cavity, S. aureus thrives with high concentrations of nitric oxide (NO) produced by the innate immune effectors and has available for growth slow metabolizing free hexoses, such as galactose. Here, we have used deep sequencing transcriptomic analysis (RNA-Seq) and 1H-NMR to uncover how S. aureus grown on galactose, a major carbon source present in the nasopharynx, survives the deleterious action of nitric oxide. We observed that, like on glucose, S. aureus withstands high concentrations of NO when using galactose. However, most likely this is achieved through a distinct metabolism that relies on the increased production of amino acids, such as glutamate, threonine and branched-chain amino acids. Moreover, we found that under these conditions the α-acetolactate synthase (ALS) enzyme, which converts pyruvate into α-acetolactate, plays a role in the resistance of S. aureus to NO. However, the role of ALS is not restricted to galactose but also extends to cells growing on glucose. The results suggest that ALS prevents intracellular acidification, promoting the production of branched-chain amino acids and activation of the TCA cycle. We show that ALS contributes to the successful infection of murine macrophages. Furthermore, ALS is also shown to contribute to the resistance of S. aureus to beta-lactam antibiotics such as methicillin and oxacillin. Overall design: mRNA profiles of S. aureus galactose-grown cells unexposed and exposed for 3h with NO were generated by deep sequencing, in duplicate, using an Ion Proton™ Sequencer.

金黄色葡萄球菌（Staphylococcus aureus）是一种全球性病原菌，可定殖于人类鼻腔，是引发呼吸道与皮肤感染的主要致病菌。在鼻腔微环境中，金黄色葡萄球菌能够在先天免疫效应分子产生的高浓度一氧化氮（nitric oxide, NO）胁迫下存活，并可利用代谢速率较慢的游离己糖（如半乳糖（galactose））作为生长碳源。本研究采用转录组深度测序（RNA-Seq）与氢核磁共振（1H-NMR）技术，解析了以鼻咽部主要碳源半乳糖为培养基生长的金黄色葡萄球菌抵御一氧化氮毒害的分子机制。研究观察到，与葡萄糖培养基培养的情况类似，金黄色葡萄球菌在利用半乳糖时同样能够耐受高浓度一氧化氮。然而，该耐受机制极有可能依托一套独特的代谢途径：通过提升谷氨酸、苏氨酸与支链氨基酸等氨基酸的合成量来实现。此外，本研究发现，在此培养条件下，可将丙酮酸转化为α-乙酰乳酸的α-乙酰乳酸合酶（α-acetolactate synthase, ALS）参与了金黄色葡萄球菌的一氧化氮耐受过程。但ALS的这一功能并非仅局限于半乳糖培养基，同样适用于葡萄糖培养的菌体。研究结果表明，ALS可通过抑制细胞内酸化、促进支链氨基酸合成以及激活三羧酸（TCA）循环来发挥作用。本研究证实，ALS可促进金黄色葡萄球菌对小鼠巨噬细胞的感染过程。此外，ALS还可提升金黄色葡萄球菌对β-内酰胺类抗生素（如甲氧西林（methicillin）与苯唑西林（oxacillin））的耐药性。实验整体设计：采用Ion Proton™测序平台，对未暴露于一氧化氮以及暴露于一氧化氮3小时的半乳糖培养金黄色葡萄球菌进行双重复转录组测序，获取其mRNA表达谱。