Metabolomics normalized ion counts in M9.

Staphylococcus aureus is an important pathogen that leads to significant disease through multiple routes of infection. We recently published a transposon sequencing (Tn-seq) screen in a mouse acute pneumonia model and identified a hypothetical gene (SAUSA300_1902, pgl) with similarity to a lactonase of Escherichia coli involved in the pentose phosphate pathway (PPP) that was conditionally essential. Limited studies have investigated the role of the PPP in physiology and pathogenesis of S. aureus. We show here that mutation of pgl significantly impacts ATP levels and respiration. RNA-seq analysis of the pgl mutant and parent strains identified compensatory changes in gene expression for glucose and gluconate as well as reductions in the pyrimidine biosynthesis locus. These differences were also evident through unbiased metabolomics studies and 13C labeling experiments that showed mutation of pgl led to reductions in pyrimidine metabolism including decreases in ribose-5P, UMP and GMP. These nucleotide reductions impacted the amount of extracellular DNA in biofilms and reduced biofilm formation. Mutation also limited the capacity of the strain to resist oxidant damage induced by hydrogen peroxide and paraquat and subsequent intracellular survival inside macrophages. Changes in wall teichoic acid impacted susceptibility to hydrogen peroxide. We demonstrated the importance of these changes on virulence in three different models of infection, covering respiratory, skin and septicemia, demonstrating the need for proper PPP function in all models. This work demonstrates the multifaceted role metabolism can play in multiple aspects of S. aureus pathogenesis.金黄色葡萄球菌（Staphylococcus aureus）是一类重要的病原菌，可通过多种感染途径引发严重疾病。本课题组近期在小鼠急性肺炎模型中开展了转座子测序（transposon sequencing, Tn-seq）筛选，并鉴定出一个与大肠杆菌（Escherichia coli）参与磷酸戊糖途径（pentose phosphate pathway, PPP）的内酯酶同源的假设基因SAUSA300_1902（pgl），该基因为条件必需基因。目前针对磷酸戊糖途径在金黄色葡萄球菌生理与致病过程中作用的研究较为有限。 本研究证实，pgl基因的突变会显著影响菌株的ATP水平与呼吸代谢功能。对pgl突变株与亲本菌株的RNA测序（RNA-seq）分析显示，菌株在葡萄糖与葡萄糖酸代谢相关基因的表达上出现了代偿性变化，同时嘧啶生物合成基因座的表达水平显著下调。通过非靶向代谢组学研究与13C标记实验，也验证了上述差异：pgl突变会导致嘧啶代谢通路受阻，具体表现为5-磷酸核糖（ribose-5P）、尿苷单磷酸（UMP）与鸟苷单磷酸（GMP）的含量降低。这类核苷酸代谢异常会影响生物被膜中的细胞外DNA含量，进而削弱生物被膜的形成能力。此外，pgl突变还会降低菌株抵抗过氧化氢与百草枯诱导的氧化损伤的能力，并削弱其在巨噬细胞内的胞内存活能力。细胞壁磷壁酸的变化会影响菌株对过氧化氢的敏感性。 本研究通过呼吸道、皮肤与败血症三种不同的感染模型，验证了上述代谢变化对金黄色葡萄球菌毒力的影响，结果显示磷酸戊糖途径的正常功能在所有感染模型中均不可或缺。本研究揭示了代谢过程在金黄色葡萄球菌致病过程的多个方面所发挥的多维度作用。