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重症疾病。我们此前发表了一项在小鼠急性肺炎模型中开展转座子测序（transposon sequencing, Tn-seq）筛选的研究，鉴定出一个与大肠杆菌（Escherichia coli）中参与磷酸戊糖途径的内酯酶相似的假设基因（SAUSA300_1902，简称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的作用，相关研究仍较为有限。本研究证实，pgl基因的突变会显著影响细菌的三磷酸腺苷（ATP）水平与呼吸活性。对pgl突变株与亲本菌株的RNA测序（RNA-seq）分析显示，葡萄糖与葡萄糖酸相关基因的表达出现代偿性变化，同时嘧啶生物合成基因座的转录水平显著下调。通过非靶向代谢组学分析与13C同位素标记实验，也验证了上述差异：pgl基因的突变会导致嘧啶代谢通路的代谢物水平下降，具体表现为5-磷酸核糖（ribose-5P）、尿苷一磷酸（UMP）及鸟苷一磷酸（GMP）的含量显著降低。这类核苷酸含量的降低会影响生物被膜（biofilms）中的胞外DNA（extracellular DNA）含量，并削弱生物被膜的形成能力。此外，pgl基因突变还会削弱菌株抵抗过氧化氢（hydrogen peroxide）与百草枯（paraquat）诱导的氧化损伤的能力，并降低其在巨噬细胞（macrophages）内的胞内存活能力。壁磷壁酸（wall teichoic acid）的含量变化会影响菌株对过氧化氢的敏感性。我们通过三类不同的感染模型——呼吸道感染、皮肤感染与败血症（septicemia）感染——验证了上述变化对菌株毒力的影响，证实了在各类感染模型中，完整的磷酸戊糖途径功能均为必需。本研究揭示了代谢过程在金黄色葡萄球菌致病过程中的多维度调控作用。