Lisher John P, Tsui Ho-Ching Tiffany, Ramos-Montañez Smirla, Hentchel Kristy L, Martin Julia E, Trinidad Jonathan C, Winkler Malcolm E, Giedroc David P
Department of Chemistry, Indiana University, Bloomington, Indiana, USA; Graduate Program in Biochemistry, Indiana University, Bloomington, Indiana, USA.
Department of Biology, Indiana University, Bloomington, Indiana, USA.
mSphere. 2017 Jan 4;2(1). doi: 10.1128/mSphere.00291-16. eCollection 2017 Jan-Feb.
The catalase-negative, facultative anaerobe D39 is naturally resistant to hydrogen peroxide (HO) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced HO. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that mutants produce significantly lower HO. In addition, both the SpxB pathway and a candidate pyruvate dehydrogenase complex (PDHC) pathway contribute to acetyl coenzyme A (acetyl-CoA) production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic versus strict anaerobic conditions shows upregulation of , a gene encoding a rhodanese-like protein (locus tag ), , , , , and an Fe-S protein biogenesis operon under HO-producing conditions. Proteome profiling of HO-induced sulfenylation reveals that sulfenylation levels correlate with cellular HO production, with endogenous sulfenylation of ≈50 proteins. Deletion increases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but targets also include pyruvate kinase, LctO, AdhE, and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated with nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated HO functions as a signaling molecule to downregulate capsule production and drive altered flux through sugar utilization pathways. Adaptation to endogenous oxidative stress is an integral aspect of colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous HO and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in .
过氧化氢酶阴性的兼性厌氧菌D39对丙酮酸氧化酶（SpxB）内源性产生的过氧化氢（H₂O₂）具有天然抗性。在此，我们研究了对内源性产生的H₂O₂的适应性反应。我们发现，将乳酸转化为丙酮酸的乳酸氧化酶对通过SpxB的丙酮酸通量有正向影响，并且突变体产生的H₂O₂显著减少。此外，SpxB途径和候选丙酮酸脱氢酶复合物（PDHC）途径在有氧生长期间都有助于乙酰辅酶A（乙酰-CoA）的产生，而丙酮酸甲酸裂解酶（PFL）途径是厌氧生长期间主要的乙酰-CoA途径。对在有氧与严格厌氧条件下培养的D39菌株进行的微阵列分析显示，在产生H₂O₂的条件下，一个编码类硫氰酸酶蛋白（基因座标签）、、、、和一个铁硫蛋白生物合成操纵子的基因上调。对H₂O₂诱导的亚磺酰化进行蛋白质组分析表明，亚磺酰化水平与细胞H₂O₂产生相关，约有50种蛋白质发生内源性亚磺酰化。缺失会使细胞亚磺酰化增加5倍，并对ATP生成有抑制作用。蛋白质亚磺酰化的两个主要靶点是甘油醛-3-磷酸脱氢酶（GapA）和SpxB本身，但靶点还包括丙酮酸激酶、LctO、AdhE和乙酸激酶（AckA）。GapA的亚磺酰化具有抑制作用，而对SpxB活性的影响可忽略不计。引人注目的是，四种荚膜多糖生物合成酶被亚磺酰化，通过5-磷酸核酮糖参与核苷酸生物合成的酶也是如此。我们提出，LctO/SpxB产生的H₂O₂作为一种信号分子来下调荚膜产生，并驱动糖利用途径中通量的改变。对内源性氧化应激的适应是肺炎链球菌定殖和毒力的一个组成部分。在这项工作中，我们确定了肺炎链球菌内源性氧化应激反应的关键转录组和蛋白质组特征。硫醇过氧化物酶TpxD在对内源性H₂O₂的适应中起关键作用，并有助于限制肺炎链球菌中糖酵解、荚膜和核苷酸生物合成酶的蛋白质亚磺酰化。
