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Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

机译:细胞内弗氏志贺氏菌的蛋白质组分析揭示了对细胞内生长重要的途径

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Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.
机译:使用三种志贺志贺氏菌2457T菌株与三种不同的生长环境相关联进行了整体蛋白质组学分析:在肉汤中生长的弗氏链球菌(体外),在上皮细胞胞质内(细胞内)生长的弗氏链球菌和与之共培养的弗氏链球菌。但并未入侵Henle细胞(细胞外)。与体外和细胞外细菌相比,细胞内细菌具有增加的侵袭和细胞间扩散所需的蛋白质,包括Ipa,Mxi和Ics蛋白质。响应细胞内环境的代谢途径的变化也很明显。糖原生物合成酶增加,糖转运蛋白的表达改变,碳存储调节剂CsrA的量减少。混合酸发酵酶在细胞内高度表达,而三羧酸(TCA)循环氧化还原酶和除CydAB外的大多数电子传输链蛋白均明显减少。这表明发酵和CydAB系统主要在细胞内维持能量产生。负责NADPH再生的PntAB水平升高,表明缺少ATP合成的还原因子。这些代谢变化可能反映了可用碳源,氧气水平和铁的利用率的变化。细胞内细菌显示出铁饥饿的强烈证据。铁捕获系统(Iut,Sit,FhuA和Feo)以及铁饥饿,与应激相关的Fe-S簇组装(Suf)蛋白大量增加。突变分析证实,混合酸发酵途径是野生型弗氏链球菌细胞内生长和传播所必需的。因此,铁胁迫和碳代谢的变化可能是弗氏链球菌从细胞外环境向细胞内环境过渡的关键因素。

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