Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA
Infect Immun. 2014 Jul;82(7):2746-55. doi: 10.1128/IAI.01575-13. Epub 2014 Apr 14.
Shigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellular S. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkAB and pykAF mutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway gene eda resulted in small plaques, but the double eda edd mutant formed normal-size plaques. This suggested that the plaque defect of the eda mutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellular S. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-type S. flexneri also formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate of S. flexneri in vitro, suggesting that it may be a preferred carbon source inside host cells.
福氏志贺菌在肠上皮细胞的细胞质中复制，可以使用 EMP、ED 或磷酸戊糖途径进行糖酵解碳代谢。为了确定细胞内福氏志贺菌使用哪种途径，构建了突变体，并在噬菌斑测定中测试了它们侵入、在细胞内复制和扩散到相邻上皮细胞的能力。EMP 途径受阻的突变体（pfkAB 和 pykAF 突变体）能够侵入细胞，但形成的噬菌斑非常小。Entner-Doudoroff 途径基因 eda 的缺失导致噬菌斑变小，但双 eda edd 突变体形成正常大小的噬菌斑。这表明 eda 突变体的噬菌斑缺陷是由于有毒中间产物 2-酮-3-脱氧-6-磷酸葡萄糖酸的积累，而不是对该途径的特定要求。磷酸戊糖途径的缺失对噬菌斑形成没有影响，表明它对细胞内福氏志贺菌不是关键的。在上皮细胞培养基中补充丙酮酸可以使糖酵解突变体形成比未补充培养基时更大的噬菌斑，这与表型微阵列（Biolog）的数据一致，表明这些突变体中丙酮酸代谢没有受到破坏。有趣的是，野生型福氏志贺菌在添加丙酮酸或葡萄糖时也形成更大的噬菌斑，其中丙酮酸产生的噬菌斑最大。对培养细胞中代谢物的分析表明，添加的化合物的细胞内水平增加。丙酮酸增加了福氏志贺菌在体外的生长速度，表明它可能是宿主细胞内的首选碳源。
