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首页> 外文期刊>Infection and immunity >Protein Kinase C Mediates Enterohemorrhagic Escherichia coli O157:H7-Induced Attaching and Effacing Lesions
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Protein Kinase C Mediates Enterohemorrhagic Escherichia coli O157:H7-Induced Attaching and Effacing Lesions

机译：蛋白激酶C介导肠出血性大肠杆菌O157：H7诱导的附着和表面损伤

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Enterohemorrhagic Escherichia coli serotype O157:H7 causes outbreaks of diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. E. coli O157:H7 intimately attaches to epithelial cells, effaces microvilli, and recruits F-actin into pedestals to form attaching and effacing lesions. Lipid rafts serve as signal transduction platforms that mediate microbe-host interactions. The aims of this study were to determine if protein kinase C (PKC) is recruited to lipid rafts in response to E. coli O157:H7 infection and what role it plays in attaching and effacing lesion formation. HEp-2 and intestine 407 tissue culture epithelial cells were challenged with E. coli O157:H7, and cell protein extracts were then separated by buoyant density ultracentrifugation to isolate lipid rafts. Immunoblotting for PKC was performed, and localization in lipid rafts was confirmed with an anti-caveolin-1 antibody. Isoform-specific PKC small interfering RNA (siRNA) was used to determine the role of PKC in E. coli O157:H7-induced attaching and effacing lesions. In contrast to uninfected cells, PKC was recruited to lipid rafts in response to E. coli O157:H7. Metabolically active bacteria and cells with intact lipid rafts were necessary for the recruitment of PKC. PKC recruitment was independent of the intimin gene, type III secretion system, and the production of Shiga toxins. Inhibition studies, using myristoylated PKCζ pseudosubstrate, revealed that atypical PKC isoforms were activated in response to the pathogen. Pretreating cells with isoform-specific PKC siRNA showed that PKCζ plays a role in E. coli O157:H7-induced attaching and effacing lesions. We concluded that lipid rafts mediate atypical PKC signal transduction responses to E. coli O157:H7. These findings contribute further to the understanding of the complex array of microbe-eukaryotic cell interactions that occur in response to infection.
机译：肠出血性大肠杆菌O157：H7血清型引起腹泻，出血性结肠炎和溶血尿毒症候群的爆发。大肠杆菌O157：H7紧密附着于上皮细胞，抹去微绒毛，并将F-肌动蛋白募集到基座上，形成附着和脱落的病变。脂质筏是介导微生物与宿主相互作用的信号转导平台。这项研究的目的是确定是否响应大肠杆菌O157：H7感染将蛋白激酶C（PKC）募集到脂质筏中，以及蛋白激酶C在附着和消除病变形成中起什么作用。用大肠杆菌O157：H7攻击HEp-2和肠道407组织培养上皮细胞，然后通过浮力密度超速离心分离细胞蛋白提取物，以分离脂质筏。进行了PKC的免疫印迹，并使用抗caveolin-1抗体确认了脂质筏中的定位。使用同工型特异性PKC小干扰RNA（siRNA）来确定PKC在大肠杆菌O157：H7诱导的附着和脱落损伤中的作用。与未感染的细胞相反，PKC被响应于大肠杆菌O157：H7募集到脂质筏中。代谢活性细菌和脂筏完整的细胞对于募集PKC是必需的。 PKC募集独立于内膜素基因，III型分泌系统和志贺毒素的产生。使用肉豆蔻基化的PKCζ伪底物进行的抑制研究表明，非典型的PKC同工型响应病原体而被激活。用同工型特异性PKC siRNA预处理细胞表明PKCζ在大肠杆菌O157：H7诱导的附着和脱落损伤中起作用。我们得出的结论是，脂筏介导了对大肠杆菌O157：H7的非典型PKC信号转导响应。这些发现进一步有助于理解响应感染而发生的微生物-真核细胞相互作用的复杂阵列。

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