Department of Medicine, Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, Chicago, IL 60612-7323, USA.
Lab Invest. 2010 Aug;90(8):1152-68. doi: 10.1038/labinvest.2010.91. Epub 2010 May 17.
Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as nonbloody diarrhea may be Stx independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria with the cecum and colon. Greater numbers of EHEC were also cultured from stool samples obtained from streptomycin-pretreated mice, as compared with those that received no antibiotics. Transmission electron microscopy shows that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of the colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in the colonic tissues of infected animals. Increased mucosal permeability to 4- kDa FITC-dextran was also observed in the colonic tissues of infected mice. Immunofluorescence microscopy showed that EHEC infection resulted in redistribution of the tight junction (TJ) proteins occludin and claudin-3 and increased the expression of claudin-2, whereas ZO-1 localization remained unaltered. Quantitative real-time PCR showed that EHEC altered mRNA transcription of OCLN, CLDN2, and CLDN3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of TJ proteins has a role in the increase in intestinal permeability.
志贺毒素(Stx)与出血性结肠炎和溶血尿毒综合征的发展有关,但肠出血性大肠杆菌(EHEC)感染的早期症状,如非血性腹泻,可能与 Stx 无关。在这项研究中,我们使用一种小鼠模型定义了在没有 Stx 的情况下 EHEC 对肠道上皮的影响。对两组未用抗生素和链霉素处理的 C57Bl/6J 小鼠的肠道定植进行了特征描述和比较。EHEC 在两组的盲肠和结肠的定植效率均高于回肠;然而,在链霉素预处理的小鼠中检测到更多的组织相关 EHEC。对感染生物发光 EHEC 的小鼠肠道组织的成像进一步证实了细菌与盲肠和结肠的紧密联系。与未接受抗生素的小鼠相比,从链霉素预处理的小鼠粪便样本中培养出的 EHEC 数量也更多。透射电子显微镜显示,EHEC 感染导致小鼠结肠上皮细胞微绒毛消失。感染小鼠结肠组织的苏木精和伊红染色显示固有层多形核白细胞数量略有增加。感染动物的结肠组织跨粘膜电阻,即上皮屏障功能的测量值降低。也观察到感染小鼠结肠组织中 4- kDa FITC-葡聚糖的粘膜通透性增加。免疫荧光显微镜显示,EHEC 感染导致紧密连接(TJ)蛋白闭合蛋白和 Claudin-3 的重新分布,并增加 Claudin-2 的表达,而 ZO-1 的定位保持不变。定量实时 PCR 显示 EHEC 改变了 OCLN、CLDN2 和 CLDN3 的 mRNA 转录。值得注意的是,Claudin-2 的表达显著增加,并与肠道通透性增加相关。我们的数据表明,C57Bl/6J 小鼠可作为研究 EHEC 感染对肠道上皮生理影响的体内模型,并表明 TJ 蛋白转录的改变在肠道通透性增加中起作用。