Department of Infectious Diseases, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark.
PLoS One. 2011;6(8):e23858. doi: 10.1371/journal.pone.0023858. Epub 2011 Aug 24.
Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t)) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t) either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6) cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and fecal C. concisus strains could mainly be assigned to apoptotic leaks together with moderate TJ changes, demonstrating a leak-flux mechanism that parallels the clinical manifestation of diarrhea.
空肠弯曲菌胃肠道感染可伴有腹泻和炎症，而人类口腔定植可能具有共生性质。我们专注于空肠弯曲菌的病理生理学，以及不同临床口腔和粪便空肠弯曲菌菌株对人 HT-29/B6 结肠细胞的影响。分离了 6 株口腔和 8 株粪便空肠弯曲菌。用空肠弯曲菌菌株感染产生粘液的 HT-29/B6 上皮单层细胞。在 Ussing 室中测量不同分子大小的跨上皮电阻（R(t)）和示踪剂通量。通过 Western blot 测定紧密连接（TJ）蛋白表达，并通过共聚焦激光扫描显微镜分析 TJ 的亚细胞分布。通过 TUNEL 染色和 caspase-3 激活的 Western blot 检测细胞凋亡诱导。所有菌株均侵袭融合的 HT-29/B6 细胞并损害上皮屏障功能，特征为 R(t) 随时间和剂量依赖性降低，无论是从顶端侧还是更从基底外侧腔感染。TJ 蛋白表达变化稀疏，仅在感染单层的凋亡区域 TJ 蛋白重新分布。仅屏障形成 TJ 蛋白 Claudin-5 的表达水平降低至 66±8%（P<0.05），通过基因表达调控。同时，乳酸脱氢酶释放升高至 3.1±0.3%，而对照为 0.7±0.1%（P<0.001），提示细胞毒性作用。此外，口腔和粪便空肠弯曲菌菌株将凋亡事件增加到 5 倍。空肠弯曲菌感染的单层细胞对 332 Da 荧光素（1.74±0.13 比对照中的 0.56±0.17 10(-6) cm/s，P<0.05）的通透性增加，但对 4 kDa FITC-右旋糖酐（FD-4）的通透性没有差异。在喜树碱暴露的单层细胞中也是如此，喜树碱用于诱导细胞凋亡。总之，口腔和粪便空肠弯曲菌菌株引起的上皮屏障功能障碍主要归因于凋亡渗漏和适度的 TJ 变化，表明渗漏通量机制与腹泻的临床表现相似。
