Clostridium difficile flagellin stimulates toll-like receptor 5, and Toxin B promotes flagellin-induced chemokine production via TLR5
Clostridium difficile flagellin stimulates toll-like receptor 5, and Toxin B promotes flagellin-induced chemokine production via TLR5
复制标题
艰难梭菌鞭毛蛋白刺激 Toll 样受体 5，毒素 B 通过 TLR5 促进鞭毛蛋白诱导的趋化因子产生
DOI：
10.1016/j.lfs.2012.11.017
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发表时间：
2013
期刊：
影响因子：
6.1
通讯作者：
Yoshino Y
中科院分区：
文献类型：
--
作者：
Wada H;Hiraoka S;Morita K;Koyanagi M;Yokoyama K;Fukuchi Y;Nitatori N;Goto H.;中村 敦;Yoshino Y
目的艰难梭菌是医院感染的重要病原体。尽管艰难梭菌毒素被认为是主要毒力因子，但艰难梭菌相关疾病的发病机制仍有待确定。在本研究中，我们研究了艰难梭菌鞭毛蛋白是否参与艰难梭菌相关疾病的发病机制。主要方法。采用超速离心和低速离心相结合的方法从细菌体中提取艰难梭菌鞭毛蛋白。将提取的艰难梭菌鞭毛蛋白添加到瞬时转染 pUNO-mcs（空载体）或 pUNO-hTLR5 的 HEK293T 细胞中，并通过双荧光素酶测定比较 NF-kappaB 激活。通过ELISA测定法测量肠上皮细胞系、HT29细胞和Caco-2细胞的培养基中艰难梭菌鞭毛蛋白诱导的炎症介质（例如白细胞介素8和CCL20）的量。通过 Caco-2 细胞中的蛋白质印迹分析检查鞭毛蛋白诱导的 p38 丝裂原激活蛋白激酶的磷酸化。还通过 ELISA 测定法测量了存在或不存在艰难梭菌毒素 B 时艰难梭菌鞭毛蛋白诱导的炎症介质的量。关键发现。艰难梭菌鞭毛蛋白通过 Toll 样受体 5 诱导 HEK293T 细胞中 NF-κB 的激活。艰难梭菌鞭毛蛋白还诱导 p38 丝裂原激活蛋白激酶的激活，并通过 Toll 样受体 5 促进肠上皮细胞中白细胞介素 8 和 CCL20 的产生。用毒素 B 预处理可增强鞭毛蛋白诱导的细胞因子产生。意义我们的结果表明，毒素 B 促进鞭毛蛋白诱导肠上皮细胞的激活，并且艰难梭菌鞭毛蛋白可能在艰难梭菌相关疾病的发生中发挥作用。
AIMSClostridium difficile is an important pathogen in nosocomial infections. Although C. difficile toxins are considered to be major virulence factors, pathogenesis of C. difficile associated diseases remains to be determined. In this study, we investigated whether C. difficile flagellin is involved in the pathogenesis of C. difficile-associated diseases.MAIN METHODSC. difficile flagellin was extracted from bacterial body by using a combination of ultracentrifugation and low speed centrifugation. Extracted C. difficile flagellin was added to HEK293T cells transiently transfected with pUNO-mcs (empty vector) or pUNO-hTLR5, and NF-kappaB activation was compared by a dual-luciferase assay. The amount of C. difficile flagellin-induced inflammatory mediators such as interleukin-8 and CCL20 was measured by ELISA assay in the culture media of intestinal epithelial cell lines, HT29 cells and Caco-2 cells. Flagellin induced phosphorylation of p38 mitogen-activated protein kinase was examined by Western blotting analysis in Caco-2 cells. The amount of C. difficile flagellin-induced inflammatory mediators in the presence, or absence of C. difficile toxin B was also measured by ELISA assay.KEY FINDINGSC. difficile flagellin induced activation of NF-kappaB in HEK293T cells via toll-like receptor 5. C. difficile flagellin also induced activation of p38 mitogen-activated protein kinase, and promoted the production of interleukin-8 and CCL20 in intestinal epithelial cells via toll-like receptor 5. Pretreatment with toxin B enhanced flagellin-induced cytokine productions.SIGNIFICANCEOur results indicate that toxin B promotes flagellin-induced activation of intestinal epithelial cells, and that C. difficile flagellin may play a role in the occurrence of C. difficile-associated diseases.