Human α-defensins inhibit Clostridium difficile toxin B
Human α-defensins inhibit Clostridium difficile toxin B
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DOI：
10.1053/j.gastro.2008.03.008
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发表时间：
2008-06-01
期刊：
影响因子：
29.4
通讯作者：
Aktories, Klaus
中科院分区：
文献类型：
--
作者：
Giesemann, Torsten;Guttenberg, Gregor;Aktories, Klaus
背景与目的：艰难梭菌毒素A和B是假膜性结肠炎和抗生素相关性腹泻的主要毒力因子。毒素是葡萄糖基转移酶，它使参与细胞信号传导的Rho蛋白失活。人类α -防御素作为先天免疫系统的一部分灭活各种微生物病原体以及特定的细菌外毒素。本研究研究了α -防御素人中性粒细胞蛋白(HNP)-1、HNP-3和肠人防御素(HD)-5对艰难梭菌毒素A和b活性的影响。方法：显微镜下观察α -防御素在体内对艰难梭菌毒素的失活，测定CaCo-2细胞单层的经上皮耐药性，分析毒素处理细胞中Rac1的糖基化。体外糖基化法测定K-m和中位抑制浓度（IC50）值。通过沉淀和浊度研究分析了防御素-毒素复合物的形成。结果：用人α -防御素处理细胞导致毒素B的细胞毒性丧失，而毒素a则没有。只有α -防御素，而不是β -防御素-1或cathelicidin LL-37，以竞争方式抑制毒素B催化的Rho鸟苷三磷酸酶的体外糖基化，将尿苷5'-二磷酸-葡萄糖的K-m值提高了10倍。α -防御素抑制毒素b催化糖基化的IC50值为0.6 ~ 1.5 μ mol/L。在高浓度下，防御素（HNP-1 >= 2 mu mol/L）引起高分子质量聚集，相当于炭疽芽孢杆菌的保护抗原和致死因子。结论：我们的数据表明，毒素B与α -防御素具有高亲和力的相互作用，表明防御素可能为某些类型的梭菌糖基化细胞毒素提供了防御机制。
Background & Aims: Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human alpha-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of alpha-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. Methods: Inactivation of C difficile toxins by alpha-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine K-m and median inhibitory concentration (IC50) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. Results: Treatment of cells with human alpha-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only alpha-defensins, but not beta-defensin-1 or cathelicidin LL-37, inhibited toxin B-catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing K-m values for uridine 5'-diphosphate-glucose up to 10-fold. The IC50 values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 mu mol/L. At high concentrations, defensins (HNP-1 >= 2 mu mol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. Conclusions: Our data indicate that toxin B interacts with high affinity with alpha-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.