Giesemann Torsten, Guttenberg Gregor, Aktories Klaus
Institut für Experimentelle and Klinische Pharmakologie und Toxikologie, Universität Freiburg, Freiburg, Germany.
Gastroenterology. 2008 Jun;134(7):2049-58. doi: 10.1053/j.gastro.2008.03.008. Epub 2008 Mar 10.
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.
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 (IC(50)) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies.
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 IC(50) values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 micromol/L. At high concentrations, defensins (HNP-1 > or = 2 micromol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor.
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.
艰难梭菌毒素A和毒素B是与假膜性结肠炎及抗生素相关性腹泻相关的主要毒力因子。这些毒素是葡糖基转移酶，可使参与细胞信号传导的Rho蛋白失活。人α-防御素作为固有免疫系统的一部分，可使多种微生物病原体以及特定细菌外毒素失活。在此，我们研究了α-防御素人中性粒细胞蛋白（HNP）-1、HNP-3和肠道人防御素（HD）-5对艰难梭菌毒素A和毒素B活性的影响。
通过显微镜观察、测定CaCo-2细胞单层的跨上皮电阻以及分析毒素处理细胞中Rac1的糖基化，监测体内α-防御素对艰难梭菌毒素的失活作用。体外糖基化用于确定米氏常数（K(m)）和半数抑制浓度（IC(50)）值。通过沉淀和浊度研究分析防御素-毒素复合物的形成。
用人α-防御素处理细胞导致毒素B的细胞毒性丧失，但毒素A未丧失。只有α-防御素，而不是β-防御素-1或cathelicidin LL-37，以竞争性方式抑制毒素B催化的Rho鸟苷三磷酸酶的体外糖基化，使尿苷5'-二磷酸葡萄糖的K(m)值增加高达10倍。α-防御素抑制毒素B催化糖基化的IC(50)值为0.6 - 1.5微摩尔/升。在高浓度下，防御素（HNP-1≥2微摩尔/升）会导致高分子量聚集体形成，类似于炭疽芽孢杆菌保护性抗原和致死因子。
我们的数据表明毒素B与α-防御素具有高亲和力相互作用，并提示防御素可能提供针对某些类型梭菌糖基化细胞毒素的防御机制。
