Demarest Stephen J, Salbato Jared, Elia Marikka, Zhong Jingping, Morrow Theresa, Holland Trevin, Kline Katie, Woodnutt Gary, Kimmel Bruce E, Hansen Geneviève
Department of Protein Therapeutics, Diversa Corp., 4955 Directors Place, San Diego, CA 92121, USA.
J Mol Biol. 2005 Mar 11;346(5):1197-206. doi: 10.1016/j.jmb.2004.12.059.
Clostridium difficile (C.difficile) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca2+-dependent fashion. Millimolar Ca2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca2+ in the sensitization of toxin A/cell-surface interactions.
艰难梭菌是一种医院获得性肠道杆菌，可引起慢性腹泻和危及生命的结肠炎。该杆菌的致病作用由两种毒素A和B的释放介导。两种毒素的C末端部分由20和30个残基重复序列组成，称为细胞壁结合（CWB）结构域。我们克隆并表达了毒素A和B的CWB结构域以及几种截短的CWB结构域构建体，以研究它们的结构和功能。以协同方式折叠的最小CWB结构域是毒素A的11重复构建体。这使毒素A和B的C末端结构域与肺炎链球菌LytA的CWB结构域有所不同，后者仅需六个重复序列即可折叠。11重复毒素A构建体以毫摩尔亲和力直接结合Ca2+，并以浓度和Ca2+依赖的方式与哺乳动物细胞表面相互作用。毫摩尔水平的Ca2+在体外细胞试验中也加速了毒素介导的CHO细胞杀伤。总之，这些数据表明细胞外Ca2+在毒素A/细胞表面相互作用的敏化中起作用。
