PURIFICATION AND MECHANISM OF C. DIFFICILE TOXIN

抗生素相关性肺炎的病原菌艰难梭菌 腹泻和结肠炎，现在被公认为主要的肠道疾病之一 病原体。艰难梭菌是一种厌氧杆菌，它能释放几种 介导受感染动物结肠组织损伤的蛋白质毒素 或者是人类。主要的毒素是毒素B，是一种不耐热的蛋白质，它能引起 在单层培养中生长的细胞呈圆形。这种毒素也是 在抗生素相关性腹泻患者的粪便中可见 和结肠炎，并可能介导组织损伤(结肠炎)， 伴随着这种病原体的感染。 这项建议的总体目标是提纯艰难梭菌毒素B和 确定其体内外作用机制。假设是为了 在这个项目中被测试的是毒素B干扰结构和 细胞骨架的功能，导致细胞变圆，而这反过来 导致大分子合成受阻，并最终导致 手机。纯化的毒素将根据组成进行表征， 分子量、亚基结构和构效关系。 纯化的毒素将被用来确定细胞的机制 舍入，更具体地说，它对细胞骨架和基质的影响 蛋白质。肌动蛋白、纤维连接蛋白、肌球蛋白等细丝的合成 蛋白质将在纤维母细胞和平滑肌细胞中进行研究 纯B毒素分级剂量对大分子物质的影响 合成将用~3H-亮氨酸和~3H-胸腺嘧啶核苷作为 蛋白质和DNA合成的前体。 这些研究的意义与艰难梭菌的重要性有关。 作为人类病原体。通过更好地了解B毒素如何 工作应该可以定义其中一个的病理生理学 人类最常见和最重要的肠道感染。从基本的 从科学的角度来看，这些研究应该为 细胞骨架和基质蛋白与细胞形态的关系 功能和增殖。

Clostridium difficile, the causative agent of antibiotic-associated diarrhea and colitis, is now recognized as one of the major enteric pathogens. C. difficile is an anaerobic bacillus which releases several protein toxins that mediate tissue damage in the colon of infected animals or humans. The major toxin, toxin B, is a heat labile protein which causes rounding of cells growing in monolayer culture. This toxin is also demonstrable in the stools of patients with antibiotic-associated diarrhea and colitis, and presumably mediates the tissue injury (colitis) which accompanies infection with this pathogen. The overall goals of this proposal are to purify C. difficile toxin B and determine its mechanism of action in vitro and in vivo. The hypothesis to be tested in this project is that toxin B interferes with the structure and function of the cytoskeleton, causing cell rounding, and that this in turn causes inhibition of macromolecular synthesis and eventually death of the cell. The purified toxin will be characterized as regards composition, molecular weight, sub-unit structure and structure-activity relationships. The purified toxin will then be used to determine the mechanism of cell rounding, more specificially its effects on cytoskeletal and matrix proteins. The syntehsis of actin, fibronectin, myosin and other filament proteins will be studied in fibroblast and smooth muscle cells exposed to graded doses of pure toxin B. The effect of toxin B on macromolecular synthesis will be quantitated using 3H-leucine and 3H-thymidine as precursors for protein and DNA syntehsis. The significance of these studies relates to the importance of C. difficile as a human pathogen. By providing a better understanding of how toxin B works it should be possible to define the pathophysiology of one of the commonest and most important enteric infections in man. From a basic science viewpoint, these studies should provide insights into the relationship of cytoskeleton and matrix proteins to cell shape, synthetic function and proliferation.