ISOLATION OF TOXINS FROM C. DIFFICILE & C. SORDELLII

从艰难梭菌中分离毒素

• 批准号: 3126416
• 负责人: Tracy D. Wilkins
• 金额: $ 24.64万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3126416
• 关键词: Clostridium; antibiotics; antireceptor antibody; antitoxins; bacterial cytopathogenic effect; bacterial genetics; bacterial toxins; carbohydrate sequence; chemical binding; colitis; diarrhea; enterotoxins; gastrointestinal disorder chemotherapy; gastrointestinal infection; gene expression; goats; hamsters; host organism interaction; human tissue; infant human (0-1 year); intestinal mucosa; laboratory mouse; laboratory rabbit; molecular cloning; monoclonal antibody; nosocomial infections; protein engineering; receptor; synthetic peptide; virulence

Clostridium difficile is a relatively "new" pathogen that causes colitis in some patients receiving antibiotic therapy. The organism and its toxins occur in about 15% of patients with diarrhea in hospitals and that is a lot of patients. If untreated some of these patients would progress to Pseudomembranous Colitis, which has a high mortality rate. The disease is commonly acquired in hospitals because the organism can only grow in the colon when the normal flora has been essentially eliminated by large doses of antibiotics. Hospitals provide an ideal environment for the spread of a spore-forming organism that causes diarrhea. The organism produces two potent toxins. Toxin A is an enterotoxin that may be responsible for the major symptoms of the disease. Toxin B is a very potent cytotoxin. We have shown that toxin A binds to a Galalpha1-3Galbeta1-4G1cNAc trisaccharide via multiple binding sites that occur on this huge toxin. The repeated binding structures will be analyzed in detail by the construction of synthetic peptides and by cloning the repeats into both E. coli and C. perfringens. We will determine whether this trisaccharide is the receptor that allows toxin A to initiate the enterotoxicity and whether this is the receptor to which the toxin binds in adult human colonic tissue. Adults may vary in the occurrence of this trisaccharide in their colons and this could account for differences in susceptibility to the toxins. We also will determine whether babies have this receptor; about half of the babies born in hospitals get colonized with this organism as "normal flora" without apparent ill effects and we want to know why the toxins do not kill them. The trisaccharide is not degraded by human intestinal enzymes so we may be able to treat the disease by giving large amounts of the receptor attached to latex beads. We have found that a lectin that binds to this trisaccharide prevents the action of toxin A so this may be useful in therapy. A monoclonal antibody specific for this carbohydrate receptor also is available and will be tested. As the active site of the toxin is sequenced we will attempt to determine the mechanism of action by comparison to sequences of other toxins and enzymes. Such hints as to the mechanisms will be confirmed by collaborations with research groups working in the respective areas. As the sequence of toxin B becomes available we will analyze it in the same manner. We will continue our collaborative efforts on the mechanism of action of the toxins and continue to be a source of pure toxins and specific antibodies.

艰难梭菌是一种相对“新的”病原体，导致 一些接受抗生素疗法的患者的结肠炎。 这 有机体及其毒素发生在约15％的患者中 医院的腹泻，这是很多患者。 如果未治疗 这些患者中有一些会发展为假膜结肠炎， 死亡率很高。 该疾病通常被接受 在医院，因为有机体只能在结肠中生长 正常的菌群已经被大剂量消除了 抗生素。 医院为传播提供了理想的环境 形成孢子的生物，导致腹泻。 有机体 产生两个有效的毒素。 毒素A是一种可能是肠毒素 负责该疾病的主要症状。 毒素B是 非常有效的细胞毒素。 我们已经表明毒素A与A结合 galalpha1-3galbeta1-4g1cnac trisaccharide通过多种结合 在这种巨大的毒素上发生的网站。 重复结合 结构将通过构建详细分析 合成肽并通过将重复序列克隆到大肠杆菌和 C.刺激。 我们将确定这种三糖是否是 允许毒素A启动肠毒性的受体和 这是否是毒素在成人中结合的受体 人类结肠组织。 成年人可能会有所不同 结肠中的三糖，这可以解释 毒素易感性的差异。 我们也会 确定婴儿是否患有这种受体；约一半 在医院出生的婴儿被这种生物体殖民 “正常植物”没有明显的效果，我们想知道为什么 毒素不会杀死它们。 三糖不会被降解 人肠酶，因此我们可以通过 给出大量附着在乳胶珠上的受体。 我们 已经发现与该三糖结合的凝集素可防止 毒素A的作用因此在治疗中可能很有用。 一个 对此碳水化合物受体的单克隆抗体也 可用并将进行测试。 作为毒素的活跃部位 测序我们将尝试确定作用机理 与其他毒素和酶的序列相比。 这样的提示 至于机制将通过与 在各个领域工作的研究小组。 作为序列 毒素B可用，我们将以相同的方式分析它。 我们将继续我们的协作努力 毒素的作用，并继续成为纯毒素的来源 特定抗体。