ISOLATION OF TOXINS FROM C. DIFFICILE & C. SORDELLII

从艰难梭菌中分离毒素

• 批准号: 3126417
• 负责人: Tracy D. Wilkins
• 金额: $ 25.87万
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3126417
• 关键词: Clostridium; antibiotics; antireceptor antibody; antitoxins; bacterial cytopathogenic effect; bacterial genetics; bacterial toxins; biological products; carbohydrate sequence; chemical binding; colitis; diarrhea; enterotoxins; gastrointestinal disorder chemotherapy; gastrointestinal infection; gene expression; genetic manipulation; goats; hamsters; host organism interaction; human tissue; infant human (0-1 year); intestinal mucosa; laboratory mouse; laboratory rabbit; lectin; 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，可导致 一些接受抗生素治疗的患者患有结肠炎。 这 约 15% 的患者体内存在微生物及其毒素 医院里腹泻的病人很多。 如果不治疗 其中一些患者会发展为伪膜性结肠炎， 其死亡率很高。 该病通常是获得性的 在医院里，因为这种生物体只能在结肠中生长 正常菌群已基本被大剂量消除 抗生素。 医院为传播提供了理想的环境 引起腹泻的孢子形成生物体。 有机体 产生两种强效毒素。 毒素 A 是一种肠毒素，可能是 负责该疾病的主要症状。 毒素B是 非常有效的细胞毒素。 我们已经证明毒素 A 与 Galalpha1-3Galbeta1-4G1cNAc 三糖通过多重结合 发生在这种巨大毒素上的位点。 重复绑定 结构将通过构造进行详细分析 合成肽并将重复序列克隆到大肠杆菌和 产气荚膜梭菌。 我们将确定该三糖是否是 允许毒素 A 引发肠毒性的受体 这是否是成人体内毒素结合的受体 人类结肠组织。 成年人发生这种情况的情况可能有所不同 他们的结肠中存在三糖，这可以解释 对毒素的敏感性不同。 我们也会 确定婴儿是否有这种受体；大约一半的 在医院出生的婴儿会被这种微生物定植为 “正常菌群”没有明显的不良影响，我们想知道为什么 毒素不会杀死他们。 三糖不被降解 人类肠道酶，因此我们可以通过以下方式治疗这种疾病 给予大量附着在乳胶珠上的受体。 我们 发现与这种三糖结合的凝集素可以防止 毒素 A 的作用，因此这可能在治疗中有用。 一个 还特异针对该碳水化合物受体的单克隆抗体 可用并将进行测试。 作为毒素的活性位点 已排序，我们将尝试确定作用机制 通过与其他毒素和酶的序列进行比较。 这样的提示 至于机制将通过与以下机构的合作来确认： 研究小组在各自领域开展工作。 作为序列 毒素 B 可用，我们将以相同的方式对其进行分析。 我们将继续在机制上进行合作 毒素的作用并继续成为纯毒素的来源 特异性抗体。

项目成果

Characterization of cross-reactive proteins detected by Culturette Brand Rapid Latex Test for Clostridium difficile.

通过 Culturette Brand 艰难梭菌快速乳胶测试检测到的交叉反应蛋白的表征。

• DOI: 10.1128/jcm.26.3.397-400.1988
• 发表时间: 1988
• 期刊: Journal of clinical microbiology
• 影响因子: 9.4
• 作者: Lyerly,DM;Ball,DW;Toth,J;Wilkins,TD
• 通讯作者: Wilkins,TD

Toxin A of Clostridium difficile is a potent cytotoxin.

艰难梭菌毒素 A 是一种强效细胞毒素。

• DOI: 10.1128/jcm.28.5.869-871.1990
• 发表时间: 1990
• 期刊: Journal of clinical microbiology
• 影响因子: 9.4
• 作者: Tucker,KD;Carrig,PE;Wilkins,TD
• 通讯作者: Wilkins,TD

Purification and characterization of Clostridium difficile glutamate dehydrogenase.

• DOI: 10.1006/abbi.1993.1065
• 发表时间: 1993
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: B. Anderson;C. D. Anderson;R. Van Tassell;D. Lyerly;T. Wilkins

Problems associated with counterimmunoelectrophoresis assays for detecting Clostridium difficile toxin.

与检测艰难梭菌毒素的反免疫电泳测定相关的问题。

• DOI: 10.1128/jcm.15.2.347-349.1982
• 发表时间: 1982
• 期刊: Journal of clinical microbiology
• 影响因子: 9.4
• 作者: West,SE;Wilkins,TD
• 通讯作者: Wilkins,TD

Purification and characterization of Clostridium perfringens iota toxin: dependence on two nonlinked proteins for biological activity.

产气荚膜梭菌 iota 毒素的纯化和表征：生物活性依赖于两种非连接蛋白。

• DOI: 10.1128/iai.54.3.683-688.1986
• 发表时间: 1986
• 期刊: Infection and immunity
• 影响因子: 3.1
• 作者: Stiles,BG;Wilkins,TD
• 通讯作者: Wilkins,TD