INTESTINAL MECHANISMS OF C DIFFICILE TOXINS

艰难梭菌毒素的肠道机制

• 批准号: 2139349
• 负责人: JOHN THOMAS LAMONT
• 金额: $ 26.56万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139349
• 关键词: CHO cells; Clostridium difficile; G protein; affinity chromatography; age difference; antireceptor antibody; brush border membrane; clostridial infection; cytotoxicity; enteritis; enterotoxins; gastrointestinal function; genetic library; high performance liquid chromatography; ileum; immunocytochemistry; laboratory mouse; laboratory rabbit; molecular cloning; nucleic acid probes; protein sequence; receptor binding; receptor coupling; receptor expression; toxicology; transfection; ulcerative colitis

Clostridium difficile enteritis is one of the commonest nosocomial infections and a cause of considerable morbidity and occasional death from perforation of megacolon. C. difficile toxin A is the major determinant of intestinal inflammation and secretion. The overall goal of this proposal is to define the structure and function of the toxin A receptor on rabbit enterocytes. Four specific aims are proposed: 1. Biochemical purification of the enterocyte receptor and amino acid sequencing of receptor peptides. 2. Developmental regulation of the toxin A receptor in neonatal intestine. 3. Association of G proteins with the toxin A receptor. 4. Cloning of the receptor cDNA and determination of the amino acid sequence and carbohydrate composition. The toxin A receptor will be solubilized in detergent from rabbit ileal brush borders and purified to homogenity by affinity chromatography and HPLC. The topographic distribution of the receptor in the rabbit intestine and along the crypt-villus axis will be determined by immunohistochemistry with biotinylated toxin A and with antireceptor antibody. The toxin A receptor is absent in infant rabbits, and immature enterocytes are less responsive to the biological effects of the toxin. Therefore, receptor gene expression will be assessed in developing rabbit ileum using cDNA probes to determine if absence of the receptor in newborns is transcriptionally regulated or is related to post-translational modification. The toxin A receptor is closely associated with membrane G proteins which are likely to be involved in cellular mechanisms of the toxin. The interaction of the receptor with G proteins will be studied in membrane fractions from rabbit intestine and in T84 intestinal cell line. Finally, the isolation and characterization of cDNA clones encoding for the purified toxin A receptor will be carried out. Synthetic oligonucleotide probes corresponding to receptor peptide sequences will be used to screen rabbit cDNA libraries and the cDNA insert of the clone will be characterized and sequenced. Sequence analysis will provide information on the structural characteristics of the receptor and will be compared with sequences of other G protein-associated receptors. Transfection of CHO cells with receptor cDNA will be carried out to determine if biologically active receptor is expressed. These studies will provide information of the cellular basis of toxin A-receptor interaction and on the mechanism of action of this important bacterial enterotoxin on the intestine.

艰难梭菌肠炎是最常见的医院之一 感染和造成相当大的发病率和偶尔死亡的原因 大巨龙的穿孔。 艰难梭菌毒素A是主要决定因素 肠炎和分泌。 该提议的总体目标 是定义兔子上毒素A受体的结构和功能 肠球菌。 提出了四个具体目标：1。生化纯化 受体肽的肠细胞受体和氨基酸测序。 2。新生儿肠受体的发育调节。 3。G蛋白与毒素A受体的关联。 4。克隆 受体cDNA和氨基酸序列和碳水化合物的测定 作品。 毒素A受体将在兔回肠的洗涤剂中溶解 刷边界，并通过亲和力色谱和 HPLC。 兔肠受体的地形分布 并沿着隐窝村轴将通过免疫组织化学确定 伴有生物素化毒素A和抗受体抗体。 毒素a 婴儿兔子没有受体，未成熟的肠细胞较少 对毒素的生物学作用有反应。 因此，受体 使用cDNA，将在开发兔回肠中评估基因表达 确定新生儿中缺乏受体的探针是 转录调节或与翻译后有关 修改。 毒素A受体与膜G密切相关 可能参与细胞机制的蛋白质 毒素。 受体与G蛋白的相互作用将在 兔肠和T84肠细胞系中的膜级分。 最后，编码cDNA克隆的隔离和表征 将执行纯化的毒素A受体。 合成寡核苷酸 对应于受体肽序列的探针将用于筛查 兔cDNA库和克隆的cDNA插入物将是 表征和测序。 序列分析将提供有关 受体的结构特征，将与 其他G蛋白相关受体的序列。 转染Cho 将执行具有受体cDNA的细胞，以确定生物学上是否 表达活性受体。 这些研究将提供有关的信息 毒素A受体相互作用的细胞基础以及 这种重要的细菌肠毒素在肠道上的作用。