INTESTINAL MECHANISMS OF C. DIFFICILE TOXINS

艰难梭状芽胞杆菌肠炎是最常见的医院感染之一。 感染和相当大的发病率和偶尔死亡的原因 巨结肠穿孔。艰难梭菌毒素A是致病因子 肠道炎症和分泌物。这项提案的总体目标是 目的是确定兔毒素A受体的结构和功能 肠道细胞。提出了四个具体目标：1.生化提纯 肠细胞受体和受体多肽的氨基酸序列。 2.新生儿肠道毒素A受体的发育调控 3.G蛋白与毒素A受体的结合。4.克隆 受体基因与氨基酸序列和糖类的测定 组成。 毒素A受体在兔回肠洗涤剂中的增溶作用 通过亲和层析将刷子边缘和纯化至均一 高效液相色谱柱。受体在兔肠道的局部分布 免疫组织化学方法测定隐窝绒毛轴方向 用生物素标记的毒素A和抗受体抗体。A毒素 幼兔体内缺乏受体，未成熟的肠细胞较少。 对毒素的生物效应作出反应。因此，受体 利用基因芯片研究兔回肠发育过程中的基因表达 用于确定新生儿中是否存在该受体缺失的探针 转录调控的或与翻译后相关的 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#年进行研究。 兔肠道和T84肠道细胞系的膜组分。 最后，分离和鉴定了编码该基因的cDNA克隆。 将进行毒素A受体的纯化。合成寡核苷酸 与受体多肽序列相对应的探针将用于筛选 兔c DNA文库及克隆的c DNA插入 进行了表征和排序。序列分析将提供有关 受体的结构特征，并将其与 其他G蛋白相关受体的序列。中国仓鼠卵巢的转基因研究 带有受体cdna的细胞将被用来确定生物学上是否 表达活性受体。这些研究将提供以下信息 毒素A与受体相互作用的细胞学基础及作用机制研究 这种重要的细菌肠毒素对肠道的作用。

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.