INTESTINAL MECHANISMS OF C DIFFICILE TOXINS

艰难梭菌肠炎是最常见的医院感染 感染和相当大的发病率和偶尔死亡的原因， 巨结肠穿孔 C.艰难梭菌毒素A是 肠道炎症和分泌物。 本提案的总体目标是 目的是明确兔A毒素受体的结构和功能 肠细胞 提出了四个具体目标：1。生物化学纯化 和受体肽的氨基酸序列。 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.的克隆 受体cDNA及氨基酸序列和碳水化合物的测定 混合物. 毒素A受体将溶解在来自兔回肠的洗涤剂中， 刷状边缘并通过亲和色谱法纯化至均质， 高效液相色谱法 受体在家兔小肠的局部分布 免疫组织化学法测定沿着隐窝-绒毛轴的细胞数量 用生物素化毒素A和抗受体抗体。 毒素A 幼兔肠上皮细胞未成熟， 对毒素的生物效应作出反应。 因此，受体 将使用cDNA在发育中的兔回肠中评估基因表达 探针，以确定是否缺乏受体的新生儿， 转录调控或与翻译后相关 改性 毒素A受体与膜G紧密相关 这些蛋白质可能参与细胞机制， 毒素 受体与G蛋白的相互作用将在 在兔肠和T84肠细胞系中的膜组分。 最后，分离和表征编码该蛋白的cDNA克隆。 将进行纯化的毒素A受体。 合成寡核苷酸 对应于受体肽序列的探针将用于筛选 兔cDNA文库和克隆的cDNA插入片段将被 特征化并测序。 序列分析将提供以下信息 受体的结构特征，并将与 其他G蛋白相关受体的序列。 转染CHO 将进行具有受体cDNA的细胞，以确定生物学上是否 活性受体表达。 这些研究将提供以下信息： 毒素A-受体相互作用的细胞基础和 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.