CONTROL OF BACTERIAL TOXINS BY VIRUSES AND PLASMIDS

通过病毒和质粒控制细菌毒素

• 批准号: 2060044
• 负责人: Randall K Holmes
• 金额: $ 20.38万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-09-30 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2060044
• 关键词: Corynebacterium diphtheriae; Escherichia coli; Vibrio cholerae; adenylate cyclase; antiserum; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; bacterial toxins; bacterial virus; biological models; cholera toxin; cholera vaccine; communicable disease control; complementary DNA; diphtheria toxin; disease /disorder proneness /risk; enterotoxins; genetic manipulation; genetic regulation; genetic transcription; genetic translation; immunity; immunochemistry; in situ hybridization; laboratory mouse; laboratory rabbit; model design /development; molecular cloning; monoclonal antibody; plasmids; protein biosynthesis; radioimmunoassay; structural genes; veterinary medicine; virus antigen; virus genetics

DESCRIPTION: (Adapted from the applicants abstract) Diphtheria toxin (DT) and the family of heat-labile enterotoxins (LTs) are well studied virulence factors of Corynebacterium diphtheriae and Escherichia coli, respectively. Each toxin has a B domain that binds to plasma membrane receptors and an A domain that mediates toxicity. DT ADP-ribosylates elongation factor 2, thereby inhibiting protein synthesis and killing target cells. LTs ADP-ribosylate the regulatory protein G, and activate adenylate cyclase. The long term objectives are to understand the genetics, regulation, and mode of action of these toxins at the molecular level. Such knowledge is essential for understanding pathogenesis of diphtheria and enterotoxic E. coli diarrhea and for development of improved vaccines, immunotoxins, hybrid toxins, or related biologic products involving these toxins. The specific aims of this proposal are to: 1) determine the mechanisms by which iron regulates production of DT and the activity of the iron uptake system in C. diphtheriae. The investigators will analyze the tox promoter sequence that interacts with the E. coli Fur protein, clone and characterize the tox repressor from C. diphtheria, isolate and characterize ferric uptake regulation (fur) mutants of C. diphtheriae, and determine whether tox repressor activity is mediated by the putative fur gene product in C. diphtheriae. 2) characterize the interactions of DT with its cellular receptor. Dr. Holmes and colleagues have developed monoclonal antibodies to membrane components of human cells that block intoxication by DT and they will characterize their mode of action. They will also purify DT receptor molecule(s), clone and characterize and corresponding cDNA(s), determine whether DT-resistant cells that lack functional receptors have genes or membrane components homologous with those for DT receptors, and characterize the binding domains of DT and its receptor. 3) analyze structure-function relationships of LTs. They will compare purified type I and type II LTs in intestinal assays in several animal species to determine whether their different receptor specificities correlate with differences in biologic activity. They will use genetic complementation with cloned A and B subunit genes, directed mutagenesis of cloned genes, chemical modification of purified toxins, and synthetic oligopeptides to analyze the molecular determinants of biologic activity of representative type I and type II LTs.

描述：（改编自申请人摘要）白喉毒素（DT） 和热能肠肠毒素（LTS）的家族进行了充分研究的毒力 二杆菌和大肠杆菌的因子。 每个毒素都有一个与质膜受体结合的B结构域，A 介导毒性的领域。 DT ADP-核糖基延伸因子2， 从而抑制蛋白质合成并杀死靶细胞。 LTS ADP-核酰胺调节蛋白G，并激活腺苷酸环化酶。 长期目标是了解遗传学，调节和 这些毒素在分子水平上的作用方式。 这样的知识是 了解白喉和肠毒性E的发病机理至关重要。 大肠杆菌腹泻，用于开发改善的疫苗，免疫毒素， 杂交毒素或涉及这些毒素的相关生物产品。 这 该提案的具体目的是：1）确定机制 铁调节DT的产生和铁吸收活性 C. diphtheriae中的系统。 调查人员将分析托克斯启动子 与大肠杆菌毛皮蛋白，克隆和 表征来自白喉梭状芽胞杆菌的毒抑制剂，分离和表征 甲状腺梭菌的铁摄取调节（毛皮）突变体，并确定 托克斯阻遏活性是否由推定的皮肤基因产物介导 在白喉梭状芽胞杆菌中。 2）表征DT与其的相互作用 细胞受体。 福尔摩斯博士和同事已经开发了单克隆 人类细胞的膜成分抗体，通过 DT，他们将表征其行动方式。 他们也会净化 DT受体分子（S），克隆和表征和相应的cDNA， 确定缺乏功能受体的DT抗性细胞是否具有 与DT受体同源的基因或膜成分，以及 表征DT及其受体的结合结构域。 3）分析 LTS的结构功能关系。 他们将比较纯化的I型 在几种动物物种中的肠测定中的II型LTS和II型LTs以确定 它们的不同受体特异性是否与差异相关 在生物活性中。 他们将使用克隆的遗传互补 和B亚基基因，定向的克隆基因的诱变，化学 修饰纯化的毒素和合成寡肽以分析 代表性I型生物学活性的分子决定因素和 II型LTS。