Integrated analysis of cellular responses to toxins from clostridium difficile

细胞对艰难梭菌毒素反应的综合分析

• 批准号: 8233381
• 负责人: ERIK L HEWLETT
• 金额: $ 32.89万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233381
• 关键词: Affect; Anaerobic Bacteria; Biochemical; Biological; Biomedical Engineering; CDC42 gene; Cells; Cessation of life; Clostridium; Clostridium difficile; Colitis; Collection; Communicable Diseases; Complex; Cultured Cells; Data; Databases; Diarrhea; Disease; Enterotoxins; Event; Family member; Functional disorder; Gene Expression; Gene Proteins; Genes; Guanosine Triphosphate Phosphohydrolases; Human; Human Cell Line; In Situ Hybridization; In Vitro; Individual; Infection; Inflammatory; Knowledge; Least-Squares Analysis; Literature; Mediating; Metric; Molecular; Molecular Profiling; Monomeric GTP-Binding Proteins; Mus; N-terminal; Pathogenesis; Pathway interactions; Phenotype; Phosphorylation; Post-Translational Protein Processing; Process; Proteins; Regression Analysis; Research; Signal Pathway; Signal Transduction; Signaling Molecule; System; Systems Biology; Testing; Tissues; Toxin; Western Blotting; analytical method; base; biodefense; cell type; cytotoxicity; enteric pathogen; ileum; in vivo; in vivo Model; insight; intestinal epithelium; microbial; novel; pathogen; programs; protein expression; receptor binding; response; rho; rho GTP-Binding Proteins

C. difficile colitis is a toxin-mediated disease, which is dependent on the actions of Toxin A (TcdA) and/or Toxin B (TcdB). Although the molecular mechanisms by which TcdA and TcdB modify Rho-subfamily GTPases are well defined, the pathways connecting those events to colitis and inflammatory diarrhea remain elusive. In this sub-project of MARCE Research Program V (Interactions of Select Agent Toxins and Toxins of Emerging Bacterial Pathogens with Host Cells), we will combine the analytical methods of systems biology with expression profiles (gene expression and protein modification) and associated biological consequences, in order to elucidate the pathways affected by the specific toxin-mediated glucosylation of one or more small GTPases. Cultured human cells alone and in combinations (intestinal epithelium and other cell types directly affected by the toxins) will be studied in vitro and mouse ligated ileal loops will be studied in vivo to characterize the phenotypes induced by the toxins and to determine the optimal conditions for collection of the gene array data. A novel pathway-based compendium analysis will be used to overlay the in vitro and in vivo transcriptional profiles on a literature-derived pathway knowledge database, thereby identifying signaling networks altered by the toxins (SA 1). Concurrently, data derived through gene expression arrays will be validated using qRT-PCR, Western blotting, phospho-protein analyses and in situ hybridization. Partial least-squares regression analysis will be applied to the collective data, in order to characterize the key signaling metrics for the phenotypes elicited by TcdA and TcdB (SA 2). New hypotheses will then be generated and tested by making biological and biochemical manipulations of the newly recognized pathways and signaling molecules and determining the biological consequences with and without the toxins (SA 3). This exciting project reflects a close interaction of three groups, representing: 1) microbial toxin action and molecular pathogenesis (Hewlett); 2) pathogenesis of diseases caused by enteric pathogens and enterotoxins (Guerrant and Warren) and 3) systems bioengineering in infectious diseases (Papin).

C.艰难梭菌结肠炎是一种毒素介导的疾病，其依赖于毒素A（TcdA）的作用。 和/或毒素B（TcdB）。虽然TcdA和TcdB修饰Rho亚家族的分子机制 GTP酶定义明确，将这些事件与结肠炎和炎性腹泻联系起来的途径仍然存在 难以捉摸。在MARCE研究计划V的这个子项目中（选择代理毒素和 新出现的细菌病原体与宿主细胞的毒素），我们将结合联合收割机的分析方法， 系统生物学与表达谱（基因表达和蛋白质修饰）和相关 生物学后果，以阐明受特定毒素介导的 一种或多种小GTP酶的葡糖基化。单独培养的人细胞和组合培养的人细胞（肠 上皮和其他直接受毒素影响的细胞类型）进行体外研究， 环将在体内研究，以表征毒素诱导的表型，并确定 基因阵列数据收集的最佳条件。一个新的路径为基础的简编分析将 用于将体外和体内转录谱叠加在文献来源的途径知识上 数据库，从而识别被毒素改变的信号网络（SA 1）。同时，数据来源 通过基因表达阵列将使用qRT-PCR、蛋白质印迹、磷蛋白 分析和原位杂交。偏最小二乘回归分析将应用于集体 数据，以表征由TcdA和TcdB引起的表型的关键信号传导度量（SA 2）。 新的假设将产生和测试，通过生物和生物化学操作， 新认识的途径和信号分子，并确定生物学后果， 没有毒素（SA 3）。这个令人兴奋的项目反映了三个团体的密切互动，代表： 1)微生物毒素作用和分子发病机理（Hewlett）; 2）由 肠道病原体和肠毒素（Guerrant和Warren）和3）传染病中的系统生物工程 疾病（Papin）。