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: 7670083
• 负责人: ERIK L HEWLETT
• 金额: $ 32.26万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-03 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7670083
• 关键词: Address; Affect; Anaerobic Bacteria; Biochemical; Biological; Biomedical Engineering; CDC42 gene; Cells; Cessation of life; Clostridium; Clostridium difficile; Colitis; Collection; Communicable Diseases; Complement; Complex; Cultured Cells; Data; Data Collection; Databases; Diarrhea; Disease; Employee Strikes; Enterotoxins; Epithelial Cells; Event; Family member; Functional disorder; Gene Expression; Genes; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Human Cell Line; In Situ Hybridization; In Vitro; Incidence; Individual; Infection; Inflammatory; Intervention; Knowledge; Lamina Propria; Least-Squares Analysis; Life; Literature; Mediating; Metric; Molecular; Molecular Profiling; Monomeric GTP-Binding Proteins; Mucous Membrane; Mus; N-terminal; Pathogenesis; Pathway interactions; Phenotype; Phosphorylation; Post-Translational Protein Processing; Process; Protein Array; Proteins; Proteomics; Regression Analysis; Research; Role; Severities; Signal Pathway; Signal Transduction; Signaling Molecule; System; Systems Biology; Testing; Time; Tissues; Toxin; Virulence; Western Blotting; analytical method; base; biodefense; cell type; cytotoxicity; design; enteric pathogen; ileum; in vivo; in vivo Model; insight; intestinal epithelium; microbial; novel; novel therapeutics; pathogen; programs; prophylactic; protein expression; protein profiling; receptor binding; research study; 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).

艰难梭菌结肠炎是一种毒素介导的疾病，依赖于毒素 A (TcdA) 的作用 和/或毒素 B (TcdB)。尽管 TcdA 和 TcdB 修饰 Rho 亚家族的分子机制 GTPases 已明确定义，但将这些事件与结肠炎和炎症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（选择毒素与毒素的相互作用） 新兴细菌病原体与宿主细胞的毒素），我们将结合以下分析方法 具有表达谱（基因表达和蛋白质修饰）和相关的系统生物学 生物学后果，以阐明受特定毒素介导影响的途径 一种或多种小 GTP 酶的糖基化。单独和组合培养的人类细胞（肠 上皮细胞和其他直接受毒素影响的细胞类型）将在体外和小鼠回肠结扎上进行研究 将在体内研究环，以表征毒素诱导的表型并确定 收集基因阵列数据的最佳条件。一种新颖的基于途径的概要分析将是 用于将体外和体内转录谱叠加到源自文献的途径知识上 数据库，从而识别被毒素改变的信号网络（SA 1）。同时，得出数据 通过基因表达阵列将使用 qRT-PCR、蛋白质印迹、磷酸化蛋白进行验证 分析和原位杂交。偏最小二乘回归分析将应用于集体 数据，以便表征 TcdA 和 TcdB (SA 2) 引发的表型的关键信号指标。 然后将通过对以下物质进行生物和生化操作来产生和测试新的假设： 新认识的途径和信号分子并确定生物学后果 并且不含毒素（SA 3）。这个令人兴奋的项目反映了三个小组的密切互动，分别代表： 1）微生物毒素作用和分子发病机制（Hewlett）； 2）引起疾病的发病机制 肠道病原体和肠毒素（Guerrant 和 Warren）和 3）感染性系统生物工程 疾病（木瓜病）。