Integrated analysis of cellular responses to toxins from clostridium difficile

艰难梭菌结肠炎是一种毒素介导的疾病，依赖A毒素(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 GTP酶被很好地定义，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的这一子项目中(选定的毒物和毒素的相互作用 新出现的细菌病原体和宿主细胞的毒素)，我们将结合 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，以阐明特定毒素介导的影响途径 一个或多个小的GTP酶的糖基化。单独培养和混合培养的人体细胞(肠道 上皮细胞和其他直接受毒素影响的细胞类型)将在体外和小鼠结扎的回肠进行研究 环路将在体内进行研究，以表征毒素诱导的表型并确定 收集基因芯片数据的最佳条件。一种新的基于途径的简明分析将是 用于将体外和体内转录图谱覆盖在文献衍生途径知识上 数据库，从而识别被毒素改变的信号网络(SA 1)。同时，数据派生 通过基因表达阵列将使用qRT-PCR，Western blotting，磷酸化蛋白 分析和原位杂交。将对集体进行偏最小二乘回归分析 数据，以表征由TcdA和TcdB(SA 2)引起的表型的关键信号指标。 然后，将通过对生物和生物化学操作来生成和测试新的假说 新发现的通路和信号分子以及决定生物后果的 不含毒素(SA 3)。这一激动人心的项目反映了三个群体的密切互动，代表： 1)微生物毒素作用和分子发病机制(休利特)；2)由 肠道病原体和肠毒素(Guerrant和Warren)和3)感染性疾病中的系统工程 疾病(Papin)。

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).