SHIGA TOXIN EFFECTS ON GENE REGULATION

描述(摘自申请者的摘要)：产志贺毒素的大肠杆菌 (STEC)已经成为发达国家的一个主要健康问题，并正在 与出血性结肠炎和溶血性尿毒症等疾病相关 综合症。志贺毒素在猪瘟发病机制中的作用 STEC相关疾病是不完整的。志贺毒素的经典思考 志贺毒素在疾病发病机制中的作用是通过引起 抑制关键蛋白合成对敏感细胞的毒性作用 细胞生存所需的蛋白质。我们发现志贺毒素能够 在肠上皮细胞中诱导和超诱导IL-8。 矛盾的是，IL-8是由这些细胞分泌的，尽管全面阻断了 信使核糖核酸翻译。我们的数据表明，这可能是通过改变 宿主信号转导。这些数据支持STX如何 导致疾病，即STX参与改变 调节一个或多个宿主细胞过程，导致合成 宿主的蛋白质参与了致病过程。 这项拟议工作的目标是：1)表征志贺毒素是如何影响 肠上皮细胞IL-8基因调控2)志贺氏菌如何 毒素能在抑制IL-8的同时增加IL-8的mRNA和蛋白质 3)研究志贺毒素对小鼠相关基因的影响。 肠和肾。 志贺毒素诱导的IL-8基因调控的变化将在 转录水平。志贺毒素糖水解酶活性在细胞周期中的作用 IL-8mRNA的诱导将被评估。志贺毒素对寄主信号的影响 将对转导进行评估。细胞内转运途径抑制物 将被用来确定特定的志贺毒素转运途径是否 是合成IL-8所必需的。使用共聚焦显微镜，共定位 IL-8mRNA和细胞内志贺毒素将决定IL-8的位置 信使核糖核酸的翻译与志贺毒素转运的地方不同。最后， 志贺毒素对上皮细胞基因调控的影响 使用基因芯片杂交技术在基因组水平上进行评估。 了解这种毒素如何通过增强宿主炎症反应 影响宿主基因调控可能会让我们更好地理解 一种疾病的发病机制，这种疾病没有疫苗，只有支持性的 治疗。通过课程作业、研讨会和监督研究，索普博士 将获得与建议的技术相关的知识和专业知识 研究。

DESCRIPTION (adapted from applicant's abstract): Shiga toxin-producing E. coli (STEC) have emerged as a major health problem in the developed world and are associated with diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Our understanding of the role of Shiga toxins in the pathogenesis of STEC-associated disease is incomplete. Classical thinking about Shiga toxins' role in disease pathogenesis is that Shiga toxins harm the host by causing toxic effects on sensitive cells by inhibiting protein synthesis of critical proteins needed for cell survival. We have found that Shiga toxins are capable of inducing and superinducing IL-8, in intestinal epithelial cells. Paradoxically, IL-8 is secreted by these cells despite overall blockade of mRNA translation. Our data suggest that this may occur through alterations in host signal transduction. These data support a new model of how Stxs may contribute to disease, namely that Stxs are involved in altering the regulation of one or more host cell processes, resulting in synthesis of proteins by the host that contribute to pathogenesis. The goals of the proposed work are: 1) to characterize how Shiga toxins affect IL-8 gene regulation in intestinal epithelial cells 2) to determine how Shiga toxins are able to increase IL-8 mRNA and protein while inhibiting translation, and 3) to assess the effects of Shiga toxins on related genes in the intestine and the kidney. Shiga toxin-induced alterations in IL-8 gene regulation will be assessed at the level of transcription. The role of Shiga toxin glycohydrolase activity in IL-8 mRNA induction will be assessed. Effects of Shiga toxins on host signal transduction will be assessed. Intracellular trafficking pathway inhibitors will be employed to determine if specific Shiga toxin trafficking pathways are necessary for IL-8 synthesis. Using confocal microscopy, co-localization of IL-8 mRNA and intracellular Shiga toxin will determine if the site of IL-8 mRNA translation is distinct from where Shiga toxin traffics. Finally, the effects of Shiga toxins on gene regulation in epithelial cells will be assessed on a genomic level using cDNA microarray hybridization techniques. Understanding how this toxin may augment a host inflammatory response through affecting host gene regulation may allow us to better understand the pathogenesis of a disease for which there is no vaccine and only supportive therapies. Through coursework, seminars, and supervised research, Dr. Thorpe will gain knowledge and expertise in techniques pertinent to the proposed research.