SHIGA TOXIN EFFECTS ON GENE REGULATION

志贺毒素对基因调控的影响

• 批准号: 6631615
• 负责人: CHELESTE M THORPE
• 金额: $ 12.45万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6631615
• 关键词: Escherichia coli; bacterial cytopathogenic effect; confocal scanning microscopy; gastrointestinal epithelium; genetic transcription; genetic translation; host organism interaction; inflammation; interleukin 8; intracellular transport; microarray technology; shiga toxin; tissue /cell culture

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.

性状（改编自申请人摘要）：产滋贺毒素E.杆菌 （STEC）已成为发达国家的一个主要健康问题， 与出血性结肠炎和溶血性尿毒症等疾病相关 综合征我们对滋贺毒素在大肠癌发病机制中的作用的理解 STEC相关疾病是不完整的。关于滋贺毒素的经典思考 滋贺毒素在疾病发病机制中的作用是通过引起 通过抑制关键蛋白质的合成对敏感细胞产生毒性作用 细胞生存所需的蛋白质。我们发现滋贺毒素能够 在肠上皮细胞中诱导和超诱导IL-8。 巧合的是，IL-8由这些细胞分泌，尽管IL-8被整体阻断。 mRNA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了一个新的模型， 有助于疾病，即Stxs参与改变 调节一个或多个宿主细胞过程，导致合成 蛋白质的宿主，有助于发病机制。 拟议工作的目标是：1）表征滋贺毒素如何影响 IL-8基因在肠上皮细胞中的调控2）如何确定滋贺 毒素能够增加IL-8 mRNA和蛋白，同时抑制 3）评估滋贺毒素对相关基因的影响， 肠道和肾脏 滋贺毒素诱导的IL-8基因调控改变将在 转录水平。滋贺毒素糖水解酶活性在大肠杆菌中的作用 将评估IL-8 mRNA诱导。滋贺毒素对宿主信号的影响 将评估转导。细胞内转运途径抑制剂 将用于确定特定的滋贺毒素运输途径是否 IL-8合成所必需的。使用共聚焦显微镜，共定位 IL-8 mRNA和细胞内滋贺毒素将决定IL-8在细胞内的表达位点， mRNA翻译与滋贺毒素运输的位置不同。最后 滋贺毒素对上皮细胞基因调控的影响将是 使用cDNA微阵列杂交技术在基因组水平上评估。 了解这种毒素如何通过以下途径增强宿主的炎症反应： 影响宿主基因调控可能使我们更好地了解 没有疫苗，只能支持的疾病的发病机制 治疗通过课程，研讨会和监督研究，索普博士 将获得与建议相关的技术知识和专业知识， research.