Shiga toxin 1:uptake mechanisms and intracellular action

描述(申请人提供)：食源性腹泻每年在美国造成76,000,000例病例。产志贺毒素大肠杆菌(EHEC)是一种重要的食源性致病菌，其引起的肠道病理包括美国约35%的出血性腹泻，未知比例的水样腹泻，以及感染、溶血性尿毒症综合征(HUS)和脑病等危及生命的全身症状(高达10%)。肠出血性大肠杆菌是目前美国儿童急性和慢性肾功能衰竭的主要原因。EHEC是一种特别令人担忧的食源性病原体，因为EHEC引起的暴发数量继续显著增加，而且没有有效的特效疗法来治疗这种疾病，这种疾病在发展为HUS的儿童中可致死高达10%。因此，迫切需要更好地了解这种感染的发病机制，以促进治疗EHEC感染及其并发症的新的治疗方法的发展。了解STX摄取和转运途径进入结肠上皮的复杂机制可能有助于识别新的药物靶点和开发新的策略，以防止毒素对人体肠道的作用。在这一应用中，我们将利用动物和人的肠上皮细胞模型来研究STX1及其B亚单位(STX1B)的摄取、转运和细胞内作用的机制。在目标1中，我们将研究脂筏(LR)在STX1/STX1B跨顶端细胞表面易位中的作用。我们建议测试与毒素受体相关的LR蛋白在毒素转运机制中的作用。在目标2中，我们将研究新发现的从质膜到核仁的STX1/STX1B内化途径。在目标3中，我们将研究STX1/STX1B诱导细胞凋亡的机制及其在毒素相关肠炎中的作用。这些研究将为理解STX介导的肠上皮细胞损伤的分子基础提供新的见解，并有助于设计预防STX1摄取和细胞内作用的策略，从而为干预EHEC相关的胃肠道病理生理找到新的靶点。

DESCRIPTION (provided by applicant): Foodborne diarrheal disease cause 76,000,000 cases each year in the United States. Intestinal pathology caused by Shiga toxin-producing Escherichia coli (EHEC), important food-borne pathogens, includes approximately 35% of all bloody diarrhea in the United States, an unknown % of watery diarrhea, and the life-threatening systemic manifestations (observed in up to 10% of cases) of infection, the hemolytic uremic syndrome (HUS) and encephalopathy. EHEC are the leading cause currently of acute and chronic renal failure in children in the USA. EHEC is a particularly worrisome foodborne pathogen, because the number of outbreaks caused by EHEC continue to significantly increase and there is no effective specific therapy for this illness which is lethal in up to 10% of children who develop HUS. Thus, there is a great need to better understand the pathogenesis of this infection to promote development of new therapeutic approaches to treat EHEC infection and its complications. Understanding the complex mechanism of Stx uptake and trafficking pathways into colonic epithelium may help to identify new drug targets and develop new strategies directed at preventing toxin action on human intestine. In this application we will study the mechanism of Stx1 and its B-subunit (Stx1B) uptake, trafficking and intracellular action using animal and human intestinal epithelial cell models. In Aim 1 we will study the role of lipid rafts (LR) in Stx1/Stx1B translocation across the apical cell surface. We propose to test the role of LR proteins, which are associated with toxin receptor in toxin translocation machinery. In Aim 2 we will study a newly recognized Stx1/Stx1B internalization pathway from plasma membrane into the nucleoli. In Aim 3 we will study the mechanisms of Stx1/Stx1B-induced apoptosis and its role in toxin-related intestinal inflammation. These proposed studies should provide new insight into the understanding of the molecular basis of Stx-mediated intestinal epithelial cell injury and facilitate the design of strategies to prevent Stx1 uptake and intracellular action, and thus serve to find new targets to interfere with EHEC-related gastrointestinal pathophysiology.