Shiga toxin uptake mechanisms and intracellular action

志贺毒素摄取机制和细胞内作用

• 批准号: 7654068
• 负责人: OLGA KOVBASNJUK
• 金额: $ 21.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-20 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654068
• 关键词: Abbreviations; Actin-Binding Protein; Actins; Adherence; Antibiotics; Apical; Apoptosis; Bacteria; Binding; Binding Proteins; Blood Circulation; Brefeldin A; Cell Culture Techniques; Cell Line; Cell model; Cells; Child; Clathrin; Cleaved cell; Clinical; Collection; Complement; Complication; Data; Development; Diarrhea; Disease; Disease Outbreaks; Disease Outcome; Distal; Drug or chemical Tissue Distribution; Endocytosis; Enterocytes; Enzymes; Epithelial; Epithelial Cells; Epithelium; Escherichia coli EHEC; Exposure to; Funding; Galectin 3; Glycosphingolipids; Grant; Hemolytic-Uremic Syndrome; Human; Ileocecal Valve; Infection; Inflammatory Response; Intestinal Diseases; Intestines; Kidney; Lamina Propria; Lead; Life; Mediating; Medical; Methods; Modeling; Molecular; Monomeric GTP-Binding Proteins; Nature; Oryctolagus cuniculus; Outcome; Pathogenesis; Pathology; Pathway interactions; Patients; Pattern; Permeability; Pharmacology; Play; Prevention; Process; Production; Publishing; Relative (related person); Reporting; Risk; Role; Sampling; Shiga Toxin; Shiga-Like Toxin I; Site; Surface; Systemic disease; Testing; Therapeutic; Tight Junctions; Time; Tissue Sample; Tissues; Toxin; Tropism; Vesicle; Work; base; caspase-3; cofilin; crypt cell; enteropathogenic Escherichia coli; foodborne pathogen; human tissue; in vivo; injured; intestinal epithelium; monomer; novel therapeutic intervention; pathogen; prevent; profilin 1; public health relevance; receptor; research study; therapy development; trafficking; transcytosis; uptake

DESCRIPTION (provided by applicant): Intestinal pathology caused by Shiga toxin (Stx)-producing enterohemorrhagic E.coli (EHEC), important food- borne pathogens, causes ~ 35% of all bloody diarrhea in the USA and life-threatening systemic complications, including the hemolytic uremic syndrome (HUS). EHEC is a particularly worrisome pathogen, because the numbers of outbreaks continue to 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 and to develop new therapeutic approaches to treat Stx-induced intestinal and systemic complications. The EHEC-induced diseases required that Stx must be endocytosed by the enterocytes, cross the intestinal epithelial barrier and enter the bloodstream. We have postulated that an antibiotic-independent method to prevent consequences of EHEC infections might be based on inhibiting Stx1 uptake and transcytosis across the intestinal epithelium. We have showed that the glycosphingolipid Gb3, the only known Stx1 receptor is not expressed in normal human intestinal epithelial cells. This recognition has required a rethinking of the pathogenesis of the toxin in EHEC diseases and provides the basis for the proposed studies. In this application using intestinal tissue from EHEC-infected humans, a well established model of rabbit cecal EHEC infection, and a cell culture model we will study the mechanisms of Stx1 uptake by intestinal epithelial cells, toxin transcytosis through the epithelium and the direct damage of epithelial cells by toxin. In Aim 1 we will determine the amount and distribution of Stx1 and Stx2 in intestinal tissue from patients with EHEC infection to establish a correlation between the outcome of disease (intestinal vs. systemic) and the toxin pattern in tissue. In Aim 2 we will characterize the molecular mechanism of Stx1 and Stx2 uptake by Gb3 receptor negative intestinal epithelial cells and toxin transcytosis. The role of Src and actin-binding proteins involved in macropinocytosis will be explored. Aim 3 will determine the Stx1 trafficking pathway that leads to apoptosis of intestinal epithelial cells and the role of secreted galectin-3 in apoptosis. The results gained from these experiments will not only help to elucidate the molecular mechanisms of Stx1 interaction with receptor free intestinal epithelial cells but will also contribute to our understanding of mechanisms of interaction between broad varieties of bacterial products. PUBLIC HEALTH RELEVANCE: Shiga toxin (Stx) producing E. coli (EHEC)-related illnesses, including hemolytic uremic syndrome (HUS), are of growing medical concern in the USA. To cause HUS, Stx must spread systemically. In this grant, we will characterize the mechanisms of Stx uptake by the intestinal epithelium and develop pharmacologic methods to stop this uptake, which should allow development of therapy against EHEC.

描述(申请人提供)：由产志贺毒素(STX)的肠出血性大肠杆菌(EHEC)引起的肠道病理，是一种重要的食源性病原体，导致美国约35%的血性腹泻和危及生命的全身并发症，包括溶血性尿毒症综合征(HUS)。EHEC是一种特别令人担忧的病原体，因为暴发数量持续增加，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的儿童中有高达10%是致命的。因此，迫切需要更好地了解这种感染的发病机制，并开发新的治疗方法来治疗STX引起的肠道和全身并发症。EHEC诱导的疾病要求STX必须被肠细胞内吞，穿过肠上皮屏障进入血液。我们推测，一种不依赖抗生素的预防EHEC感染后果的方法可能是基于抑制STX1摄取和跨肠上皮细胞转运。我们已经证明，唯一已知的STX1受体--神经鞘糖脂Gb3在正常人肠上皮细胞中不表达。这一认识需要重新思考EHEC疾病中毒素的发病机制，并为拟议的研究提供基础。在这项应用中，我们将利用感染EHEC的人的肠道组织、建立的兔盲肠EHEC感染模型和细胞培养模型来研究肠上皮细胞摄取STX1、毒素通过上皮转运以及毒素对上皮细胞的直接损伤的机制。在目标1中，我们将测定肠出血性大肠杆菌感染患者肠道组织中STX1和STX2的数量和分布，以建立疾病结局(肠道与全身)与组织中毒素模式之间的相关性。在目标2中，我们将研究Gb3受体阴性的肠上皮细胞摄取STX1和STX2的分子机制和毒素转胞作用。本课程将探讨Src和肌动蛋白结合蛋白在巨噬细胞吞噬中的作用。目的3研究STX1导致肠上皮细胞凋亡的转运途径以及分泌型半乳糖凝集素-3在细胞凋亡中的作用。这些实验结果不仅有助于阐明STX1与无受体的肠上皮细胞相互作用的分子机制，而且有助于我们理解各种细菌产物之间的相互作用机制。公共卫生相关性：志贺毒素(STX)产生的大肠杆菌(EHEC)相关疾病，包括溶血性尿毒症综合征(HUS)，在美国越来越受到医疗关注。要导致HUS，STX必须在系统内传播。在这笔赠款中，我们将描述肠道上皮细胞摄取STX的机制，并开发药物方法来阻止这种摄取，这将使治疗EHEC的发展成为可能。