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（由申请人提供）：由产生志贺毒素 (Stx) 的肠出血性大肠杆菌 (EHEC) 引起的肠道病理学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，因为爆发次数不断增加，而且这种疾病尚无有效的特异性治疗方法，导致高达 10% 的 HUS 儿童死亡。因此，非常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引起的疾病要求Stx必须被肠上皮细胞内吞，穿过肠上皮屏障并进入血流。我们假设，一种不依赖抗生素的方法来预防 EHEC 感染的后果可能是基于抑制 Stx1 摄取和跨肠上皮的转胞吞作用。我们已经证明，唯一已知的 Stx1 受体——鞘糖脂 Gb3 在正常人肠上皮细胞中不表达。这一认识要求重新思考肠出血性大肠杆菌疾病中毒素的发病机制，并为拟议的研究提供了基础。在本应用中，我们将使用感染 EHEC 的人类肠道组织、完善的兔盲肠 EHEC 感染模型和细胞培养模型来研究肠上皮细胞摄取 Stx1、毒素通过上皮的转胞吞作用以及毒素对上皮细胞的直接损伤的机制。在目标 1 中，我们将确定 EHEC 感染患者肠道组织中 Stx1 和 Stx2 的数量和分布，以建立疾病结果（肠道与全身）和组织中毒素模式之间的相关性。在目标 2 中，我们将描述 Gb3 受体阴性肠上皮细胞摄取 Stx1 和 Stx2 以及毒素转胞吞作用的分子机制。将探讨 Src 和肌动蛋白结合蛋白在巨胞饮作用中的作用。目标 3 将确定导致肠上皮细胞凋亡的 Stx1 运输途径以及分泌型半乳糖凝集素 3 在细胞凋亡中的作用。从这些实验中获得的结果不仅有助于阐明 Stx1 与无受体肠上皮细胞相互作用的分子机制，而且还将有助于我们理解多种细菌产物之间的相互作用机制。公共卫生相关性：产志贺毒素 (Stx) 的大肠杆菌 (EHEC) 相关疾病，包括溶血性尿毒症综合征 (HUS)，在美国日益受到医学关注。为了引起 HUS，Stx 必须进行系统性传播。在这笔资助中，我们将描述肠上皮细胞摄取 Stx 的机制，并开发阻止这种摄取的药理学方法，这将有助于开发针对肠出血性大肠杆菌的疗法。