Enterotoxin targeting and delivery mechanisms

肠毒素的靶向和递送机制

基本信息

批准号：
7810638
负责人：
META J KUEHN
金额：
$ 29.42万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-06-15 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7810638
关键词：
Address Affect Bacterial Adhesins Binding Biochemical Genetics Biopsy Cell Communication Cell Culture Techniques Cells Child Clinical Data Developing Countries Diarrhea Disease Electron Microscopy Enterotoxins Epithelial Cells Escherichia coli Fluorescence Food Genes Gram-Negative Bacteria Heating Human In Situ In Vitro Infection Intervention Intestines Intoxication Investigation Knowledge Laboratories Lipids Location Mammalian Cell Measures Mediating Membrane Military Personnel Modeling Mutation Patients Pharmacotherapy Physiological Process Production Proteins Research Role Source Sterility Surface Techniques Toxic effect Toxin Vesicle Virulence Virulence Factors Water biodefense enterotoxigenic Escherichia coli human disease in vivo in vivo Model insight luminal membrane mutant novel pathogen pathogenic bacteria periplasm receptor research study targeted delivery trafficking transmission process

项目摘要

DESCRIPTION (provided by applicant): Enterotoxigenic Escherichia coli (ETEC) is an important diarrheagenic pathogen in third world countries and is a biodefense threat since military personnel are likely to acquire this pathogen from non-sterile water and food sources in the field. ETEC is also responsible for a large number of fatalities in children. Vesicles derived from the outer membrane of Gram-negative bacteria have been detected in biopsies from human patients. Vesicle production is not unique to ETEC, in fact all gram-negative bacteria (including other biothreatening pathogens) produce vesicles, however they have been poorly characterized and little is known about the roles of vesicle components in disease. Whereas vesicles from nonpathogens are relatively harmless "empty shells", virulence proteins are associated with vesicles from pathogens such as ETEC. We have previously found that the majority of secreted physiologically active heat-labile enterotoxin (LT) is associated with ETEC vesicles. It is enriched in vesicles as compared to the cell and is present both inside and bound to the outside of the vesicle. Vesicles transfer virulence factors, such as LT, from the pathogen directly into host cells. LT on the surface of ETEC vesicles mediates vesicle binding, and binding causes subsequent internalization of vesicles by gut epithelial cells, leading to toxicity. The clinical consequence of enterotoxin entry into human intestinal cells is sudden water efflux or debilitating diarrhea. The overall objective of this research is to characterize the roles of vesicles and vesicle components in ETEC enterotoxin targeting and delivery into human intestinal cells. The aims of this proposal are to characterize the host cell interactions of purified ETEC vesicles, understand how the lipid and protein components of the vesicles affect cellular intoxication, and assess the impact of vesiculation levels in disease. We will utilize cell culture and confocal localization techniques, as well as an established in situ and in vivo models to sensitively and quantitatively characterize the roles of the toxic vesicle components. Previously identified mutations in E. coli genes that cause over- and underproduction of vesicles will be used in the models to evaluate how vesicle production correlates with toxicity. The data resulting from these aims will reveal key features of bacterial vesicles that function in human disease. These results will also broadly impact understanding the role of toxic vesicles produced by other pathogenic bacteria that threaten our defense capabilities.

描述（由申请人提供）：产肠毒素大肠杆菌 (ETEC) 是第三世界国家的一种重要的腹泻病原体，并且是一种生物防御威胁，因为军事人员可能从野外的非无菌水和食物来源获取这种病原体。 ETEC 还造成大量儿童死亡。在人类患者的活组织检查中检测到源自革兰氏阴性细菌外膜的囊泡。囊泡的产生并非 ETEC 所独有，事实上，所有革兰氏阴性细菌（包括其他具有生物威胁的病原体）都会产生囊泡，但对它们的特征了解甚少，并且人们对囊泡成分在疾病中的作用知之甚少。非病原体的囊泡是相对无害的“空壳”，而毒力蛋白则与 ETEC 等病原体的囊泡相关。我们之前发现，大多数分泌的具有生理活性的不耐热肠毒素（LT）与 ETEC 囊泡有关。与细胞相比，它在囊泡中富集，并且存在于囊泡内部并与囊泡外部结合。囊泡将毒力因子（例如 LT）从病原体直接转移到宿主细胞中。 ETEC囊泡表面的LT介导囊泡结合，结合导致囊泡随后被肠上皮细胞内化，从而导致毒性。肠毒素进入人体肠道细胞的临床后果是突然的水流出或使人衰弱的腹泻。本研究的总体目标是表征囊泡和囊泡成分在 ETEC 肠毒素靶向和递送至人类肠道细胞中的作用。该提案的目的是表征纯化 ETEC 囊泡的宿主细胞相互作用，了解囊泡的脂质和蛋白质成分如何影响细胞中毒，并评估囊泡水平对疾病的影响。我们将利用细胞培养和共聚焦定位技术，以及已建立的原位和体内模型来敏感和定量地表征有毒囊泡成分的作用。先前发现的大肠杆菌基因突变会导致囊泡生成过多和生成不足，这些突变将用于模型中，以评估囊泡生成与毒性之间的关系。这些目标产生的数据将揭示在人类疾病中发挥作用的细菌囊泡的关键特征。这些结果还将广泛影响对其他威胁我们防御能力的病原菌产生的有毒囊泡的作用的理解。