Intestinal Disease-enterocyte toxin interaction

肠道疾病-肠细胞毒素相互作用

• 批准号: 9263933
• 负责人: WAYNE I LENCER
• 金额: $ 71.69万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-20 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9263933
• 关键词: Address; Affect; Architecture; Area; Bacterial Proteins; Bacterial Toxins; Binding; Biochemical; Biological; Biological Assay; Biology; Bordetella pertussis; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Structures; Cellular biology; Ceramides; Cholera; Cholera Toxin; Cholesterol; Complement; Complex; Coupled; Cytosol; Data; Destinations; Diarrhea; Disease; Drug Delivery Systems; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Enterocytes; Environment; Epithelial; Epithelial Cells; Escherichia coli; Eukaryotic Cell; Family; Fatty Acids; Ganglioside GM1; Genes; Genetic; Genetic Screening; Genetic study; Glycocalyx; Glycolipids; Glycosphingolipids; Goals; Grant; Head; Health; Host Defense; Immunomodulators; Inflammatory; Inflammatory Response; Innate Immune Response; Intestinal Diseases; Intestines; Intoxication; Invaded; Laboratories; Lipids; Mammalian Cell; Maps; Mediating; Membrane; Membrane Biology; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Microbe; Modeling; Molecular; Molecular Chaperones; Movement; Mucous body substance; Mus; Natural Immunity; Oligosaccharides; Outcome; Pathogenesis; Pathway interactions; Peptides; Pertussis Toxin; Phenotype; Preparation; Problem Solving; Process; Protein Isoforms; Proteins; Quality Control; RNA Interference; Reaction; Reagent; Reporter; Research; Resistance; Shigella; Signal Transduction; Sorting - Cell Movement; Sphingolipids; Sphingomyelins; Structure; Surface; Symbiosis; Testing; Tight Junctions; Tissues; Toxin; Zebrafish; base; clinically relevant; design; endoplasmic reticulum stress; genetic approach; genome-wide; germ free condition; gut microbiota; human disease; lipid transport; microbial host; microbiome; misfolded protein; multicatalytic endopeptidase complex; mutant; novel; pathogen; protein degradation; receptor; reverse genetics; sensor; trafficking; vaccine delivery

The goal of this application is to elucidate the molecular basis for invasion and intoxication of intestinal cells by cholera toxin (CT), the causative agent of Asiatic cholera, and for induction of innate immunity. Mucosal surfaces represent vast areas where host tissues are separated from the environment only by a delicate but highly effective single layer of columnar epithelial cells, joined by tight junctions that are impermeable to proteins and even small peptides. Here, we study how a bacterial protein breeches this barrier to enter the endoplasmic reticulum (ER), and then cytosol, of host intestinal cells. To do this, the toxin co-opts a sphingolipid receptor (ganglioside GM1) and endogenous mechanisms of membrane and lipid trafficking for entry into the ER. Once in the ER, a fragment of CT, the A1-chain, then enters the cytosol by hijacking the machinery essential for protein quality control in the biosynthetic pathway, which senses and eventually degrades (by retro-translocation to the cytosol) all terminally-misfolded proteins in the ER lumen. We recently found that the intestinal cell senses entry of the A1-chain into the ER to induce an innate immune response, even when the toxin is rendered enzymatically inert, suggesting a general mechanism of innate immunity. Signal transduction in this pathway appears to be mediated by canonical sensors of ER stress, which are associated with the pathogenesis of IBD. The biology co-opted by CT to enter host cells is fundamental to intestinal cell structure and function, and clinically relevant for diverse human diseases in addition to the toxigenic diarrheas. This project proposes to continue 22 years of focused research. We will use biochemical, molecular, cell biological, and genetic approaches to: explain how GM1 sphingolipids and CT-GM1 complexes traffic to the ER and other destinations (Aim 1); analyze the processing of the toxin by the ER, and elucidate the mechanisms for transport to the cytosol, and for its induction of an innate immune response (Aim 2); and identify novel molecular components involved in all the toxin pathways using unbiased forward and reverse genetic approaches (Aim 3). We have established novel reagents and approaches to solve these problems, including: synthesis of GM1 structural isoforms for direct structure-function studies on sphingolipid trafficking; and preparation of novel CT mutants designed to isolate the fraction of toxin within the ER lumen or to trap it in intermediate reactions to understand how the ER processes the toxin for transport to the cytosol and for induction of innate immunity. We have also developed the zebrafish for genetic studies and identified 13 families by forward screen as resistant to intoxication. The mutant genes in these families will be identified by positional-mapping and their function studied.

该应用的目的是阐明通过 霍乱毒素（CT），亚洲霍乱的病因，以及诱导先天免疫。 粘膜表面代表了广阔的区域，其中宿主组织仅与环境分开 精致但高效的柱状上皮细胞的单层，由紧密连接连接 不受蛋白质甚至小肽的渗透。在这里，我们研究了细菌蛋白马裤如何这种障碍 进入宿主肠细胞的内质网（ER），然后进入细胞质。为此，毒素合作 鞘脂受体（神经节苷脂GM1）和膜和脂质运输的内源性机制 进入急诊室。一旦进入ER，A1链的CT片段，然后通过劫持该细胞质进入细胞质 生物合成途径中蛋白质质量控​​制必不可少的机械，该途径感官并最终 ER管腔中的所有终末不满意的蛋白质降解（通过恢复到细胞质）降解。我们最近 发现肠细胞会感觉到A1链进入ER，以诱导先天免疫反应， 即使毒素被酶惰性呈惰性，也表明了先天免疫的一般机制。 该途径中的信号转导似乎是由ER应力的规范传感器介导的， 与IBD的发病机理有关。 CT选择进入宿主细胞的生物学对 肠道细胞结构和功能，以及临床上与多种人类疾病相关的 毒素性腹泻。 该项目建议继续进行22年的重点研究。我们将使用生化，分子，细胞 生物学和遗传方法：解释GM1鞘脂和CT-GM1如何将流量与 ER和其他目的地（AIM 1）；分析ER的毒素加工，并阐明 转运到细胞质的机制，以及诱导先天免疫反应的机制（AIM 2）；和 使用公正的前进和反向确定所有参与所有毒素途径中涉及的新分子成分 遗传方法（目标3）。我们已经建立了新的试剂和方法来解决这些问题， 包括：GM1结构同工型的合成，用于鞘脂运输的直接结构功能研究； 并制备新型CT突变体，旨在隔离ER管腔内毒素的分数或将其捕获 中间反应以了解ER如何处理毒素以将其运输到细胞质和 诱导先天免疫。我们还开发了斑马鱼进行遗传研究，并确定了13 通过向前屏幕的家庭对中毒具有抵抗力。这些家族中的突变基因将通过 位置映射及其功能研究。