Intestinal Disease-enterocyte toxin interaction

描述（由申请人提供）：本申请的目的是阐明霍乱毒素（霍乱的病原体）侵入和中毒肠细胞的分子基础，以及诱导先天免疫。粘膜表面代表了宿主组织与环境分离的广阔区域，宿主组织与环境仅通过一个精致但高效的单层柱状上皮细胞分离，由蛋白质甚至小肽无法渗透的紧密连接连接在一起。在这里，我们研究了细菌蛋白如何突破这一屏障进入宿主肠细胞的内质网（ER），然后进入细胞质。为此，毒素选择鞘脂受体（神经节苷脂GM1）和内源性膜和脂质运输机制进入内质网。一旦进入内质网，一个CT片段，a1链，然后通过劫持生物合成途径中蛋白质质量控制的基本机制进入细胞质，该途径感知并最终降解（通过逆行转位到细胞质溶胶）内质网管中所有最终错误折叠的蛋白质。我们最近发现肠细胞感知a1链进入内质网诱导先天免疫反应，甚至当毒素被酶惰性化时，提示先天免疫的一般机制。这一途径的信号转导似乎是由内质网应激的典型传感器介导的，这与IBD的发病机制有关。CT进入宿主细胞的生物学特性是肠细胞结构和功能的基础，除产毒素腹泻外，还与多种人类疾病具有临床相关性。该项目计划继续进行22年的重点研究。我们将使用生化、分子、细胞生物学和遗传学方法来解释GM1鞘脂和CT-GM1复合物如何运输到内质网和其他目的地（目的1）；分析内质网对毒素的处理，阐明向细胞质转运及其诱导先天免疫反应的机制（目标2）；并使用无偏正向和反向遗传方法识别涉及所有毒素途径的新分子成分（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：合成GM1结构异构体，用于直接研究鞘脂运输的结构-功能；以及新型CT突变体的制备，旨在分离内质网腔内的毒素部分或将其困在中间反应中，以了解内质网如何处理毒素以运输到细胞质并诱导先天免疫。我们还开发了斑马鱼进行遗传研究，并通过正向筛选确定了13个具有抗中毒能力的家族。这些家族的突变基因将被定位定位并研究其功能。

DESCRIPTION (provided by applicant): 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.