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（CT）（亚细亚霍乱的病原体）侵入和中毒肠细胞以及诱导先天性免疫的分子基础。粘液表面代表了宿主组织与环境仅通过精细但高效的单层柱状上皮细胞分离的广阔区域，所述柱状上皮细胞通过蛋白质甚至小肽不可渗透的紧密连接连接。在这里，我们研究了细菌蛋白如何突破这一屏障进入内质网（ER），然后胞质溶胶，宿主肠细胞。为此，毒素选择鞘脂受体（神经节苷脂GM 1）和膜和脂质运输进入ER的内源性机制。一旦在ER中，CT的片段，即A1链，然后通过劫持生物合成途径中蛋白质质量控制所必需的机制进入胞质溶胶，其感测并最终降解（通过逆向易位至胞质溶胶）ER腔中的所有末端错误折叠的蛋白质。我们最近发现，肠细胞的感觉进入ER的A1链诱导先天免疫反应，即使当毒素是酶惰性，这表明先天免疫的一般机制。该通路中的信号转导似乎是由ER应激的典型传感器介导的，其与IBD的发病机制相关。由CT选择进入宿主细胞的生物学是肠细胞结构和功能的基础，并且除了致炎性大肠杆菌之外，与多种人类疾病临床相关。该项目计划继续进行22年的重点研究。我们将使用生物化学，分子，细胞生物学和遗传学方法：解释GM 1鞘脂和CT-GM 1复合物如何运输到ER和其他目的地（目标1）;分析ER对毒素的处理，并阐明运输到胞质溶胶的机制，以及诱导先天性免疫应答（目标2）;并使用无偏正向和反向遗传方法鉴定所有毒素途径中涉及的新分子组分（Aim 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GM 1结构异构体的鞘脂运输的直接结构-功能研究;和制备新的CT突变体，旨在分离ER内腔内的毒素部分或将其捕获在中间反应中，以了解ER如何处理毒素运输到胞质溶胶和诱导先天免疫。我们还开发了用于遗传研究的斑马鱼，并通过正向筛选确定了13个耐中毒家族。这些家族中的突变基因将被定位并研究其功能。

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.