Intestinal Disease-enterocyte toxin interaction

本申请的目的是阐明肠道细胞入侵和中毒的分子基础 霍乱毒素 (CT)，亚洲霍乱的病原体，用于诱导先天免疫。 粘膜表面代表宿主组织与环境仅通过 精致但高效的单层柱状上皮细胞，通过紧密连接连接在一起 蛋白质甚至小肽都无法渗透。在这里，我们研究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，毒素会选择 鞘脂受体（神经节苷脂 GM1）以及膜和脂质运输的内源机制 进入急诊室。一旦进入内质网，CT 片段（即 A1 链）就会通过劫持 生物合成途径中蛋白质质量控制所必需的机械，它能够感知并最终 降解（通过逆向易位至胞质溶胶）内质网腔中所有末端错误折叠的蛋白质。我们最近 发现肠细胞感知 A1 链进入内质网以诱导先天免疫反应， 即使毒素呈酶惰性，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 应激的经典传感器介导的，这些传感器是 与 IBD 的发病机制有关。 CT 选择进入宿主细胞的生物学特性是 肠细胞的结构和功能，以及除 毒性腹泻。 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：解释 GM1 鞘脂和 CT-GM1 复合物如何运输至 急诊室和其他目的地（目标 1）；分析 ER 对毒素的加工，并阐明 转运至细胞质并诱导先天免疫反应的机制（目标 2）；和 使用无偏正向和反向识别涉及所有毒素途径的新分子成分 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 结构异构体的合成，用于鞘脂运输的直接结构功能研究； 以及新型 CT 突变体的制备，旨在分离 ER 腔内的毒素部分或将其捕获在 中间反应，以了解 ER 如何处理毒素以转运至细胞质并 诱导先天免疫。我们还培育了用于遗传学研究的斑马鱼，并鉴定了 13 家庭通过向前筛选作为抵抗中毒。这些家族中的突变基因将通过以下方式鉴定： 研究了位置映射及其功能。

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.