Intestinal Disease-enterocyte toxin interaction

这项应用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，用于诱导天然免疫。 粘膜表面代表宿主组织与环境仅有一段距离的广阔区域。 精致但高效的单层柱状上皮细胞，由紧密的连接连接在一起 对蛋白质甚至小肽都是不透水的。在这里，我们研究细菌蛋白质是如何跨越这一屏障的 进入宿主肠道细胞的内质网(ER)，然后进入胞浆。为了做到这一点，毒素选择了 鞘磷脂受体(神经节苷脂GM1)及其内源性膜和脂类转运机制 进入急诊室。一旦进入内质网，CT的一个片段，A1链，然后通过劫持 在生物合成途径中，蛋白质质量控制所必需的机械，它感觉并最终 降解内质网管腔中所有末端错误折叠的蛋白质(通过逆转移位到胞浆中)。我们最近 研究发现，肠道细胞感觉A1链进入内质网，以诱导先天免疫反应， 即使当毒素在酶作用下变得惰性时，也表明了一种天然免疫的一般机制。 在这一途径中的信号转导似乎是由典型的内质网应激传感器介导的，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是 肠道细胞的结构和功能，临床上与多种人类疾病有关，除了 毒素性腹泻。 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复合体是如何向 ER和其他目的地(目标1)；分析ER对毒素的处理，并阐明 转运到细胞质及其诱导先天免疫反应的机制(目标2)； 利用无偏正向和反向确定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结构异构体的合成，用于鞘磷脂运输的直接结构-功能研究； 以及新型CT突变体的制备，该突变体旨在分离内质网管腔内的毒素部分或将其捕获 中间反应，以了解内质网如何处理毒素运输到细胞质和 先天免疫的诱导。我们还开发了用于遗传研究的斑马鱼，并鉴定了13种 家庭由前向筛选为耐醉酒。这些家族中的突变基因将通过 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.