Intestinal Disease-enterocyte toxin interaction

本申请的目的是阐明肠细胞侵袭和中毒的分子基础， 霍乱毒素（CT），亚细亚霍乱的病原体，并用于诱导先天性免疫。 粘膜表面代表了宿主组织仅通过膜与环境分离的广阔区域。 精细但高效的单层柱状上皮细胞，由紧密连接连接， 蛋白质甚至小肽都无法渗透。在这里，我们研究细菌蛋白质如何突破这一屏障 进入宿主肠细胞的内质网（ER），然后进入胞质溶胶。为了做到这一点，毒素 鞘脂受体（神经节苷脂GM 1）和膜和脂质运输的内源性机制， 进入急诊室。一旦进入内质网，CT的一个片段，A1链，然后通过劫持细胞质进入细胞质。 在生物合成途径中，蛋白质质量控制所必需的机械， 降解（通过逆向易位到胞质溶胶）ER腔中的所有末端错误折叠的蛋白质。我们最近 发现肠细胞感觉到A1链进入ER以诱导先天性免疫应答， 即使当毒素被赋予酶惰性时也是如此，这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应激的典型传感器介导的， 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，临床上与各种人类疾病有关， 产纤维的美洲狮。 该项目计划继续进行22年的重点研究。我们会用生化分子细胞 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（目的1）;分析ER对毒素的加工，并阐明 转运至胞质溶胶和诱导先天性免疫应答的机制（目的2）;以及 使用无偏正向和反向鉴定参与所有毒素途径的新分子组分 遗传学方法（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：合成GM 1结构异构体，用于鞘脂运输的直接结构-功能研究; 以及制备新的CT突变体，其设计用于分离ER腔内的毒素部分或将其捕获在ER腔内， 中间反应，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， 家庭通过向前屏幕抵抗中毒。这些家族中的突变基因将通过 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.