Intestinal Disease-enterocyte toxin interaction

描述(申请人提供)：本申请的目的是阐明亚洲霍乱病原体霍乱毒素(CT)入侵和中毒肠道细胞的分子基础，以及诱导天然免疫的分子基础。粘膜表面代表着广阔的区域，宿主组织与环境之间只有一层微妙但高效的柱状上皮细胞，由不能渗透到蛋白质甚至小肽的紧密连接连接在一起。在这里，我们研究细菌蛋白质如何越过这一屏障进入宿主肠道细胞的内质网(ER)，然后进入胞浆。为此，毒素利用鞘糖脂受体(神经节苷脂GM1)和内源性膜机制和脂类转运机制进入内质网。一旦进入内质网，CT的一段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制进入胞浆，生物合成途径感知并最终降解内质网管腔中所有末端错误折叠的蛋白质(通过逆转位到胞浆中)。我们最近发现，即使毒素在酶作用下处于惰性状态，肠道细胞也能感觉到A1链进入内质网，从而诱导天然免疫反应，这表明了天然免疫的一般机制。这一途径中的信号转导似乎是由内质网应激的典型感受器介导的，这些感受器与IBD的发病机制有关。CT选择的进入宿主细胞的生物学是肠道细胞结构和功能的基础，在临床上与除毒素性腹泻外的各种人类疾病有关。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复合体如何向内质网和其他目的地运输(目标1)；分析内质网对毒素的处理，并阐明转运到细胞质和其诱导先天性免疫反应的机制(目标2)；以及使用无偏见的正向和反向遗传方法确定参与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，用于直接研究鞘磷脂运输的结构和功能；以及制备新型CT突变体，旨在分离内质网管腔内的毒素片段或将其捕获到中间反应中，以了解内质网如何处理毒素，将其运输到胞浆和诱导先天性免疫。我们还开发了用于遗传研究的斑马鱼，并通过前向筛选鉴定出13个耐醉酒的家族。这些家族中的突变基因将通过定位图谱进行鉴定，并对其功能进行研究。 公共卫生相关性：这项应用的目标是了解细菌蛋白如何突破肠道屏障，导致腹泻疾病，并诱导先天免疫反应。该途径模拟了一些正常的肠道微生物在健康和疾病中如何与宿主相互作用。该主题还涉及用于疾病治疗的药物、疫苗和免疫调节剂的粘膜递送。

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. PUBLIC HEALTH RELEVANCE: The goal of this application is to understand how a bacterial protein can breech the intestinal barrier to cause diarrheal disease and to induce an innate immune response. The pathway models how some normal gut microbes might interact with the host in both health and disease. The topic is also relevant to mucosal delivery of drugs, vaccines, and immunomodulators for disease treatment.