Glycolipid Trafficking of Cholera Toxin by Lipid Rafts

描述（由申请人提供）： 本申请的目的是了解肠粘膜上皮细胞如何将膜糖脂分类到从质膜（PM）到内质网（ER）的逆行运输途径中，这是各种细菌毒素和病毒利用的途径。我们的假设是，这种排序取决于膜微区的结构/功能称为脂筏。霍乱毒素（CT），亚细亚霍乱的病原体，典型的结构和功能的AB 5-亚基毒素，进入宿主细胞的筏相关的糖脂受体上逆行在这条途径。这些过程的分子机制尚未完全理解，甚至尚未完全确定。提出了两种互补的方法来描述脂筏在毒素入侵的逆行运输途径中的作用和生理学：第一种方法将利用T84和A431上皮细胞培养系统，再现CT和相关E.大肠杆菌毒素LTIIb。与CT不同，LTIIb不能分配到脂筏中，逆行到ER，并且不会在人类中诱发疾病。我们将研究筏结构/功能，因为它涉及到CT和LTIIb的内吞作用的第一步，以及开发体外运输试验。这些研究将检测CT从早期内体转运至高尔基体/ER所需的内源性因子。我们的目标是发现参与CT和LTIIb到不同途径的分区的宿主细胞分子，从而解释真核细胞如何利用脂筏进行亚细胞组织。第二种方法将使用斑马鱼作为遗传模型来阐明CT细胞侵袭的分子基础。我们最近发现，斑马鱼胚胎表现出视觉和生化表型响应CT中毒，这是特定的和完整的人口。这些因素将允许采用无偏倚的正向遗传筛选，旨在鉴定由于相关基因中的病变而表现出对CT的减弱应答的突变体。将在我们的肠细胞模型系统中鉴定和研究来自任何此类突变体的相关基因。我们还建议在斑马鱼中进行反向遗传实验，以测试参与逆行贩运的特定基因产物。 该奖项将为申请人提供上皮细胞生物学和遗传学职业发展的重要时期，以及追求具有很高潜力的项目的资源，该项目将过渡到候选人的独立研究计划。 霍乱毒素是亚洲霍乱的病原体，导致大规模的严重腹泻。以霍乱为代表的粘液菌感染是全世界5岁以下儿童死亡的主要原因。这项拟议中的研究将发现新的宿主细胞因子参与霍乱的发病机制尚未归因于这样的作用，并可能导致新的治疗和预防方法

DESCRIPTION (provided by applicant): The goal of this application is to understand how epithelial cells of the intestinal mucosa sort membrane glycolipids into the retrograde trafficking pathway from the plasma membrane (PM) to the endoplasmic reticulum (ER), a route exploited by various bacterial toxins and viruses. Our hypothesis is that such sorting depends on the structure/function of membrane microdomains termed lipid rafts. Cholera toxin (CT), the causative agent of Asiatic cholera, typifies the structure and function of the AB5-subunit toxins that enter host cells by traveling retrograde in this pathway on raft-associated glycolipid receptors. The molecular mechanisms of these processes are not fully understood or even completely identified. Two complementary approaches are proposed to delineate the role and physiology of lipid rafts in the retrograde trafficking pathway with respect to toxin invasion: The first approach will utilize T84 and A431 epithelial cell culture systems that reproduce the differential trafficking/sorting of CT and the related E. coli toxin LTIIb in the human intestine. Unlike CT, LTIIb is unable to partition into lipid rafts, travel retrograde to the ER, and does not induce disease in humans. We will investigate raft structure/function as it pertains to the first steps of endocytosis of CT and LTIIb as well as develop in vitro trafficking assays. These studies will test endogenous factors required for the transport of CT from early endosomes to the Golgi/ER. We aim to discover host cell molecules involved in the partitioning of CT and LTIIb into divergent pathways, thus explaining how eukaryotic cells utilize lipid rafts for subcellular organization. The second approach will use the zebrafish as a genetic model to elucidate the molecular basis for cell invasion by CT. We have recently found that zebrafish embryos demonstrate a visual and biochemical phenotype in response to CT intoxication that is both specific and complete within the population. These factors will allow for the employment of an unbiased forward genetic screen aimed at identifying mutants that demonstrate an attenuated response to CT due to a lesion in an involved gene. The relevant gene(s) from any such mutants will be identified and studied in our intestinal cell model systems. We also propose reverse genetic experiments in the zebrafish that will test specific gene products for involvement in retrograde trafficking. This, award will provide the applicant with an essential period of career development in epithelial cell biology and genetics as well as the resources to pursue a project that has high potential for transitioning into an independent research program for the candidate. Cholera toxin is the causative agent of Asiatic cholera and results in massive seretory [sic] diarrhea. Mucosal infections, typified by cholera, are the leading cause of death in children less than 5 years old around the world. This proposed research will discover novel host cell factors involved in the pathogenesis of cholera not yet ascribed to such a role, and may lead to new approaches for treatment and prevention