Glycolipid Trafficking of Cholera Toxin by Lipid Rafts

描述(由申请人提供)： 这项应用的目的是了解肠粘膜上皮细胞如何将膜糖脂分类进入从质膜(PM)到内质网(ER)的逆行运输途径，这是一条被各种细菌毒素和病毒利用的途径。我们的假设是，这种分类取决于被称为脂筏的膜微域的结构/功能。霍乱毒素是亚洲霍乱的病原体，其典型的结构和功能是AB5亚单位毒素通过RAFT相关的糖脂受体逆行进入宿主细胞。这些过程的分子机制还没有完全了解，甚至还没有完全确定。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：第一种方法将利用T84和A431上皮细胞培养系统来再现CT和相关的大肠杆菌毒素LTIIb在人体肠道中的差异运输/分选。与CT不同，LTIIb不能分割成脂筏，逆行到ER，并且不会在人类中诱发疾病。我们将研究RAFT的结构/功能，因为它与CT和LTIIb的内吞作用的第一步有关，并建立了体外转运试验。这些研究将测试CT从早期内体转运到高尔基体/内质网所需的内源性因素。我们的目标是发现参与CT和LTIIb分裂成不同途径的宿主细胞分子，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，以阐明CT入侵细胞的分子基础。我们最近发现，斑马鱼胚胎对CT中毒的反应表现出视觉和生化表型，这种表型在种群中是特异的和完整的。这些因素将允许采用无偏见的正向遗传筛查，目的是识别由于相关基因的损伤而对CT反应减弱的突变。任何此类突变体的相关基因(S)都将在我们的肠道细胞模型系统中进行鉴定和研究。我们还建议在斑马鱼中进行反向基因实验，以测试参与逆行贩运的特定基因产品。 这一奖项将为申请者提供上皮细胞生物学和遗传学职业发展的关键时期，以及追求一个具有很高潜力转变为候选人独立研究计划的项目的资源。 霍乱毒素是亚洲霍乱的病原体，会导致大量的严重腹泻。以霍乱为代表的粘膜感染是世界各地5岁以下儿童的主要死亡原因。这项拟议的研究将发现新的宿主细胞因子，涉及霍乱的发病机制，但尚未归因于这种作用，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