Glycolipid Trafficking of Cholera Toxin by Lipid Rafts

本申请的目的是了解肠粘膜上皮细胞如何分类膜 糖脂进入从质膜（PM）到内质网的逆行运输途径 内质网（ER），各种细菌毒素和病毒利用的途径。我们的假设是， 取决于被称为脂筏的膜微区的结构/功能。霍乱毒素 亚洲霍乱的病原体，代表进入宿主的AB 5亚单位毒素的结构和功能 通过在筏相关糖脂受体上逆行这一途径来刺激细胞。分子 这些过程的机制尚未完全理解，甚至尚未完全确定。两个互补 提出了描述脂筏在逆行运输中的作用和生理学的方法 第一种方法将利用T84和A431上皮细胞培养物 复制CT和相关E.大肠杆菌毒素LTIIb 人体肠道与CT不同，LTIIb不能分隔成脂筏，逆行至ER， 而不是诱发人类疾病。我们将调查筏的结构/功能，因为它涉及到的第一步， CT和LTIIb的内吞作用以及开发体外运输测定。这些研究将测试 CT从早期内体运输到高尔基体/ER所需的内源性因子。我们的目标是 发现参与CT和LTIIb分配到不同途径的宿主细胞分子， 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 以斑马鱼为遗传模型，通过CT阐明细胞侵袭的分子基础。我们最近 发现斑马鱼胚胎对CT表现出视觉和生化表型反应 中毒是特定的和完全的人群。这些因素将使 采用无偏的正向遗传筛选，旨在鉴定表现出 由于相关基因的损伤而减弱对CT的反应。来自任何此类突变体的相关基因 将在我们的肠道细胞模型系统中被识别和研究。我们还提出了反向遗传 在斑马鱼中进行的实验将测试特定的基因产物是否参与逆行贩运。 该奖项将为申请人提供一个重要的职业发展时期^上皮细胞 生物学和遗传学，以及资源，以追求一个项目，有很大的潜力过渡到 为候选人提供独立的研究计划。 霍乱毒素是引起亚洲霍乱的病原体，可引起大规模的严重腹泻。粘膜 以霍乱为代表的感染是世界各地5岁以下儿童死亡的主要原因。 世界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 目前还没有人将其归因于这种作用，并可能导致新的治疗和预防方法。

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 ceHmodelsystems. 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^ 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 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.