Glycolipid Trafficking of Cholera Toxin by Lipid Rafts

这项应用的目标是了解肠粘膜上皮细胞如何对膜进行分类 糖脂进入从质膜到内质的逆行运输途径 网状(ER)，一种被各种细菌毒素和病毒利用的途径。我们的假设是，这种排序 取决于膜微域的结构/功能，称为脂筏。霍乱毒素(CT)， 亚洲霍乱的病原体，代表进入宿主的AB5亚单位毒素的结构和功能 细胞通过RAFT相关的糖脂受体在这一途径上逆行。分子 这些过程的机制还没有完全了解，甚至还没有完全确定。两个互补性 提出了描述脂筏在逆行运输中的作用和生理学的方法。 毒素侵袭途径：第一种途径是利用T84和A431上皮细胞培养 复制CT和相关的大肠杆菌毒素LTIIb在 人的肠子。与CT不同的是，LTIIb不能分割成脂筏，逆行到内质网，并且 不会在人类身上引发疾病。我们将研究筏子结构/功能，因为它与以下第一步有关 CT和LTIIb的内吞作用以及体外转运试验的建立。这些研究将检验 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 以斑马鱼为遗传模型，用CT阐明细胞侵袭的分子基础。我们最近做了 发现斑马鱼胚胎对CT的反应表现出视觉和生化表型 在人群中既特定又完全的陶醉。这些因素将允许 使用无偏正向遗传筛查的目的是识别表现出 由于相关基因的损伤，对CT的反应减弱。来自任何此类突变体的相关基因(S) 将在我们的肠道CEH模型系统中被识别和研究。我们还提出了反向遗传 在斑马鱼身上进行实验，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职业发展的关键时期？上皮细胞 生物学和遗传学，以及进行一个有很高潜力过渡到 候选人的独立研究计划。 霍乱毒素是亚洲霍乱的病原体，会导致大量的严重腹泻。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。这项拟议的研究将发现与霍乱发病机制有关的新宿主细胞因子。 还没有被归因于这种作用，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.