Discovery of novel cholera toxin receptors

描述（由申请人提供）：霍乱毒素亚基B （CTxB）与宿主肠上皮细胞表面的分子结合。在最初的结合事件之后，全毒侵入细胞，劫持细胞机制，导致大量腹泻，这是霍乱感染的特征。全面了解霍乱毒性的机制细节对于制定抗击霍乱暴发的新战略是必要的。虽然神经节苷脂GM1a被广泛描述为CTxB识别的单一受体，但我们实验室和其他实验室的最新数据表明存在其他霍乱毒素结合伙伴，可能是糖蛋白。本提案的总体目标是确定新的CTxB结合伙伴，并发现它们是否在霍乱中毒中起作用。Aim 1的目标是利用新型光交联技术和现代质谱方法，以及传统的亲和纯化方法，鉴定T84细胞中被CTxB识别的细胞表面分子。额外的CTxB结合伙伴可以发挥辅助作用，协助形成典型的CTxB-GM1a复合物，或者它们可能作为替代受体，使霍乱毒素在缺乏GM1a的情况下结合细胞。Aim 2的目标是区分这些可能性，通过使用shrna来短暂地敲低这些替代结合伙伴的表达和糖基化抑制剂来干扰它们的翻译后修饰。这些实验的结果将使我们能够确定CTxB与T84细胞结合是否需要新的受体或受体。虽然其他结合伙伴可能促进CTxB的结合，但一个更关键的问题是它们是否在毒素的内化中起作用。Aim 3中提出的免疫荧光显微镜实验和氯离子分泌测定的结果将揭示以前未确定的CtxB受体是否促进霍乱中毒过程。该项目挑战了现有的范式，即GM1a是CTxB绑定和内部化的必要和充分条件。随着我们对霍乱疾病进展的理解的影响，所提出的实验结果将对理解和区分不同的内吞作用机制非常重要。此外，由于CTxB通常用于识别和可视化脂筏，因此CTxB结合伙伴的完整知识对于定义膜微结构域的组成和特征至关重要。

DESCRIPTION (provided by applicant): Cholera toxin subunit B (CTxB) binds to molecules presented on the surface of host intestinal epithelial cells. After the initial binding event, the holotoxin invades the cells, hijacking the cellular machinery to cause the profuse diarrhea that characterizes cholera infection. A complete understanding of the mechanistic details of cholera toxicity is necessary to facilitate the development of novel strategies to combat cholera outbreaks. While the ganglioside GM1a is widely described as the single receptor recognized by CTxB, recent data from our lab and others implies the existence of additional cholera toxin binding partners, which may be glycoproteins. The broad goals of this proposal are to identify novel CTxB binding partners and discover whether they function in cholera intoxication. The goal of Aim 1 is to identify cell surface molecules in T84 cells that are recognized by CTxB, making use of a novel photocrosslinking technique and modern mass spectrometry methods, along with traditional affinity purification approaches. Additional CTxB binding partners could play an auxiliary role, assisting in formation of the canonical CTxB-GM1a complex, or they might function as alternative receptors, enabling cholera toxin to bind cells in the absence of GM1a. The goal of Aim 2 is to distinguish between these possibilities, by using shRNAs to transiently knockdown expression of these alternative binding partners and glycosylation inhibitors to interfere with their post- translational modification. The results of these experiments will allow us to determine if the novel receptor or receptors are required for CTxB binding to T84 cells. While alternative binding partners might facilitate CTxB binding, a more critical question is whether they function in internalization of the toxin. The results of the immunofluorescence microscopy experiments and chloride ion secretion assays, proposed in Aim 3, will reveal whether previously unidentified CtxB receptors facilitate the process of cholera intoxication. This project challenges the existing paradigm that GM1a is both necessary and sufficient for CTxB binding and internalization. Along with the impact on our understanding of cholera disease progression, the results of the proposed experiments will be important for understanding and distinguishing among different mechanisms of endocytosis. Furthermore, because CTxB is commonly used to identify and visualize lipid rafts, a complete knowledge of CTxB binding partners will be vital to defining the composition and characteristics of membrane microdomains. PUBLIC HEALTH RELEVANCE: The bacteria Vibrio cholerae produce a toxin that binds to and invades human intestinal cells, causing the profuse diarrhea that characterizes cholera infection. The cholera toxin has long been believed to interact with a molecule known as GM1a, but our data suggest that the toxin may also interact with an additional molecule or molecules. We propose to identify the additional molecules with which cholera toxin interacts and to determine whether these interactions are critical to disease progression.