Discovery of novel cholera toxin receptors

描述(申请人提供)：霍乱毒素B亚单位(CTxB)与宿主肠道上皮细胞表面呈现的分子结合。在最初的结合事件之后，全息毒素侵入细胞，劫持细胞机器，导致以霍乱感染为特征的大量腹泻。完全了解霍乱毒性的机制细节对于促进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，但来自我们实验室和其他实验室的最新数据表明，存在额外的霍乱毒素结合伙伴，可能是糖蛋白。这项提议的广泛目标是寻找新的CTxB结合伙伴，并发现它们是否在霍乱中毒中发挥作用。目标1的目标是利用一种新的光交联技术和现代质谱学方法，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。更多的CTxB结合伙伴可以发挥辅助作用，帮助形成规范的CTxB-GM1a复合体，或者它们可能作为替代受体发挥作用，使霍乱毒素能够在缺乏GM1a的情况下结合细胞。目标2的目标是区分这些可能性，使用shRNA瞬时击倒这些替代结合伙伴的表达，并使用糖基化抑制剂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细胞结合是否需要一个或多个新的受体。虽然替代结合伙伴可能促进CTxB结合，但更关键的问题是它们是否在毒素内化中发挥作用。目标3中提出的免疫荧光显微镜实验和氯离子分泌试验的结果将揭示以前未知的CtxB受体是否促进了霍乱中毒的过程。该项目挑战了现有的范式，即GM1a对于CTxB的结合和内化既是必要的，也是充分的。除了影响我们对霍乱疾病进展的理解外，拟议的实验结果将对理解和区分不同的内吞作用机制具有重要意义。此外，由于CTxB通常用于识别和可视化脂筏，对CTxB结合伙伴的完整了解对于确定膜微域的组成和特征将是至关重要的。 与公共卫生相关：霍乱弧菌产生一种毒素，与人类肠道细胞结合并入侵，导致以霍乱感染为特征的大量腹泻。长期以来，霍乱毒素一直被认为与一种名为GM1a的分子相互作用，但我们的数据表明，这种毒素也可能与另一种或多种分子相互作用。我们建议确定霍乱毒素与之相互作用的其他分子，并确定这些相互作用是否对疾病进展至关重要。

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.