Functional characterization of Rho inactivation by Vibro cholerae RTX toxin

霍乱弧菌 RTX 毒素导致 Rho 失活的功能特征

• 批准号: 7950483
• 负责人: Brett L Geissler
• 金额: $ 5.38万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7950483
• 关键词: Actins; Animals; Antibiotics; Bacterial Toxins; Binding; Cell Line; Cell membrane; Cells; Cessation of life; Cholera; Cholera Toxin; Co-Immunoprecipitations; Cysteine Protease; Developing Countries; Disease; Eukaryotic Cell; Family; Gram-Negative Bacteria; Hela Cells; Higher Order Chromatin Structure; Human; Intoxication; Knowledge; Laboratories; Lead; Life; Mammalian Cell; Membrane; Modeling; Pathogenesis; Play; Precipitation; Production; Protease Domain; Proteins; Regulation; Research; Role; Sanitation; Signal Pathway; Signal Transduction; Structure; Toxin; Vaccines; Vibrio cholerae; Virulence; Virulence Factors; World Health Organization; cell type; crosslink; genetic regulatory protein; graduate student; member; monomer; pathogen; public health relevance; response; rho; rho GTP-Binding Proteins; toxin V

DESCRIPTION (provided by applicant): Vibrio cholerae is responsible for causing the life-threatening diarrheal disease cholera. The World Health Organization confirmed 236,896 cases and 6,311 deaths from cholera in 2006. V. cholerae uses a multitude of virulence factors to colonize and infect humans. Although not directly responsible for eliciting the massive diarrheal response, the V. cholerae RTX toxin (MARTXVc) is involved in pathogenesis and contributes to disease. Incubation of various mammalian cell types with MARTXVc+ strains of V. cholerae causes cell rounding, cross-linking of actin monomers into higher order structures, and inactivation of the Rho GTPases. Recent evidence shows that MARTXVc is composed of at least three functionally distinct domains: the actin cross-linking domain (ACD) that is necessary and sufficient for cross-linking actin, the Rho-inactivation domain (RID) that is sufficient for inactivating Rho, and the cysteine protease domain (CPD) whose function is unknown. The isolation of the MARTXVc domain responsible for inactivating Rho presents an exciting research opportunity. Preliminary evidence gathered by a graduate student in our laboratory indicates that the mechanism for Rho-inactivation by RID is unlike any used by known bacterial toxins. That RID may utilize a unique approach to inactivate Rho further emphasizes the importance of characterizing its mechanism of action because it may help to elucidate host-cell signaling pathways. The focus of my application is to further characterize the mechanism of Rho inactivation by RID. My two specific aims are to: 1) Isolate regions and residues of the MARTXVc RID membrane localization domain, which are important for function. I will carry out a structure/function analysis to establish the portions of RID that are required for localization to the plasma membrane of HeLa cells. 2) Identify binding partners for MARTXVc RID within eukaryotic cells. I will perform co-immunoprecipitations on intoxicated cells to isolate proteins from HeLa cells that interact with RID. PUBLIC HEALTH RELEVANCE: By characterizing the mechanism of action of RID (and MARTXVc), we will enhance our understanding of the strategies V. cholerae utilizes to cause disease. This knowledge may assist in the production of a vaccine or antibiotic that is able to protect those in afflicted regions from cholera. A domain found within RID is found in numerous toxins from many human and animal pathogens, thereby increasing the applicability of our findings to other diseases. The further characterization of components involved in the regulation of Rho GTPases from the proposed research will also benefit eukaryotic cell signaling research.

描述（由申请人提供）：霍乱弧菌是导致危及生命的腹泻病霍乱的原因。世界卫生组织在 2006 年确认了 236,896 例霍乱病例和 6,311 人死亡。霍乱弧菌利用多种毒力因子来定殖并感染人类。虽然霍乱弧菌 RTX 毒素 (MARTXVc) 不直接引起大规模腹泻反应，但它参与了发病机制并导致疾病。将各种哺乳动物细胞类型与霍乱弧菌 MARTXVc+ 菌株一起孵育会导致细胞变圆、肌动蛋白单体交联成更高级的结构以及 Rho GTP 酶失活。最近的证据表明，MARTXVc 由至少三个功能不同的结构域组成：肌动蛋白交联结构域 (ACD)，对于交联肌动蛋白来说是必要且充分的；Rho 失活结构域 (RID)，足以使 Rho 失活；以及功能未知的半胱氨酸蛋白酶结构域 (CPD)。分离负责使 Rho 失活的 MARTXVc 结构域提供了一个令人兴奋的研究机会。我们实验室的一名研究生收集的初步证据表明，RID 导致 Rho 失活的机制与已知细菌毒素所使用的机制不同。 RID 可能利用独特的方法来灭活 Rho，这进一步强调了表征其作用机制的重要性，因为它可能有助于阐明宿主细胞信号传导途径。我的应用重点是进一步表征 RID 灭活 Rho 的机制。我的两个具体目标是：1) 分离 MARTXVc RID 膜定位结构域的区域和残基，这对于功能很重要。我将进行结构/功能分析，以确定定位到 HeLa 细胞质膜所需的 RID 部分。 2) 鉴定真核细胞内 MARTXVc RID 的结合伴侣。我将对中毒细胞进行免疫共沉淀，以从 HeLa 细胞中分离出与 RID 相互作用的蛋白质。公共卫生相关性：通过表征 RID（和 MARTXVc）的作用机制，我们将加深对霍乱弧菌引起疾病的策略的理解。这些知识可能有助于生产能够保护受影响地区的人们免受霍乱侵害的疫苗或抗生素。 RID 中的一个结构域存在于许多人类和动物病原体的多种毒素中，从而增加了我们的发现对其他疾病的适用性。拟议研究中涉及 Rho GTPases 调节的成分的进一步表征也将有​​利于真核细胞信号传导研究。