Functional characterization of Rho inactivation by Vibro cholerae RTX toxin

描述(申请人提供)：霍乱弧菌是导致危及生命的腹泻疾病霍乱的元凶。2006年，世界卫生组织确认了236,896例霍乱病例和6,311例死亡。霍乱弧菌使用多种毒力因子来定植和感染人类。霍乱弧菌RTX毒素(MARTXVc)虽然不是引起大规模腹泻反应的直接原因，但它参与了发病机制并导致了疾病。不同的哺乳动物细胞类型与MARTXVc+霍乱弧菌菌株孵育会导致细胞变圆，肌动蛋白单体交联化成更高级别的结构，并使Rho GTP酶失活。最近的研究表明，MARTXVc至少由三个功能不同的结构域组成：肌动蛋白交联域(ACD)是交联肌动蛋白的必要条件和充分条件，Rho失活结构域(RID)是足以灭活Rho的结构域，半胱氨酸蛋白酶结构域(CPD)的功能未知。分离出负责灭活Rho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灭活Rho的机制不同于已知的细菌毒素使用的任何机制。RID可能利用一种独特的方法来灭活RHO，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通路。我申请的重点是进一步表征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，它们是功能上的重要部分。我将进行结构/功能分析，以确定定位到HeLa细胞质膜上所需的RID部分。2)确定真核细胞内MARTXVc RID的结合伙伴。我将对中毒的细胞进行免疫共沉淀，从与RID相互作用的HeLa细胞中分离出蛋白质。公共卫生相关性：通过描述RID(和MARTXVc)的作用机制，我们将加强对霍乱弧菌致病策略的理解。这些知识可能有助于生产一种疫苗或抗生素，能够保护受霍乱影响地区的人们。在RID中发现的一个结构域在来自许多人类和动物病原体的大量毒素中被发现，从而增加了我们的发现对其他疾病的适用性。对Rho GTP酶调控组分的进一步鉴定也将有助于真核细胞信号转导的研究。

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.