Novel effector protein functions encoded by T3SS positive V. cholerae

项目摘要 霍乱弧菌导致严重的腹泻疾病霍乱，霍乱在亚洲、非洲和南部的大部分地区流行 美国，最近又被重新引入海地。该物种高度多样化，尽管只有O1或 O139血清群菌株可引起流行病。然而，报告的散发性疾病在#年有所增加。 流行地区，由属于非O1/非O139血清群的菌株引起，这些血清群呈现公众 在发达国家和工业化国家，包括美国，都存在健康威胁。与致病O1不同 和O139菌株，绝大多数致病的非O1/非O139菌株没有很好的携带特性 侵染毒力因子(TCP)和毒素产生(CT)以及使用的毒力机制 这些菌株还没有被很好地理解。我们开始了对致病性非O1/非O139血清群菌株的研究 对临床分离的O39血清群菌株AM-19226进行了基因组测序，揭示了一种类型 三种分泌系统(T3SS)，在其他霍乱弧菌中是保守的。我们的后续行动 实验确定了13种通过T3SS仪器转运到真核细胞中的效应蛋白， 以及编码在T3SS基因组岛上的两个转录调控因子。我们假设有一个子集 效应器和T3SS编码的非效应器蛋白在定植中起着重要作用，而另一种独特的 霍乱弧菌效应器协同作用扰乱宿主细胞信号，导致腹泻反应 细胞-细胞连接中断、离子转运失衡和/或细胞应激反应。我们建议使用 相互补充的体外和体内方法，以确定和表征定植和 霍乱弧菌效应蛋白靶向的宿主细胞蛋白和途径。初步研究将确定 最少一组T3SS编码的定植所必需的基因，并定义它们在对宿主的依附中的作用 细胞。效应蛋白分析将集中在总共五种蛋白质及其在不同阶段的作用 感染/疾病：VopZZ、VopX、VopM、VopF和VopK。VopZZ和VopM对定居至关重要， VOPs F和M具有与细胞骨架重塑相关的功能，这将在我们的研究中进行研究。 VopsX和K是霍乱弧菌T3SS、VopsX和K所独有的。我们将用我们的经验来研究酿酒酵母 发现作为效应器活性目标的宿主细胞蛋白的模型系统，并指导我们的研究 哺乳动物细胞。我们还将使用哺乳动物在体外检测宿主细胞对AM-19226感染的反应 共文化和表达模式。共培养实验将被用作分析效应器如何相互作用的工具。 在感染期间通过哺乳动物的信号转导途径来破坏体内平衡，而基于病毒的 表达系统将被用来生化鉴定效应蛋白活性的靶标和对 哺乳动物细胞向效应器表达。我们期望我们的结果能够揭示TCP/CT的分子机制 编码T3SS的非O1/非O139菌株亚群的独立致病机制。

Project Summary Vibrio cholerae causes the severe diarrheal disease cholera that is endemic in much of Asia, Africa, and South America, and has recently been reintroduced into Haiti. The species is highly diverse, although only O1 or O139 serogroup strains cause epidemic disease. However, increasing sporadic disease has been reported in endemic areas, and is caused by strains belonging to non-O1/non-O139 serogroups that present a public health threat both in developed and industrialized nations, including the United States. Unlike pathogenic O1 and O139 strains, the vast majority of pathogenic non-O1/non-O139 strains do not carry the well characterized virulence factors for colonization (TCP) and toxin production (CT), and the virulence mechanisms used by these strains are not well understood. Our study of pathogenic non-O1/non-O139 serogroup strains began with genomic sequencing of the clinically isolated O39 serogroup strain, AM-19226, which revealed a Type Three Secretion System (T3SS) that is conserved among other V. cholerae isolates. Our subsequent experiments identified 13 effector proteins that are translocated into the eukaryotic cell by the T3SS apparatus, and two transcriptional regulators encoded within the T3SS genomic island. We hypothesize that a subset of effectors and a T3SS encoded non-effector protein play important roles in colonization, and that other, unique V. cholerae effectors cooperate to disrupt host cell signaling to result in the diarrheal response that arises from cell-cell junction disruption, ionic transport imbalance and/or cellular stress responses. We propose to use complimentary in vitro and in vivo approaches to identify and characterize the mechanism of colonization and the host cell proteins and pathways targeted by V. cholerae effector proteins. Initial studies will define the minimum set of T3SS encoded genes necessary for colonization and define their roles in adherence to host cells. Effector protein analysis will focus on a total of five proteins and their roles in different stages of infection/disease: VopZZ, VopX, VopM, VopF, and VopK. VopZZ and VopM are critical for colonization, and Vops F and M have functions associated with cytoskeletal remodeling, which will be investigated in our studies. VopsX and K are unique to the V. cholerae T3SS, VopsX and K. We will use our experience the S. cerevisiae model system to discover host cell proteins that are the targets of effector activity and direct our studies in mammalian cells. We will also examine the host cell response to AM-19226 infection in vitro using mammalian co-culture and expression models. The co-culture assay will be used as a tool to dissect how effectors interact with mammalian signal transduction pathways during infection to disrupt homeostasis, whereas a viral-based expression system will be used to biochemically identify targets of effector protein activity and the response of mammalian cells to effector expression. We expect our results to reveal the molecular mechanisms of TCP/CT independent pathogenesis in the subset of non-O1/non-O139 strains that encode a T3SS.