Novel effector protein functions encoded by T3SS positive V. cholerae

项目摘要 霍乱弧菌引起严重的霍乱，是亚洲、非洲和南非洲大部分地区的地方病。 美国，最近被重新引入海地。该物种是高度多样化的，虽然只有O1或 O139血清群菌株引起流行性疾病。然而，据报道， 流行区，由属于非O1/非O139血清群的菌株引起， 健康威胁在发达国家和工业化国家，包括美国。与致病性O1不同 和O139菌株，绝大多数致病性非O1/非O139菌株不携带良好表征的 定殖毒力因子（TCP）和毒素产生（CT），以及 这些菌株还没有被很好地理解。我们对致病性非O1/非O139血清群菌株的研究开始于 对临床分离的O39血清群菌株AM-19226进行基因组测序， 三个分泌系统（T3SS）是其他霍乱弧菌分离株中保守的。我们随后 实验鉴定了通过T3SS装置易位到真核细胞中的13种效应蛋白， 和T3SS基因组岛内编码的两个转录调节因子。我们假设， 效应子和T3SS编码的非效应子蛋白在定殖中起重要作用，而另一个，独特的 诉胆固醇效应物协同破坏宿主细胞信号传导，导致由以下引起的肠道反应： 细胞-细胞连接破坏、离子转运不平衡和/或细胞应激反应。我们建议使用 互补的体外和体内方法，以确定和表征定殖机制， 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 最小的一组T3SS编码的基因必需的殖民和定义他们的作用，坚持主机 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对殖民至关重要， 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所特有的。我们将利用我们的经验，S。酿酒酵母 模型系统，以发现宿主细胞蛋白质是效应活性的目标，并指导我们的研究， 哺乳动物细胞我们还将使用哺乳动物细胞在体外检查宿主细胞对AM-19226感染的应答。 共培养和表达模式。共培养试验将用作剖析效应物如何相互作用的工具。 与哺乳动物的信号转导途径在感染破坏稳态，而基于病毒的 表达系统将用于生物化学鉴定效应蛋白活性的靶点和 哺乳动物细胞的效应表达。我们希望我们的研究结果能够揭示TCP/CT的分子机制 在编码T3SS的非O1/非O139菌株亚群中的独立发病机制。

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.