Novel effector protein functions encoded by T3SS positive V. cholerae

T3SS 阳性霍乱弧菌编码的新效应蛋白功能

• 批准号: 10199931
• 负责人: MICHELLE DZIEJMAN
• 金额: $ 43.72万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-14 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10199931
• 关键词: Actins; Address; Adherence; Africa; Animal Model; Architecture; Area; Asia; Attenuated; Binding; Biochemical; Biological; Biological Assay; Biological Models; Bioterrorism; Caco-2 Cells; Categories; Cell Communication; Cell Death; Cell physiology; Cells; Cellular Stress Response; Cellular Structures; Cellular biology; Centers for Disease Control and Prevention (U.S.); Cholera; Cholera Toxin; Clinical; Coculture Techniques; Country; Data; Defect; Developed Countries; Developing Countries; Diarrhea; Disease; Endemic Diseases; Epidemic; Eukaryotic Cell; Event; Fluids and Secretions; Food Contamination; Genes; Genetic; Genetic Transcription; Genomic Islands; Genomics; Goals; Growth; Haiti; Health; Homeostasis; In Vitro; Individual; Infection; Infrastructure; Intercellular Junctions; Intestines; Knowledge; Laboratories; Mammalian Cell; Mediating; Modeling; Modernization; Molecular; Molecular Target; Mus; Names; Natural Disasters; Oryctolagus cuniculus; Pathogenesis; Pathogenicity; Pathogenicity Island; Pathway interactions; Permeability; Pilum; Play; Population; Production; Protein Analysis; Proteins; Public Health; Refugee Camp; Reporting; Research; Role; Saccharomyces cerevisiae; Sanitation; Signal Pathway; Signal Transduction; Signal Transduction Pathway; South America; Structure; Structure-Activity Relationship; Study models; System; Tertiary Protein Structure; Testing; Tissues; Toxin; Travel; United States; Vaccines; Vibrio cholerae; Vibrio cholerae O1; Viral; Virulence; Virulence Factors; War; Work; World Health Organization; Yeasts; base; clinical sequencing; contaminated water; diarrheal disease; experience; experimental study; gene product; genetic approach; gut colonization; in vivo; novel; protein function; response; tool; yeast genetics

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.

项目摘要 弧菌霍乱引起严重的腹泻病霍乱，在亚洲，非洲和南部的大部分地区是地方性的 美国，最近被重新引入了海地。该物种高度多样，尽管只有O1或 O139血清群菌株引起流行病。然而，据报道，偶发性疾病的增加 流行区域，是由属于非O1/非O139血清群的菌株引起的 包括美国在内的发达国家和工业化国家中的健康威胁。与致病性O1不同 和O139菌株，绝大多数致病性非O1/non-O139菌株都不具有良好的特征 定植（TCP）和毒素产生（CT）的毒力因子以及使用的毒力机制 这些菌株尚不清楚。我们对致病性非O1/非O139血清群菌株的研究开始了 随着临床分离的O39血清群菌株的基因组测序，AM-19226，揭示了一种类型 在其他V.霍乱分离株中保守的三个分泌系统（T3S）。我们随后的 实验确定了13种效应子蛋白，这些效应蛋白被T3SS设备易位到真核细胞中， 以及在T3SS基因组岛内编码的两个转录调节器。我们假设一个子集 效应子和T3SS编码的非效应蛋白在定植中起重要作用，而其他独特的 V.霍乱效应子合作以破坏宿主细胞信号传导，从而导致腹泻反应。 细胞 - 细胞连接破坏，离子转运失衡和/或细胞应激反应。我们建议使用 在体外和体内互补的方法，以识别和表征定植的机制和 霍乱梭菌效应蛋白靶向的宿主细胞蛋白和途径。初步研究将定义 最低限度的T3SS编码基因，用于定殖并定义其在遵守中的作用 细胞。效应子蛋白分析将集中于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对于殖民化至关重要，并且 VOPS F和M具有与细胞骨架重塑有关的功能，这将在我们的研究中进行研究。 VOPSX和K是V.霍乱T3SS，VOPSX和K。我们将利用我们的经验S. cerevisiae 模型系统发现宿主细胞蛋白是效应活动的靶标，并将我们的研究指导 哺乳动物细胞。我们还将使用哺乳动物在体外检查宿主细胞对AM-19226感染的反应 共培养和表达模型。共培养测定将用作剖析效应子相互作用的工具 感染期间哺乳动物信号转导途径破坏稳态，而基于病毒的稳态 表达系统将用于生化识别效应蛋白活性的靶标和 哺乳动物细胞具有效应子表达。我们希望我们的结果揭示了TCP/CT的分子机制 编码T3SS的非O1/非O139菌株子集中的独立发病机理。