Vibrio Cholerae Colonization of Chitin Surfaces

霍乱弧菌在甲壳质表面的定植

• 批准号: 6804055
• 负责人: Gary K Schoolnik
• 金额: $ 36万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6804055
• 关键词: N acetylglucosamine; Vibrio cholerae; aquatic organism; biofilm; chemotaxis; chitin; cholera; confocal scanning microscopy; diarrhea; fluorescence microscopy; genetic regulatory element; green fluorescent proteins; histidine; infection; invertebrate cuticle; microarray technology; oligosaccharides

DESCRIPTION (provided by applicant): Vibrio cholerae O1, a cause of epidemic diarrheal disease, normally resides as an indigenous component of riverine, estuarine and marine ecosystems. In these habitats, it associates with the chitinous exoskeletons of zooplankton. The principal objective of this research project is to characterize the interaction of this human pathogen with a chitin surface. Chitin is an insoluble polymer of N-acetylglucosamine; through the secretion of chitinases, V. cholerae can use chitin as a sole source of carbon and nitrogen in nutrient-poor aquatic habitats. This project will explore the hypothesis that chitin utilization by V. cholerae is a four step process: chemotaxis toward and attachment to the chitin surface; horizontal dispersal of attached bacteria across the surface; vertical growth of the attached population as a biofilm community; and detachment from the biofilm surface and resumption of the planktonic mode-of-growth. The work proposed here will explore each of these hypothesized steps through the combined use of genomic, genetic and cell imaging methods. In collaboration with Professor Gill Geesey at Montana State University, reflected differential interference contrast (DIC) and epifluorescence microscopy will be used to image individual cells as they attach to and spread across a synthetic chitin membrane attached to a laminar flow cell experimental system. Scanning confocal laser microscopy (SCLM) will be employed at the Stanford Biofilm Center to capture the temporal and spatial features of biofilm development on the chitin surface. Microarray expression profiling will be used to identify genes, which comprise a hypothesized detachment regulon. Studies undertaken in collaboration with Professor Saul Roseman at Johns Hopkins University will examine the role of a newly identified chitin sensor histidine kinase protein in each of the four chitin utilization steps. Green fluorescent protein (GFP) promoter fusions will be used to disclose when and where individual genes are expressed on the chitin surface and mutants will be sought that are defective in the chitin utilization program. In the last Specific Aim of the proposed work, these results will be examined using experimental systems that more closely resemble conditions in natural aquatic ecosystems including their relevance for the colonization of swimming copepods. If successful, this project should generate new information about the persistence and control of infectious agents in aquatic reservoirs.

描述（由申请人提供）：霍乱弧菌O 1，一种流行性腹泻病的病因，通常作为河流、河口和海洋生态系统的固有成分存在。在这些栖息地，它与浮游动物的几丁质外骨骼。本研究项目的主要目的是表征这种人类病原体与几丁质表面的相互作用。几丁质是N-乙酰葡糖胺的不溶性聚合物;通过分泌几丁质酶，霍乱弧菌可以在营养贫乏的水生栖息地中将几丁质作为唯一的碳和氮来源。本项目将探讨以下假设：霍乱弧菌利用几丁质是一个四步过程：趋化性和附着到几丁质表面;附着细菌在表面的水平扩散;附着种群作为生物膜群落的垂直生长;从生物膜表面脱离并恢复非稳态生长模式。这里提出的工作将通过基因组，遗传和细胞成像方法的组合使用来探索这些假设的步骤。与蒙大拿州立大学的Gill Geesey教授合作，将使用反射微分干涉对比（DIC）和落射荧光显微镜对单个细胞进行成像，因为它们附着在连接到层流细胞实验系统的合成几丁质膜上并在其上扩散。扫描共聚焦激光显微镜（SCLM）将在斯坦福大学生物膜中心捕获的时间和空间特征的甲壳素表面上的生物膜发展。微阵列表达谱将用于鉴定基因，其包含假设的分离调节子。与约翰霍普金斯大学的扫罗罗斯曼教授合作进行的研究将研究一种新发现的几丁质传感器组氨酸激酶蛋白在四个几丁质利用步骤中的作用。将使用绿色荧光蛋白（GFP）启动子融合体来揭示单个基因在几丁质表面上表达的时间和位置，并寻找在几丁质利用程序中有缺陷的突变体。在拟议工作的最后一个具体目标中，将使用更接近自然水生生态系统条件的实验系统来检查这些结果，包括它们与游泳桡足类定殖的相关性。如果成功，该项目将产生关于水生水库中传染性病原体的持久性和控制的新信息。