Vibrio Cholerae Colonization of Chitin Surfaces

描述（由申请人提供）：霍乱弧菌O1是一种引起流行性腹泻的病原体，通常作为河流、河口和海洋生态系统的本地组成部分存在。在这些栖息地中，它与浮游动物的几丁质外骨骼有关。本研究项目的主要目的是表征这种人类病原体与甲壳素表面的相互作用。几丁质是n -乙酰氨基葡萄糖的不溶性聚合物；通过分泌几丁质酶，霍乱弧菌可以利用几丁质作为营养贫乏的水生生境中碳和氮的唯一来源。本项目将探讨霍乱弧菌对甲壳素的利用是一个四步过程：对甲壳素表面的趋化和附着；附着细菌在表面的水平扩散；附着菌作为生物膜群落的垂直生长研究脱离生物膜表面，恢复浮游生物的生长模式。这里提出的工作将通过结合使用基因组、遗传和细胞成像方法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，反射差干涉对比（DIC）和荧光显微镜将用于成像单个细胞，因为它们附着并扩散到附着在层流细胞实验系统上的合成甲壳素膜上。斯坦福生物膜中心将使用扫描共聚焦激光显微镜（SCLM）来捕捉几丁质表面生物膜发育的时空特征。微阵列表达谱分析将用于鉴定基因，其中包括一个假设的分离调控。与约翰霍普金斯大学的Saul Roseman教授合作进行的研究将检查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。绿色荧光蛋白（GFP）启动子融合将用于揭示个体基因在几丁质表面表达的时间和位置，并将寻找在几丁质利用程序中存在缺陷的突变体。在本研究的最后一个具体目标中，这些结果将使用更接近于自然水生生态系统条件的实验系统进行检验，包括它们与游泳桡足类动物定殖的相关性。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。

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.