Vibrio Cholerae Colonization of Chitin Surfaces

说明(申请人提供)：O1霍乱弧菌是引起流行性腹泻的原因之一，通常存在于河流、河口和海洋生态系统中。在这些栖息地，它与浮游动物的几丁质外骨骼联系在一起。这项研究项目的主要目的是描述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；霍乱弧菌通过分泌几丁质酶，可以在营养不良的水生生境中将几丁质作为碳和氮的唯一来源。本项目将探索霍乱弧菌利用甲壳素是一个四步过程：对甲壳素表面的趋化和附着；附着细菌在表面的水平扩散；附着的种群作为生物膜群落的垂直生长；从生物膜表面脱离和恢复浮游生长模式。这里提出的工作将通过组合使用基因组、遗传和细胞成像方法来探索这些假设步骤中的每一个。与蒙大拿州立大学的Gill Geesey教授合作，反射微分干涉对比(DIC)和荧光显微镜将被用来对单个细胞附着在层流细胞实验系统上的合成几丁质膜并在其上扩散时进行成像。斯坦福生物膜中心将使用扫描共聚焦激光显微镜(SCLM)来捕捉甲壳素表面生物膜发育的时间和空间特征。微阵列表达谱将被用来识别基因，这些基因包括一个假想的脱离调节子。与约翰·霍普金斯大学的索尔·罗斯曼教授合作进行的研究将检验新发现的甲壳素传感器组氨酸蛋白在甲壳素利用的四个步骤中的每一个步骤中的作用。绿色荧光蛋白(GFP)启动子的融合将用于揭示个别基因在甲壳素表面表达的时间和地点，并将寻找在甲壳素利用计划中存在缺陷的突变体。在拟议工作的最后一个具体目标中，将使用与自然水生生态系统中的条件更相似的实验系统来检验这些结果，包括它们与游泳的桡足类动物定殖化的相关性。如果成功，这个项目应该会产生关于水生水库中感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.