ANALYSIS OF V CHOLERAE GENES INVOLVED IN COLONIZATION

本提案中概述的研究旨在提供一个更好的 了解霍乱弧菌的毒力决定因素， 参与肠道定植。该项目的目标是 对霍乱弧菌染色体的一个区域进行分子分析， 含有一组四个协同调节基因（acf基因座，辅助基因， 定殖因子），它们参与了 诉胆汁。这四个基因中任何一个的插入突变 在乳鼠中产生数量上相同的定植缺陷。 拟议的实验旨在阐明组织， 表达和指定ACF表型的基因的功能。 编码Acf决定簇的四个基因将被分离并进行 DNA序列分析和计算机辅助氨基酸相似性 搜索这一区域的DNA将受到转座子和删除 突变分析，以便：确定每个ACF基因的限度; 定位启动子和控制元件;并鉴定其他基因 参与ACF表达。Acf基因产物将被鉴定， 特征，并定位于特定的细菌隔室， 针对PhoA融合蛋白、纯化的acf蛋白产生的抗血清，或 使用免疫印迹分析、荧光显微镜和 胶体金标记抗体免疫电镜。V型胆固醇 将在ctxA缺失中构建具有确定acf突变的菌株 通过体内标记物交换作为潜在疫苗使用的背景 候选人将评估成年兔动物模型的 表征的适用性（保护性？）宿主对Acf的免疫应答 决定因素E1菌株的霍乱弧菌（负责 目前的大流行）将以ACF为特征;迄今为止 这些基因仅在经典菌株中得到表征。 从拟议的调查中获得的知识可为以下方面提供依据： 研制有效的霍乱疫苗。

The studies outlined in this proposal are aimed at providing a better understanding of Vibrio cholerae virulence determinants which are involved in intestinal colonization. The goal of this project is the molecular analysis of a region of the V. cholerae chromosome which contains a set of four coordinately regulated genes (acf loci, accessory colonization factor) that are involved in the colonization properties of V. cholerae. Insertional mutations in any one of these four genes produces a quantitatively identical colonization defect in suckling mice. The proposed experiments are intended to elucidate the organization, expression, and function of genes specifying the ACF phenotype. The four genes encoding Acf determinants will be isolated and subjected to both DNA sequence analysis and computer aided amino acid similarity search. This region of DNA will be subjected to transposon and deletion mutational analysis in order to: define the limits of each acf gene; locate promoters and controlling elements:; and identify additional genes involved in ACF expression. Acf gene products will be identified, characterized, and localized to specific bacterial compartments with antisera raised against PhoA fusion proteins, purified acf proteins, or synthetic peptides using immunoblot analysis, fluorescence microscopy and colloidal gold labeled antibody immuno-electronmicroscopy. V cholerae strains with defined acf mutations will be constructed in ctxA deletion backgrounds by in vivo marker exchange for use as potential vaccine candidates. An adult rabbit animal model will be assessed for its suitability in characterizing (protective?) host immune responses to Acf determinants. E1 tor strains of V. cholerae (the biotype responsible for the current pandemic) will be characterized with respect to ACF; to date these genes have been characterized only for a classical strain. Knowledge gained from the proposed investigation may provide a basis for the development of efficacious cholera vaccines.