ANALYSIS OF V CHOLERAE GENES INVOLVED IN COLONIZATION

这项提案中概述的研究旨在提供更好的 霍乱弧菌毒力决定因素的了解 与肠道定植有关。这个项目的目标是 霍乱弧菌染色体上一个区域的分子分析 包含一组四个协调调节的基因(ACF基因座、附属 定植因素)，它们与定植特性有关 霍乱弧菌。这四个基因中任何一个基因的插入突变 在哺乳小鼠身上产生数量上相同的定植缺陷。 拟议的实验旨在阐明这种组织， ACF表型基因的表达和功能。 将分离编码ACF决定子的四个基因并接受 DNA序列分析和计算机辅助氨基酸相似性 搜索。这一DNA区域将受到转座子和缺失的影响 突变分析以：确定每个ACF基因的界限； 定位启动子和控制元件：；并识别其他基因 参与ACF的表达。ACF基因产物将被鉴定， 特征的，并定位到特定的细菌隔间 针对PhoA融合蛋白、纯化的ACF蛋白或 利用免疫印迹分析、荧光显微镜和 胶体金标记抗体免疫电子显微镜。霍乱弧菌 具有明确ACF突变的菌株将在ctxA缺失中构建 背景通过体内标记交换作为潜在的疫苗 候选人。将对成年兔动物模型进行评估 是否适合刻画(保护性？)宿主对ACF的免疫应答 决定因素。E1or霍乱弧菌(霍乱弧菌的生物型 目前的大流行)将以ACF为特征；迄今为止 这些基因只针对一种经典菌株进行了表征。 从拟议的调查中获得的知识可为 有效霍乱疫苗的开发。

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.