ANALYSIS OF V CHOLERAE GENES INVOLVED IN COLONIZATION

霍乱弧菌定植相关基因分析

• 批准号: 3455254
• 负责人: Kenneth Milan Peterson
• 金额: $ 9.51万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455254
• 关键词: Vibrio cholerae; antibody formation; bacterial genetics; bacterial proteins; chimeric proteins; gene expression; gene induction /repression; genetic manipulation; genetic strain; genetic transcription; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; laboratory rabbit; microorganism growth; molecular cloning; mutant; synthetic protein; virulence; western blottings

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.

本提案中概述的研究旨在提供更好的 了解霍乱弧菌毒力决定因素 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 包含一组四个协调调节基因（acf 基因座、辅助基因座） 定植因子）涉及定植特性 霍乱弧菌。这四个基因中任何一个的插入突变 在乳鼠中产生数量相同的定植缺陷。 拟议的实验旨在阐明组织， 指定 ACF 表型的基因的表达和功能。 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。该DNA区域将被转座子和删除 突变分析，以便： 定义每个 acf 基因的限制； 定位启动子和控制元件：；并识别额外的基因 参与 ACF 表达。 Acf基因产物将被鉴定， 特征化并定位于特定的细菌区室 针对 PhoA 融合蛋白、纯化的 acf 蛋白或 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 通过体内标记物交换作为潜在疫苗的背景 候选人。将评估成年兔动物模型的 表征（保护性？）宿主对 Acf 的免疫反应的适用性 决定因素。霍乱弧菌 E1 tor 菌株（负责霍乱弧菌的生物型） 当前的大流行）将根据 ACF 进行表征；迄今为止 这些基因仅针对经典菌株进行了表征。 从拟议的调查中获得的知识可以为 开发有效的霍乱疫苗。