M TUBERCULOSIS SURVIVAL IN MACROPHAGES

结核分枝杆菌在巨噬细胞中的存活

• 批准号: 2880995
• 负责人: Richard Lee Friedman
• 金额: $ 25.46万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2880995
• 关键词: Mycobacterium smegmatis; Mycobacterium tuberculosis; bacteria infection mechanism; cell death; gene expression; genetic library; genetic mapping; macrophage; molecular cloning; nucleic acid sequence; plasmids; reporter genes; virulence

DESCRIPTION (Adapted from the Applicant's Abstract): The infection of macrophages by Mycobacterium tuberculosis can be divided into four steps: adherence, entry, intracellular survival, and multiplication. The aim of the proposed research is to identify, clone, and characterize genes of M. tuberculosis which are required for intracellular survival within macrophages. Potential virulence factor genes of M. tuberculosis will be cloned by first constructing a recombinant library and incorporating it into the nonpathogenic Mycobacterium smegmatis, a rapidly-growing mycobacterium that is otherwise killed by macrophages. Clones with enhanced survival in macrophages will be identified and examined for the presence of M. tuberculosis genes involved in survival. In initial experiments, the applicant has demonstrated the feasibility of the proposed studies by isolating a plasmid clone from an M. tuberculosis library that reproducibly confers enhanced survival in U937 cells, and has isolated several cosmid clones, using the same strategy. The specific aims are: 1) To screen M. smegmatis clones containing an M. tuberculosis H37Rv recombinant library (either a plasmid or cosmid library) for survival in monolayers of the human macrophage-like cell line, U937. 2) To isolate M. smegmatis clones that survive within the U937 macrophage monolayers for further study. Plasmids or cosmids from these clones will be recovered and the M. tuberculosis genes contained will be restriction-mapped, sequenced, and compared to the published genome sequence of M. tuberculosis H37Rv as well as to other databases, and analyzed with other molecular techniques to characterize the cloned genes. 3) Genes identified and characterized in Specific Aim 2 which enhance M. smegmatis survival within macrophages will be selected and used to evaluate the role of these genes in virulent M. tuberculosis. Mutations will be constructed in these genes and introduced into the chromosome of virulent M. tuberculosis H37Rv by allelic exchange. The ability of the mutants to survive, persist, and replicate in the U937 macrophage survival assay will be tested and compared to the virulent wild-type parental strain. 4) Genes identified from the screening of the plasmid and cosmid libraries will be used in gene expression studies. Levels of gene expression will be monitored in vitro and within macrophages using integrating reporter gene vectors.

描述（改编自申请人的摘要）：感染 结核分枝杆菌的巨噬细胞可以分为四个步骤： 依从性，进入，细胞内存活和乘法。目的 拟议的研究是识别，克隆和表征M的基因。 巨噬细胞内细胞内存活所必需的结核病。 结核分枝杆菌的潜在毒力因子基因将被首次克隆 构建重组库并将其纳入非对病原 分枝杆菌Smegmatis，一种快速生长的分枝杆菌 被巨噬细胞杀死。在巨噬细胞中生存增强的克隆将是 鉴定并检查了是否存在涉及的结核分枝杆菌基因 生存。在最初的实验中，申请人证明了 提出的研究的可行性是通过从M中分离出质粒克隆的可行性。 结核病文库可重复地赋予U937细胞的生存增强， 并使用相同的策略隔离了几个宇宙克隆。具体 目的是：1）筛选含有结核分枝杆菌H37RV的Smegmatis克隆。 重组文库（质粒或宇宙库），用于生存 人类巨噬细胞样细胞系的单层，U937。 2）分离M。 在U937巨噬细胞单层中生存的Smegmatis克隆，以进一步 学习。将回收这些克隆的质粒或宇宙。 结核基因所包含的基因将被限制映射，测序，并且 与发表的结核分枝杆菌H37RV的基因组序列以及 到其他数据库，并用其他分子技术进行分析 特征克隆的基因。 3）鉴定和表征的基因 具体的目标2，增强巨噬细胞中Smegmatis存活的特定目标将是 选择并用于评估这些基因在毒物中的作用。 结核。突变将在这些基因中构建，并引入 等位基因交换的毒性结核分枝杆菌H37RV的染色体。这 突变体在U937中生存，持久和复制的能力 将测试巨噬细胞生存测定法，并将其与有毒的野生型进行比较 父母菌株。 4）质粒筛选和 宇宙文库将用于基因表达研究。基因水平 表达式将在体外和巨噬细胞中通过整合进行监测 记者基因载体。