M. tuberculosis survival in Macrophages

结核分枝杆菌在巨噬细胞中的存活

• 批准号: 6511005
• 负责人: Richard Lee Friedman
• 金额: $ 30.3万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511005
• 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 (provided by the applicant): Central to the disease process in tuberculosis are the interactions of the bacilli with host macrophages. The infection of macrophages by Mycobacterium tuberculosis can be divided into four steps: adherence, entry, intracellular survival, and multiplication. This proposal will concentrate on one of these steps, survival. The role virulence factors of M. tuberculosis play in these complex interactions is virtually unknown. The aim of the proposed research is to identify, clone, and characterize genes and their protein products 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 non-pathogenic Mycobacterium smegmatis. This rapidly growing mycobacterium is internalized and killed by macrophages. Clones with enhanced survival in macrophages will be identified and examined for the presence of M tuberculosis genes involved in survival. Using this system we have already isolated a M. tuberculosis gene, named eis (enhanced intracellular survival gene), which does enhance intracellular survival of M. smegmatis within macrophages. The primary focus of this grant application is the further characterization of the eis gene and its protein product Eis. The specific aims are: 1.Effect of eis Gene Inactivation on Survival and Multiplication of M tuberculosis in Macrophages and Mice. Mutations will be constructed in eis and introduced into the chromosome of both avirulent and virulent M tuberculosis (H37Ra and H37Rv) by allelic exchange. The ability of the eis knockout mutants to survive and replicate in the U-937 macrophage survival assay and in human mononuclear phagocytes will be tested and compared to the parental strain. Additionally, the ability of the eis mutants to persist and replicate in vivo in a mouse intravenous infection model will also be evaluated. 2.Mechanism(s) Whereby eis Enhances Survival and Multiplication of M tuberculosis in Unactivated and Interferon-gamma-Activated Macrophages. To learn how eis may enhance intracellular survival of mycobacteria in macrophages, survival in interferon-gamma-activated U-937 cells and human monocytes will be evaluated. The ability of M. smegmatis with and without eis, as well as wild-type M. tuberculosis and eis knock-out mutants, constructed in Specific Aim No.1, to survive/multiply in both unactivated and interferon-gamma-activated U-937 cells and human monocytes will be determined. Studies will also be done to determine what role Eis may play in the ability of M. tuberculosis to resist known killing mechanisms operating in macrophages. 3.Properties of the Eis Protein and their Relationship to its Survival-Increasing Action. These studies will include: (I) intracellular localization of Eis in M. tuberculosis and M. smegmatis, (2) purification of the Eis protein, (3) screening of sera from tuberculosis patients for presence of antibody to Eis, and (4) measurement of eis gene expression in vitro and within macrophages using integrative reporter gene vectors. 4.Identification of Non-eis Survival Genes in a New M. tuberculosis DNA Library. In initial studies, eis-containing clones were the predominate clones isolated after the sixth passage in the U-937 macrophage survival assay. Such eis-containing clones are preferentially selected and appear to out-compete/dominate other M. tuberculosis genes which may also play a role in intracellular survival. Thus, in order to identify these other potential genes, a new M. tuberculosis plasmid library will be constructed with larger (10-12 kb) DNA inserts of genomic DNA from an H37Rv eis knockout mutant. This eis knockout library will then be screened for survival in the U-937 macrophage survival assay. Clones with enhanced survival will be isolated and further characterized.

描述（由申请人提供）：疾病过程的核心 结核病是杆菌与宿主巨噬细胞的相互作用。这 结核杆菌对巨噬细胞的感染可分为四种 步骤：粘附、进入、细胞内存活和增殖。这 提案将集中于这些步骤之一：生存。作用毒力 结核分枝杆菌在这些复杂的相互作用中发挥作用的因素实际上是 未知。拟议研究的目的是识别、克隆和 描述结核分枝杆菌的基因及其蛋白质产物，它们是 巨噬细胞内细胞内生存所需的。潜在毒力 结核分枝杆菌的因子基因将通过首先构建 重组文库并将其整合到非致病性分枝杆菌中 包皮垢。这种快速生长的分枝杆菌被内化并杀死 巨噬细胞。将鉴定出在巨噬细胞中具有增强存活能力的克隆 并检查了与生存有关的结核分枝杆菌基因的存在。 使用该系统，我们已经分离出结核分枝杆菌基因，命名为 eis （增强细胞内存活基因），它确实增强了细胞内 耻垢分枝杆菌在巨噬细胞内的存活。本次资助的主要重点 应用是eis基因及其蛋白质的进一步表征 产品Eis。具体目标是： 1.eis基因失活对M存活和增殖的影响 巨噬细胞和小鼠中的结核病。突变将在 eis 中构建， 引入无毒力和强毒力结核分枝杆菌的染色体 （H37Ra 和 H37Rv）通过等位基因交换。 eis敲除突变体的能力 在 U-937 巨噬细胞存活测定和人体中存活和复制 将测试单核吞噬细胞并与亲本菌株进行比较。 此外，eis突变体在体内持续存在和复制的能力 还将在小鼠静脉感染模型中进行评估。 2.eis 增强 M 存活和增殖的机制 未激活和干扰素γ激活的巨噬细胞中的结核病。到 了解 eis 如何增强分枝杆菌的细胞内存活 巨噬细胞，在干扰素γ激活的U-937细胞和人类中的存活 将评估单核细胞。有或没有 eis 的耻垢分枝杆菌的能力， 以及野生型结核分枝杆菌和 eis 敲除突变体，构建于 具体目标一，在未激活和未激活的环境中生存/繁殖 将测定干扰素-γ激活的U-937细胞和人单核细胞。 还将进行研究以确定 Eis 在能力方面可能发挥什么作用 结核分枝杆菌能够抵抗巨噬细胞中已知的杀伤机制。 3.Eis蛋白的性质及其与它的关系 增加生存的行动。这些研究将包括：（I）细胞内 Eis 在结核分枝杆菌和耻垢分枝杆菌中的定位，（2）纯化 Eis蛋白，(3)筛查结核病患者血清中是否存在 Eis抗体的制备，以及(4)体外eis基因表达的测量和 使用整合报告基因载体在巨噬细胞内。 4.新结核分枝杆菌DNA中非eis生存基因的鉴定 图书馆。在最初的研究中，含有 eis 的克隆是主要的克隆 在 U-937 巨噬细胞存活测定中第六次传代后分离。这样的 优先选择含有 eis 的克隆，并且似乎 竞争/支配其他结核分枝杆菌基因，这些基因也可能在 细胞内存活。因此，为了识别这些其他潜在基因， 将构建一个新的结核分枝杆菌质粒文库，具有更大的（10-12 kb) H37Rv eis 敲除突变体基因组 DNA 的 DNA 插入片段。这个是 然后将筛选敲除文库在 U-937 巨噬细胞中的存活情况 生存测定。存活率提高的克隆将被分离并进一步 特点。