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（申请人提供）：疾病过程中的核心 结核病是杆菌与宿主巨噬细胞的相互作用。这 结核分枝杆菌感染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。这 提案将集中于这些步骤之一，即生存。角色毒力 在这些复杂相互作用中发挥结核分枝杆菌的因素实际上是 未知。拟议研究的目的是识别，克隆和 表征基因及其结核分枝杆菌的蛋白质产物 巨噬细胞内细胞内存活所必需的。潜在的毒力 首先构造结核分枝杆菌的因子基因将克隆 重组库，并将其纳入非致病分枝杆菌 Smegmatis。这种快速生长的分枝杆菌被内在化和杀死 巨噬细胞。将确定具有增强生存率的克隆 并检查了是否存在与生存有关的M结核基因。 使用该系统，我们已经分离了结核分枝杆菌基因，称为EIS （增强细胞内存活基因），它确实增强了细胞内 巨噬细胞中Smegmatis的生存。这笔赠款的主要重点 应用是EIS基因及其蛋白的进一步表征 产品EIS。具体目的是： 1. EIS基因失活对M的生存和繁殖的影响 巨噬细胞和小鼠的结核病。突变将在EIS和 引入无毒和有毒的M结核的染色体 （H37RA和H37RV）通过等位基因交换。 EIS敲除突变体的能力 在U-937巨噬细胞存活分析和人类中生存和复制 将测试单核吞噬细胞并将其与亲本菌株进行比较。 另外，EIS突变体在体内持续和复制的能力 在小鼠中，还将评估静脉感染模型。 2. EIS增强M的生存和繁殖的机制 未激活和干扰素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率 巨噬细胞，干扰素 - 伽马活化的U-937细胞中的生存和人类 将评估单核细胞。 Smegmatis带有和不带EIS的能力， 以及野生型结核病和EIS敲除突变体 特定的目标1，以在未激活和繁殖中生存/繁殖 将确定干扰素 - γ激活的U-937细胞和人类单核细胞。 还将进行研究以确定EIS在能力中可能发挥的作用 结核病。抗拒巨噬细胞运行的已知杀戮机制。 3. EIS蛋白的专业及其与之的关系 生存的动作。这些研究将包括：（i）细胞内 EIS在结核分枝杆菌和Smegmatis中的定位，（2）纯化 EIS蛋白质，（3）筛查结核病患者的血清蛋白 抗EIS的抗体，以及（4）在体外和 在巨噬细胞中使用综合报告基因载体。 4.在新的结核分枝杆菌DNA中识别非EIS生存基因 图书馆。在最初的研究中，含EIS的克隆是主要的克隆 在U-937巨噬细胞存活测定法中第六通道后分离。这样的 优先选择含EIS的克隆，并看起来 竞争/统治其他结核病基因，这也可能在 细胞内存活。因此，为了识别这些其他潜在基因， 将使用较大的结核分枝杆菌质粒文库建造（10-12 KB）从H37RV EIS基因敲除突变体中基因组DNA的DNA插入物。这个EIS 然后，将筛选淘汰图书馆在U-937巨噬细胞中生存 生存分析。具有增强生存的克隆将被隔离，进一步 特征。