Genetic Screens for Virulence Factors of Mycobacteria

描述（由申请人提供）： 结核分枝杆菌（M.tb）感染了世界上三分之一的 它每年夺走两到三百万人的生命。其 成功是通过其在敌对的细胞内持续存在的能力来实现的。 感染的巨噬细胞的环境。在入侵之后，结核分枝杆菌操纵了 宿主细胞的吞噬途径，以抑制吞噬体的成熟 变成吞噬溶酶体此外，结核分枝杆菌抑制细胞凋亡反应， 感染的巨噬细胞这些影响很可能代表了一种高度进化的 结核分枝杆菌用来逃避宿主免疫反应的策略。尽管一些 细胞蛋白质已被表征为结核分枝杆菌的靶点， 不清楚哪些细菌蛋白质或脂质介导相互作用。 拟议的研究项目旨在填补我们知识中的这一空白， 重点是鉴定结核分枝杆菌的蛋白质或脂质， 涉及抑制细菌吞噬体的成熟， 抑制宿主细胞的凋亡反应。的最新进展 分枝杆菌的遗传操作将用于随机诱变 绿色荧光蛋白（GFP）标记的结核分枝杆菌和筛选突变体缺陷 在抑制吞噬体成熟使用新开发的基于流式细胞仪，高- 通量测定。在第二种方法中，耻垢分枝杆菌， 缺乏抑制吞噬体成熟的非致病性分枝杆菌， 宿主细胞的凋亡，将与结核分枝杆菌基因和克隆互补 将选择获得这种能力的人。结核分枝杆菌基因的鉴定 对于病原体的持久性至关重要，这将提供重要的目标， 治疗结核病的新药物的开发， 开发可用作疫苗的新的结核分枝杆菌减毒株。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (M.tb) infects one-third of the world's population and claims the lives of two to three million people each year. Its success is achieved by its ability to persist in the hostile intracellular environment of infected macrophages. After invasion, M.tb manipulates the phagocytic pathway of the host cell to inhibit the maturation of the phagosome into a phagolysosome. In addition, M.tb inhibits the apoptotic response of infected macrophages. These effects are likely to represent a highly evolved strategy that is used by M.tb to evade the host immune response. Although some cellular proteins have been characterized as targets of M.tb, it is currently unclear which bacterial proteins or lipids mediate the interactions. The proposed research project aims at filling this gap in our knowledge by focusing on the identification of proteins or lipids of M.tb that are implicated in either inhibition of maturation of the bacterial phagosome or inhibition of the apoptotic response of the host cell. The recent advances in the genetic manipulations of mycobacteria will be used to randomly mutagenize green fluorescence protein (GFP)-labeled M.tb and screen for mutants deficient in inhibiting phagosome maturation using a newly developed FACS-based, high- throughput assay. In a second approach, Mycobacterium smegmatis, a nonpathogenic mycobacterium deficient in inhibiting phagosome maturation and apoptosis of the host cell, will be complemented with M.tb genes and clones that have gained this capacity will be selected. Identification of M.tb genes essential for persistence of the pathogen will provide important targets for the development of new drugs for the treatment of tuberculosis and the development of new attenuated strains of M.tb that may be used as vaccines.