Mycobacterial genes mediating resistance to bactericidal ubiquitin peptides

描述（由申请人提供）：结核病已重新成为全球健康问题，世界卫生组织估计结核分枝杆菌感染了世界三分之一的人口。通过阻止吞噬体成熟而在宿主巨噬细胞内存活和繁殖的能力是M.结核病致病性然而，当细菌不能阻止吞噬体成熟时，例如在活化的巨噬细胞中，它们被递送到溶酶体并被杀死。从原代巨噬细胞分离的溶解的溶酶体具有与泛素和泛素衍生肽相关的有效抗分枝杆菌特性。从用溶酶体蛋白酶消化全长泛素或合成肽Ub 2获得的泛素衍生肽在体外具有杀分枝杆菌作用。对合成的抗分枝杆菌肽Ub2的进一步研究对于确定这些分子的靶点和特异性是必要的。 推测存在与分枝杆菌对宿主源性抗菌肽的敏感性和耐药性有关的因素，这些因素有助于分枝杆菌对宿主源性抗菌肽的耐药性。结核病毒力本项目将通过鉴定和表征分枝杆菌超敏感和超耐药突变体来阐明泛素衍生肽的作用模式，并将确定宿主抗菌肽在分枝杆菌中的作用。肺结核感染。在具体目标1中，我们将确定参与泛素衍生的抗菌肽的易感性和耐药性的分枝杆菌因子。已经分离出对泛素衍生肽Ub2的抗分枝杆菌作用超敏感和超抗性的分枝杆菌突变体。这些突变体中被破坏的遗传位点将被鉴定并在功能上分类。预测编码外排泵、ABC转运系统和膜蛋白的突变体将优先用于后续实验。在具体目标2中，我们将确定对M的贡献。结核病毒力的分枝杆菌因素参与的敏感性和耐药性的泛素衍生的抗菌肽。静止、活化和自噬的巨噬细胞将被M.结核病抗微生物剂抗性突变体，以确定它们是否表现出改变的表型。小鼠感染M.结核病抗微生物剂抗性突变体将决定这些因素对毒力的贡献。

DESCRIPTION (provided by applicant): Tuberculosis has re-emerged as a global health concern and the World Health Organization estimates that Mycobacterium tuberculosis infects one third of the world population. The ability to survive and multiply within the host macrophage by arresting phagosome maturation is a hallmark of M. tuberculosis pathogenicity. However, when bacteria are unable to arrest phagosome maturation, such as in activated macrophages, they are delivered to the lysosome and killed. Solubilized lysosomes isolated from primary macrophages possess potent antimycobacterial properties that are associated with ubiquitin and ubiquitin-derived peptides. Ubiquitin-derived peptides obtained from digestion of full-length ubiquitin with lysosomal proteinases or the synthetic peptide Ub2 were mycobactericidal in vitro. Further study of the synthetic antimycobacterial peptide Ub2 is necessary to determine the target and specificity of these molecules. It is hypothesized that there are mycobacterial factors involved in susceptibility and resistance to host-derived antimicrobial peptides and these factors contribute to M. tuberculosis virulence. This project will elucidate the mode of action of ubiquitin-derived peptides through identification and characterization of mycobacterial hyper-susceptible and hyper-resistant mutants and will define the role of host antimicrobial peptides in M. tuberculosis infection. In Specific Aim 1, we will identify mycobacterial factors involved in susceptibility and resistance to ubiquitin-derived antimicrobial peptides. Mycobacterium mutants that are hyper-susceptible and hyper-resistant to the antimycobacterial action of the ubiquitin-derived peptide Ub2 have been isolated. The genetic locus disrupted in these mutants will be identified and classified functionally. Mutants predicted to encode efflux pumps, ABC transport systems, and membrane proteins will be prioritized for subsequent experiments. In Specific Aim 2, we will determine contribution to M. tuberculosis virulence of mycobacterial factors involved in susceptibility and resistance to ubiquitin-derived antimicrobial peptides. Resting, activated, and autophagic macrophages will be infected with M. tuberculosis antimicrobial resistance mutants to determine if they exhibit altered phenotypes. Mouse infection studies with M. tuberculosis antimicrobial resistance mutants will determine the contribution of these factors toward virulence.