Mycobacterial genes mediating resistance to bactericidal ubiquitin peptides

描述(申请人提供)：结核病再次成为全球关注的健康问题，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。通过阻止吞噬小体成熟在宿主巨噬细胞内生存和繁殖的能力是结核分枝杆菌致病性的一个标志。然而，当细菌不能阻止吞噬小体成熟时，例如在激活的巨噬细胞中，它们被输送到溶酶体并被杀死。从原代巨噬细胞中分离出的溶解溶酶体具有与泛素和泛素衍生肽相关的强大的抗分枝杆菌特性。用溶酶体蛋白酶或合成肽UB2消化全长泛素得到的泛素衍生的多肽在体外具有抗分枝杆菌的作用。为了确定这些分子的靶点和特异性，有必要对合成的抗分枝杆菌多肽UB2进行进一步研究。 推测存在分枝杆菌因子参与宿主来源抗菌肽的敏感性和耐药性，这些因子参与了结核分枝杆菌毒力的形成。该项目将通过鉴定和鉴定分枝杆菌超敏感和高耐药突变体来阐明泛素衍生多肽的作用方式，并将确定宿主抗菌肽在结核分枝杆菌感染中的作用。在具体目标1中，我们将确定与泛素衍生抗菌肽的敏感性和耐药性有关的分枝杆菌因子。已经分离出对泛素衍生多肽UB2的抗分枝杆菌作用高度敏感和高度抵抗的分枝杆菌突变体。在这些突变体中被破坏的遗传位点将被识别并从功能上进行分类。预计编码外排泵、ABC运输系统和膜蛋白的突变体将被优先用于后续实验。在特定目标2中，我们将确定与泛素衍生抗菌肽的敏感性和耐药性有关的分枝杆菌因子对结核分枝杆菌毒力的贡献。静息、激活和自噬的巨噬细胞将被结核分枝杆菌耐药突变株感染，以确定它们是否表现出改变的表型。用结核分枝杆菌抗药性突变体进行的小鼠感染研究将确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.