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。通过阻止吞噬体成熟在宿主巨噬细胞内存活和繁殖的能力是结核分枝杆菌致病性的标志。然而，当细菌无法阻止吞噬体成熟时，例如在活化的巨噬细胞中，它们被运送到溶酶体并被杀死。从原代巨噬细胞中分离的溶解溶酶体具有与泛素和泛素衍生肽相关的强效抗真菌特性。用溶酶体蛋白酶消化全长泛素或合成肽Ub2获得的泛素衍生肽在体外具有抗菌作用。为了确定这些分子的靶点和特异性，有必要进一步研究合成的抗菌肽Ub2。

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.