Mycobacterium tuberculosis, a major threat to health in South Africa : intracellular survival after treatment with novel drugs designed against the mycothiol pathway

[摘要] ENGLISH ABSTRACT: Mycothiol (MSH) is unique to mycobacteria as the major low molecular weight cellular thiol responsible for protection of bacteria against oxidative stress. The design of drugs and inhibitors against enzymes of the mycothiol pathway was based on the premise that mycothiol is unique to mycobacteria, and is thus important for its survival. A total of 80 inhibitors designed against enzymes of the mycothiol pathway were screened for inhibition of growth on in vitro growing M. tuberculosis using the BACTEC 460TM assay. The most active compounds were further tested for inhibitory potential of M. tuberculosis within macrophages. Initial screening in the macrophage system was done using the human-like THP1 cell line and then mouse bone marrow-derived macrophages. In this investigation we established that phenothiazine can be exploited as an inhibitor of enzymes of the mycothiol pathway. Although tunicamycin significantly inhibited the growth of M. tuberculosis both in vitro and ex vivo; it was found to be cytotoxic to host macrophages. To this end we provide proof-of-concept that compounds which can inhibit the expression of mycothiol enzymes have potential as anti-tubercular drugs.The response of M. tuberculosis to stress conditions was assessed via LC-MS in which maximal levels of mycothiol were produced during the early time points of exposure to isoniazid. We used mycothiol-deficient (mshA) M. tuberculosis to investigate the role of mycothiol for survival as well as the resultant phenotype when such mutants are exposed to stress conditions. The mshA deletion mutants in M. tuberculosis were resistant to INH at concentrations which inhibited growth in the wild-type strains. We postulated that katG and inhA, the genes involved in INH metabolism, required mycothiol for their activation.Morphological alterations of M. tuberculosis within macrophages were assessed using electron microscopy approaches. In this way we attempted to follow the fate of M. tuberculosis within the phagosomes, and how mycobacteria is processed in phagosomes in terms of replication, survival and degradation. The establishment of a successful infection by M. tuberculosis depends on the initial encounter with hostmacrophages, which represent the first line of cellular defense against microbial invasion. At the interface between mycobacteria and macrophages, the complex outermost layer of the mycobacterial cell wall probably plays a role in facilitating host cell entry. Under normal conditions (i.e. ingestion of non pathogenic microorganisms), newly formed phagosomes intermingle contents and membrane with the successive compartments of the endocytic pathway (early endosomes, late endosomes, lysosomes) through a complex series of fusion and fission. As they are processed into phagolysosomes, they undergo gradual modifications by specific addition and removal of membrane constituents. In addition, they become acidified due to the vacuolar proton pump ATPase located in the membrane and acquire toxic constituents, including hydrolases that will ultimately destroy bacteria.

[发布机构] Stellenbosch University