Browsing by Author "Ymele Soko, Vivette"

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    Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads.
    (Stellenbosch : Stellenbosch University, 2023-03) Ymele Soko, Vivette; Mavumengwana, Vuyo; Loxton, Andre G.; Smith, Liezel; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Apoptosis is a natural immune protective mechanism that allows the host to clean up nonfunctional cells. Not surprisingly, blocking apoptosis is presented as one of the strategies Mycobacterium tuberculosis (M.tb) uses to avoid host immune defence. In the absence of apoptosis, infected cells are taken captive to support the growth of their invader and eventually progress to an uncontrolled death (necrosis), which in the case of M.tb infection is called caseous necrosis and is characteristic of pathology observed in tuberculosis patients. In this project, host-directed therapy (HDT) was exploited and involved using pro-apoptotic drugs to induce and restore the normal process of controlled cell death (apoptosis) in infected macrophages. The advantage of this strategy is that, unlike current tuberculosis (TB) therapy which is not specific to certain forms of bacteria. Including dormant and multi-resistant M.tb, HDT could affect all forms of bacteria because it does not directly target the pathogen. This project used Mycobacterium smegmatis mc2 155 (M. smegmatis)-infected macrophages as a model to assess the efficacy of selected apoptotic inducer (API) drugs in controlling the intracellular growth of M. smegmatis. The model has been structured so that its product can be cultured on agar plates to evaluate the growth of bacteria representative of the inhibition or growth of intracellular bacteria. Preliminary tests were performed to evaluate the antimicrobial susceptibility of mycobacterium against the selected (API) drugs. Amongst the 12 drugs that were assessed, cepharanthine, CP31398 dihydrochloride hydrate, marinopyrrole A, and nutlin-3a showed activity against M. tuberculosis and M. smegmatis mc2 155. These four drugs have a minimum inhibitory concentration (MIC) of 3.1 - 6.2 µg/mL, 6.2 – 12.5 µg/mL, 25 – 50 µg/mL and 50–100 µg/mL, respectively, against M. smegmatis mc2 155. Also, when tested against M.tb H37Rv, the MIC value of these drugs was one-fold lower than their respective MIC values observed on M. smegmatis. Furthermore, the minimum bactericidal concentration (MBC) test revealed that cepharanthine or CP-31398 dihydrochloride hydrate could kill M. smegmatis at 12.5 µg/mL or 25 µg/mL, respectively. Ex-vivo treatment of M. smegmatis-infected macrophages with tolerated concentrations of API drugs indicated there was some limitations in this host model due to spontaneous inhibition of M. smegmatis growth in THP-1 macrophages. Nonetheless, it was possible to test the efficacy of drugs intended for HDT using the difference in the relative survival of intracellular bacteria as indicators. Cepharanthine and CP-31398 dihydrochloride hydrate inhibited the intracellular growth of the bacteria after 12hrs treatments at a concentration representing half of their MIC value or one-quarter of their MBC value. Higher concentrations of these drugs, for instance, 6.2 µg/mL of cepharanthine; or 9.2 µg/mL of CP-31398 dihydrochloride hydrate, resulted in the absence of growth of intracellular bacteria after only 6 hrs treatment. Multi-cytokines analysis on the cell culture supernatants sampled during the treatment of macrophages indicated that mild expression of IFN-gamma, TNF-alpha, IL-1beta, IL-5, combined with the inhibition of IL-6 and IL-22 was required to eliminate intracellular M. smegmatis mc2 155. However, high levels of TNF-alpha, IL-1beta, IL-6, and IL-22 coupled with the absence of expression of IFN-gamma and IL-5 correlated with a high burden of intracellular bacteria.