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Towards identifying intracellular drug targets of Mycobacterium tuberculosis against hit compounds in defined growth media Bojang, Adama

Abstract

With the emergence of drug-resistant strains and widespread HIV epidemics, Mycobacterium tuberculosis (Mtb), the causative agent of TB, remains a number one global health problem. Human alveolar macrophages are the natural host and reservoir of Mtb and as an intracellular pathogen, Mtb relies on host lipids as growth substrates. However, little is known about the in vivo availability of carbon and energy sources as substrates to support Mtb growth and how its intracellular environment influences anti-mycobacterial compound activity. In this study, we aimed to use carbon restriction to mimic intracellular environment of Mtb and determined in vitro activities of hit compounds against Mtb. We also aimed to use “chemical genetic” approach to screen for resistant mutants against novel inhibitors and identify intracellular drug targets of Mtb against hit compounds in defined growth substrate media. Using resazurin and cytotoxicity assays, compound’s activities against Mtb as well as against host cells were determined. Resistant mutants were isolated on a defined growth substrate 7H10 solid media supplemented with hit compounds. Genomic DNA of all resistant mutants were extracted and sequenced to identify mutated genes. Less than 30% of compound were active in albumin dextrose catalase as rich-media. Anti-mycobacterial compounds were all active in single carbon substrate media. Acetate or glycerol in growth media without hit compounds had an inhibitory effect on Mtb. Hit compounds at the highest concentration did not have cytotoxicity effect against host cells. A total of 8 mutants resistant to various novel inhibitors were isolated, with more than a single gene mutation identified in each strain. We showed albumin dextrose catalase as rich-media is not ideal for compound screening. Media supplemented with glucose alone or cholesterol plus glucose or acetate closely resembles the MIC profile of the intracellular environment. Resistant mutants isolated in vitro showed similar resistant phenotype to intracellularly grown bacteria, suggesting a link between the resistant phenotype and the mutated genes identified. Identifying intracellular druggable targets will facilitate the development of better approaches in TB treatment. The findings from this project will improve knowledge of the mode of action of particular compounds against Mtb.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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