Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most devastating infectious agents in the world. Chemical-genetic characterization through in vitro evolution combined with whole genome sequencing analysis was used identify novel drug targets and drug resistance genes in Mtb associated with its intracellular growth in human macrophages. We performed a genome analysis of 53 Mtb mutants resistant to 15 different hit compounds. We found nonsynonymous mutations/indels in 30 genes that may be associated with drug resistance acquisitions. Beyond confirming previously identified drug resistance mechanisms such as rpoB and lead targets reported in novel anti-tuberculosis drug screenings such as mmpL3, ethA, and mbtA, we have discovered several unrecognized candidate drug targets including prrB. The exploration of the Mtb chemical mutant genomes could help novel drug discovery and the structural biology of compounds and associated mechanisms of action relevant to tuberculosis treatment.
