Identifying a novel mechanism of L-leucine uptake in Mycobacterium tuberculosis using a chemical genomic approach

Amino acid biosynthesis is vital for Mycobacterium tuberculosis (Mtb) proliferation and tuberculosis (TB) pathogenesis. However, it is not clear how amino acids are transported in Mtb, particularly the branched-chain amino acids (BCAAs) that contribute to the production of the cell-wall lipid component precursors, such as acetyl-CoA and propionyl-CoA. While performing the screening of an FDA-approved repurposed library of small molecule inhibitors against the auxotrophic strain Mtb mc² 6206, which lacks leuC-leuD and panC-panD genes, we identified a molecule, namely semapimod, which exclusively inhibits the growth of the auxotrophic strain, whereas no effect is observed against the wild-type Mtb H₃₇Rv. Interestingly, 24 hr of exposure of Mtb mc² 6206 to semapimod causes massive transcriptional reprogramming with differential expression of >450 genes associated with a myriad of metabolic activities. By performing a series of experiments, we affirm that semapimod indeed inhibits the L-leucine uptake in Mtb mc² 6206 by targeting a protein involved in the cell-wall lipid biosynthesis pathway. Remarkably, semapimod treatment of mice infected with Mtb H₃₇Rv causes a significant reduction of bacterial load in lungs and spleen, despite showing no efficacy against the pathogenic strain in vitro. Overall findings of our study reveal that together with an endogenous pathway for L-leucine biosynthesis, a well-orchestrated machinery for its uptake is functional in Mtb, which is important for intracellular survival of the TB pathogen.