ACA kills Mycobacterium tuberculosis and M. bovis by targeting cell wall core assembling protein CpsA

Tuberculosis (TB), caused by Mycobacterium tuberculosis, still globally threatens human health. With the emergence of multidrug-resistant strains, there is an imperative need to discover new anti-TB agents and identify novel drug targets. Here, we report that the compound ACA, namely 3-azidothiophene-2-carboxylic acid, can kill M. tuberculosis by targeting the bacterial cell wall core assembling process. Whole-genome sequencing of spontaneous ACA-resistant mutants identified single-nucleotide variants in the cpsA2 ligase gene responsible for the covalent attachment of arabinogalactan and peptidoglycan. The cell wall of cpsA2 or its homolog cpsA1-deleted mutant H37Ra strains show increased permeability and drug sensitivity, which is similar to the ACA-treated mycobacteria. Both cpsA1 and cpsA2 could reverse the resistant phenotype of ACA-resistant mutants and the growth defects of the ΔcpsA1 H37Ra strain. ACA can directly bind CpsA1 and CpsA2 to inhibit significantly the pyrophosphatase activity of CpsA1 and CpsA2. Our results suggest that ACA may disturb the cell wall core assembling process and kill M. tuberculosis by targeting CpsA1 and CpsA2, which provide potential candidate drug target for controlling drug-resistant TB.