Background: Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), is the world’s leading infections disease. Because of increasing emergence of drug-resistant Mtb, development of a novel anti-TB drug is required. Bacterial proteases are maintaining proteastasis and essential for bacterial survival. Here, we investigated the feasibility of bacterial proteases as drug targets. 
Methods and Results:  Inducible knockdown strains of proteases (clp, ftsH or htrA) in Mycobacterium smegmatis mc²-155 were created by using CRISPR interference. Bacterial growth was not suppressed by gene knockdown, but knockdown of clp increased the susceptibility against isoniazid and rifampicin, traditional anti-TB drugs. Furthermore, checkerboad assay revealed the synergistic effect between cyclomarin A, a novel clp inhibitor, and anti-TB drugs.
Conclusion: Clp is a promising drug target for development of a novel anti-TB drug.