Abstract Description

Tuberculosis (TB) is a highly infectious disease, caused by Mycobacterium tuberculosis (Mtb). TB is one of the top ten causes of death globally and is the leading cause of death by a single source of infection. Of the 10.8 million global new TB infections in 2023, approximately 25 % were recorded in Africa. Drug resistant Mtb strains arise from mutations in drug target proteins, which often lead to alterations in protein conformation and general protein stability. One of the first-line TB drug target proteins is Mtb InhA, a transenoyl-acyl – carrier protein. InhA is essential in the FAS-II pathway for biosynthesis of mycolic acids which form a major part of the mycobacterial cell wall. Mutations in InhA may influence the overall protein stability and increase reliance on cellular protein folding systems. The Mtb chaperone protein DnaK, an HSP70 homologue is an essential protein involved in protein folding, refolding, and degradation. This study evaluates InhA as a DnaK client (interactor) and subsequently, whether DnaK stabilises mutant variants of InhA. Using in vitro chaperone complexes reconstituted from purified proteins, we demonstrate that InhA is heat labile and that DnaK enhances the thermal stability of InhA and its mutant variant S94A. Subsequently, our protein-protein interaction studies show that DnaK bind to InhA and its mutant variants with different affinities. Taken together, our analyses demonstrate dependence of InhA on DnaK for stability. Consequently, our study provides a platform to determine whether DnaK inhibition may reverse resistance driven by mutations in InhA.