ESAT-6 modulates Calcimycin-induced autophagy through microRNA-30a in mycobacteria infected macrophages

Abstract

ObjectiveMycobacterium tuberculosis (M. tb) has a sumptuous repertoire of effector molecules to counter host defenses. Some of these antigens inhibit autophagy but the exact mechanism of this inhibition is poorly understood. MethodsPurified protein derivative (PPD) was fractionated using 10 (PPD 10, antigenic molecular weight > 10 kDa) and 3 (PPD 3, mol. weight > 3 kDa) kDa cutters. Effect of these fractions on Calcimycin-induced autophagy and intracellular mycobacterial viability was then studied using different experimental approaches. ResultWe found significant downregulation of autophagy by PPD 3 pre-treatment in Calcimycin-treated dTHP-1 cells compared to PPD 10. This reduction in autophagy also corroborated with the enhanced survival of mycobacteria in macrophages. We demonstrate that recombinant early secreted antigenic target 6 (rESAT-6) is responsible to inhibit Calcimycin-induced autophagy and enhance intracellular survival of mycobacteria. We also show that pre-treatment with rESAT-6 upregulates microRNA (miR)-30a-3p expression and vis-a-vis downregulates miR-30a-5p expression in Calcimycin-treated dTHP-1 cells. Transfection studies with either miR-30a-3p inhibitor or miR-30a-5p mimic clearly elucidated the opposing roles of miR-30a-3p and miR-30a-5p in rESAT-6 mediated mycobacterial survival through autophagy inhibition. ConclusionTaken together, our result evidently highlights that rESAT-6 enhances intracellular survival of mycobacteria by modulating miR-30a-3p and miR-30a-5p expression.