Mechanistic Insight on the Role of ESAT-6 in Modulating Host Defensive Pathways Through MicroRNA-30a in Mycobacteria Infected Macrophages

Abstract

The weaponry possessed by Mycobacterium tuberculosis (M. tb) in the form of immunodominant antigens hijack the host defense system to give a survival advantage to this intracellular fiend, but the mechanism of this control is not entirely known. The present study was undertaken to understand the effect of mycobacterial antigens on the anti-mycobacterial effect of Calcimycin. We found significant downregulation of autophagy by purified protein derivative (PPD) 3 (PPD fraction with a molecular weight of antigens > 3 kDa) pre-treatment in Calcimycin-treated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 (dTHP-1) cells compared to PPD 10 (antigenic mol. weight > 10 kDa). This reduction in autophagy also corroborated with the enhanced survival of M. smegmatis and M. bovis BCG in macrophages. We further demonstrate that recombinant early secreted antigenic target 6 (rESAT-6), an immunodominant antigen of M. tb, is responsible for inhibiting Calcimycin-induced autophagy and enhancing intracellular survival of mycobacteria. We also show that pre-treatment with rESAT-6 upregulates microRNA (miR)-30a-3p expression and vis-à-vis downregulates miR-30a-5p expression in Calcimycin-treated dTHP-1 cells. Further, transfection studies using miR-30a-3p inhibitor or -5p mimic highlighted the contrary roles of different arms of the same miRNA in regulating autophagy and IL-18 response by rESAT-6 in Calcimycin-treated dTHP-1 cells. By using either IL-18 neutralizing antibody or inhibitors of phosphoinositide 3-kinase (PI3K)/NF-κB/phagosome-lysosome fusion in the miRNA-30a transfected background, IL-18 mediated signaling and intracellular killing of mycobacteria was reversed in the presence of rESAT-6. Overall, the results of this study conclusively prove the contrary roles of miR-30a-3p and miR-30a-5p in regulating autophagy and IL-18-mediated phagosome-lysosome fusion by rESAT-6 in dTHP-1 cells upon Calcimycin treatment that affected intracellular survival of mycobacteria.