Furamidine-induced autophagy exerts an anti-mycobacterial effect in a SIRT 1-FOXO3a-dependent manner by elevation of intracellular Ca 2+ level expression

Abstract

Mycobacterium tuberculosis (M. tb), the causative agent of human tuberculosis (TB), continues to be a significant cause of mortality worldwide. The latest findings have highlighted autophagy as a host-defence mechanism that eradicates many invading bacteria, including M. tb. Thus, novel approaches like the stimulation of autophagy using various pharmaceutical drugs can be undertaken to deal with this noxious pathogen. The present study has been formulated to evaluate the antimycobacterial potential of Furamidine, a pharmaceutical drug from the LOPAC library. Initially, a non-cytotoxic concentration of Furamidine (10µM) was used to evaluate its effect on intracellular mycobacterial viability in dTHP-1 cells. Furamidine treatment compromised intracellular mycobacterial fitness compared to control cells. Autophagy, a well-known host defensive strategy was investigated as a possible contributor to reveal the mechanism of action. Multiparametric approaches were employed to study autophagic response that conclusively suggested the autophagy induction potential of Furamidine in dTHP-1 cells. Further, elevated LC3-II expression and increased autophagic vacuole accumulation in the presence of Baf-A1 demonstrated the positive regulation of autophagic flux upon Furamidine treatment. Pre-treatment of Wortmannin abrogated autophagy in treated cells, indicating the specificity of autophagy induction. Mechanistic investigations showed increased intracellular Ca2+ level expression, SIRT 1 and FOXO3a activation upon its treatment. Inhibition of Ca 2+ level expression suppressed calcium-mediatedFOXO3a level in Furamidine-treated cells. Furthermore, the administration of various inhibitors hampered the Furamidine-induced autophagy, reducing intracellular mycobacteria clearance. These results conclude that Furamidine triggered the Ca 2+ /SIRT 1/FOXO3a pathway, causing less mycobacterial load in dTHP-1 cells.