SIRT1-FOXO3a-Mediated Autophagy Elicited by Furamidine Halts Mycobacterial Growth by Raising Intracellular Calcium Levels.

Abstract

In the ongoing global battle against tuberculosis (TB), which remains a significant contributor to worldwide mortality, Mycobacterium tuberculosis (M. tb), continues to pose formidable challenges. To effectively combat this pandemic, TB control has to be enhanced in several areas. The present study has been formulated to evaluate the anti-mycobacterial potential of Furamidine, a pharmaceutical drug from the LOPAC library. Initially, a non-cytotoxic concentration of Furamidine (lOµM) was used to evaluate its effect on intracellular mycobacterial growth 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 revealing the mechanism of action. Multiparametric approaches such as LC3-I to II conversion, protein level expression of different autophagic markers, and MDC staining were employed to study the autophagic response, which 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-Al demonstrated the positive regulation of autophagic flux upon Furamidine treatment. Pre­treatment with Wortmannin abrogated autophagy in treated cells, indicating the specificity of autophagy induction. Mechanistic investigations showed increased intracellular Ca2+ level expression, SIRT 1, and FOX03a activation upon its treatment. Inhibition of Ca2+ level expression suppressed calcium-mediated FOX03a level in Furamidine-treated cells. Furthermore, administering various inhibitors hampered the Furamidine-induced autophagy that impacted intracellular mycobacterial clearance. These results conclude that Furamidine triggered the Ca2+/SIRT 1/FOX03a pathway, causing less mycobacterial load in dTHP-1 cells.