Furamidine-induced autophagy exerts an anti-mycobacterial effect in a SIRT1-pAMPK-FOXO3a-dependent manner by elevation of intracellular Ca2+ level expression

Abstract

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), continues to be a major contributor to global mortality rates. To effectively combat this pandemic, TB control has to be enhanced in several areas, including point-of-care diagnostics, shorter and safer drug regimens, and preventative vaccination. The latest findings have highlighted autophagy as a host-defense 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 anti-mycobacterial potential of Furamidine, a DNA minor groove binder (MGB). Initially, a non-cytotoxic concentration of Furamidine (10 µM) was used to assess its impact on the intracellular persistence of mycobacteria in differentiated THP-1 (dTHP-1) cells. Furamidine treatment compromised intracellular mycobacterial growth 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 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 under Baf-A1 treatment demonstrated the positive regulation of autophagic flux upon Furamidine treatment. Mechanistic investigations showed increased intracellular calcium (Ca²⁺) level expression, SIRT1, pAMPK, and FOXO3a activation upon its treatment. Inhibition of Ca²⁺ level expression suppressed Ca²⁺-mediated-FOXO3a level in Furamidine-treated cells. Furthermore, administering various inhibitors hampered the Furamidine-induced autophagy that impacted intracellular mycobacteria clearance. These results conclude that Furamidine triggered the Ca²⁺/pAMPK/SIRT1/FOXO3a pathway, causing less mycobacterial load in dTHP-1 cells.