Calcimycin Inhibits Intracellular Mycobacterial Growth by Regulating Autophagy

Abstract

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis resides and multiplies in macrophages by inhibiting various host defensive pathways like autophagy. Increased autophagy by different inducers like adenosine triphosphate (ATP) has been shown to be antimycobacterial through calcium-dependent mechanism that decreased M. bovis BCG viability. Our preliminary screening experiment using whole cell based approach has identified Calcimycin, which increases intracellular calcium as a potent inhibitor of M. tb growth. We observed that Calcimycin was bactericidal in action and inhibited M. tb growth in vitro by 99% at 1.25 mM. So, present study was undertaken to decipher the role of Calcimycin on intracellular mycobacteria in THP-1 cells. We found by MTT assay that 0.4 μM of Calcimycin is non-toxic even after 72 h of treatment. Viability findings were further corroborated by Trypan blue dye exclusion assay where Calcimycin treatment for 72 h reduced cell viability to 86.4 ± 1.8%, 73.0 ± 11.9% and 62.2 ± 10.2% at 0.6 μM, 0.8 μM and 1 μM respectively. Time kinetic experiment was performed with 0.4 μM of Calcimycin to study its effect on the autophagy in THP-1 cells. We found optimum up-regulation of different autophagy markers like Beclin-1, Atg 7 and Atg 3 after 12 h of treatment through western blotting. Increased conversion of LC3-I to LC3-II, widely used marker of autophagy induction was also observed in treated cells suggesting increased autophagy. Addition of 3- methyl adenine (3-MA), an autophagy inhibitor, abrogated the effect of Calcimycin on autophagy. We found down-regulation of Beclin-1, Atg 7, Atg 3 and LC3-II expression in Calcimycin treated cells in the presence of 3-MA. Increased autophagy in Calcimycin treated cells led to decrease in intracellular M. smegmatis viability and addition of 3-MA reversed this effect. Studies are currently underway to determine the mechanism of how Calcimycin induced autophagy exerts its antimycobacterial effect