Modulation of Autophagy and Its Effect on Intracellular Mycobacteria by Calcimycin

Abstract

Calcium ionophores like Calcimycin are known for their antimicrobial activity in vitro against gram-positive bacteria and fungi but its antimycobacterial effect is in infancy. The present study was undertaken in THP-1 cells to deduce the effect of Calcimycin on internalized mycobacteria. In our study, we observed that Calcimycin is antimycobacterial in action by inducing autophagy, one of the prominent host-defence pathways. Then, we deciphered the mechanism of autophagy induction leading to the killing of intracellular microbe and observed that Calcimycin exerts its effect through purinergic receptor P2X7 (P2RX7) that controls autophagy by regulating intracellular calcium-modulated ATP release. Further, we looked at the role of immune modulators in autophagy-dependent antimycobacterial effect of Calcimycin. We found significant IL-12 mRNA expression followed by its release in Calcimycin treated cells compared to other pro- and anti-inflammatory cytokines with concomitant expression of IL-12 receptor on the treated cell surface. P2RX7 inhibitors like 1-[N, O-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and P2RX7 siRNA abrogated IL-12 release upon Calcimycin treatment by affecting Jun N-terminal kinase (JNK) – NF-κB signalling pathway. Specific inhibitors against JNK and NF-κB molecules showed decreased IL-12 production and less autophagy in Calcimycin treated cells that lead to a spurt in intracellular mycobacterial growth. Experiment with IL-12 neutralizing antibody conclusively proved that IL-12 acts in an autocrine fashion to regulate autophagy-dependent intracellular mycobacterial growth in Calcimycin-treated cells. So, overall our study delineated the mechanistic pathway of autophagy induction by Calcimycin that may provide an attractive target for the control of mycobacterial infection, which will help in developing better therapeutic interventions against tuberculosis (TB).