Calcimycin induced autophagy decreases mycobacterial growth in THP-1 cells through P2RX7 dependent pathway mediated by intracellular calcium

Abstract

Introduction – Calcimycin, calcium ionophore, show in vitro antimicrobial activity against gram-positive bacteria and fungi. In our study we found that it is bactericidal in action against Mycobacterium tuberculosis (M.tb) and it induces autophagy as well as kill intracellular mycobacteria at a low concentration due to which we want to decipher the mechanism of autophagy induction and its effect on survival of intracellular mycobacteria in THP-1 cells. Methods – We checked the expression of major autophagy genes in calcimycin treated and infected samples by western blotting. To understand the mechanism of autophagy induction we check the expression of ATP receptor, purinergic receptor P2X7 (P2RX7), in calcimycin treated samples by qRT-PCR and measure extracellular ATP release by fluorescence to validate P2RX7 activation. We checked intracellular calcium level by fluorescence and confocal microscopy. We also checked the interaction of calcimycin with P2RX7 by performing docking experiment. To validate P2RX7 role in autophagy induction we use P2RX7 inhibitor, 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) or intracellular chelator, 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) and checked the expression of major autopghay markers by western blotting and confocal microscopy. Results - We noticed that treatment with calcimycin led to up-regulation of major autophagy markers like Beclin-1, autophagy-related gene (Atg) 7, Atg 3 and enhanced microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion in infected macrophages. We also demonstrate that calcimycin binding with P2RX7 led to increase in intracellular calcium level that regulates the extracellular release of ATP. Blocking of either P2RX7 expression by KN-62 or reducing intracellular calcium levels by BAPTA-AM abrogated the antimycobacterial activity of calcimycin. Conclusion - Calcimycin exerts its antimycobacterial effect by regulating intracellular calcium-dependent ATP release that induces autophagy in a P2RX7 dependent manner.