Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages

Mycobacterium fortuitum causes ‘mycobacteriosis’ in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca +2 )-signalling cascade on M . fortuitum- induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M . fortuitum could trigger intracellular-Ca +2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKII g ). Gene silencing and inhibitor studies established the role of CaM in M . fortuitum pathogenesis. We noted that CaMKII g activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKII g in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKII g to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M . fortuitum pathogenesis. We propose that M . fortuitum triggers intracellular Ca +2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKII g resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M . fortuitum -infected HKM.