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

ABSTRACT: 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 (PKC?) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKC?-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg 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 PKC?-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg 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.

SUBMITTER: Datta D

PROVIDER: S-EPMC4713470 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

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

Datta Debika D Khatri Preeti P Banerjee Chaitali C Singh Ambika A Meena Ramavatar R Saha Dhira Rani DR Raman Rajagopal R Rajamani Paulraj P Mitra Abhijit A Mazumder Shibnath S

PloS one 20160111 1

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 (CaMKIIg). Gene silencing and inhibitor studies established the role of ...[more]

PMID: 26752289

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Project description:Initiation of nitric oxide (NO.)-mediated apoptotic cell death in RAW 264.7 macrophages is associated with up-regulation of mitochondrial manganese superoxide dismutase (MnSOD; SOD2) and down-regulation of cytosolic copper zinc superoxide dismutase (CuZnSOD; SOD1) at their individual mRNA and protein levels. To evaluate the decreased CuZnSOD expression and the initiation of apoptosis we stably transfected macrophages to overexpress human CuZnSOD. Individual clones revealed a 2-fold increase in CuZnSOD activity. Expression of a functional and thus protective CuZnSOD was verified by attenuated superoxide (O2(.)-)-mediated apoptotic as well as necrotic cell death. In this study we showed that SOD-overexpressing macrophages (R-SOD1-12) were also protected against NO.-initiated programmed cell death. Protection was substantial towards NO. derived from exogenously added NO donors or when NO. was generated by inducible NO synthase activation, and was evident at the level of p53 accumulation, caspase activation and DNA fragmentation. Stimulation of parent and SOD-overexpressing cells with a combination of lipopolysaccharide and murine interferon gamma produced equivalent amounts of nitrite/nitrate, which ruled out attenuated inducible NO. synthase activity during protection. Because protection by a O2(.)--scavenging system during NO. -intoxication implies a role of NO. and O2(.)- in the progression of cell damage, we used uric acid to delineate the role of peroxynitrite during NO.-elicited apoptosis. The peroxynitrite scavenger uric acid left S-nitrosoglutathione or spermine-NO-elicited apoptosis unaltered, blocking only 3-morpholinosydnonimine-mediated cell death. As a result we exclude peroxynitrite from contributing, to any major extent, to NO. -mediated apoptosis. Therefore protection observed with CuZnSOD overexpression is unlikely to stem from interference with peroxynitrite formation and/or action. Unequivocally, the down-regulation of CuZnSOD is associated with NO. cytotoxicity, whereas CuZnSOD overexpression protects macrophages from apoptosis.

Dataset Information

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

Publications

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

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