title: 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 (PKCα) 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 PKCα-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 PKCα-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.
authors.affiliation: Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
authors.forename: Debika
authors.initials: D
authors.lastname: Datta
authors.affiliation: Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
authors.forename: Preeti
authors.initials: P
authors.lastname: Khatri
authors.affiliation: Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
authors.forename: Chaitali
authors.initials: C
authors.lastname: Banerjee
authors.affiliation: Gut Biology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
authors.forename: Ambika
authors.initials: A
authors.lastname: Singh
authors.affiliation: School of Environmental Sciences, Jawaharlal Nehru University, Delhi, India.
authors.forename: Ramavatar
authors.initials: R
authors.lastname: Meena
authors.affiliation: Microscopy Laboratory, National Institute of Cholera and Enteric Diseases, Kolkata, India.
authors.forename: Dhira Rani
authors.initials: DR
authors.lastname: Saha
authors.affiliation: Gut Biology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
authors.forename: Rajagopal
authors.initials: R
authors.lastname: Raman
authors.affiliation: School of Environmental Sciences, Jawaharlal Nehru University, Delhi, India.
authors.forename: Paulraj
authors.initials: P
authors.lastname: Rajamani
authors.affiliation: Genome Analysis Laboratory, Animal Division, Indian Veterinary Research Institute, Izatnagar, Bareilly, India.
authors.forename: Abhijit
authors.initials: A
authors.lastname: Mitra
authors.affiliation: Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi, India.
authors.forename: Shibnath
authors.initials: S
authors.lastname: Mazumder
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002397:Catfishes / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008264:Macrophages / Q000378:metabolism*; D019907:Mycobacterium fortuitum / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
chemical_list: D013481:Superoxides; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D002118:Calcium