Date Published: April 24, 2014
Publisher: Public Library of Science
Author(s): Chaitali Banerjee, Preeti Khatri, Rajagopal Raman, Himanshi Bhatia, Malabika Datta, Shibnath Mazumder, Steven R. Blanke.
The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.
Aeromonas hydrophila, a Gram-negative, rod-shaped, facultatively intracellular bacterium is commonly found as part of the normal microbial flora in the aquatic environment . The pathogenicity of A. hydrophila is complex and multi-factorial. It induces a plethora of symptoms in fish characterized by severe open dermal ulcers, anaemia, visceral granulomata, septicaemia, failure of osmoregulatory balance and death, which together comprise the ulcerative disease syndrome or UDS . A. hydrophila is also known for its wide range of host tropism that includes amphibians, reptiles as well as mammals . In humans, this bacterium is frequently associated with individuals suffering from gastroenteritis, wound infections, septicemia and immunodeficiency disorders .
Calmodulin plays a significant role in microbial pathogenicity. Its role has been implicated in the pathogenicity of Mycobacterium sp. , Clostridium perfingens and for survival of Pneumocystis-infected alveolar macrophages . The presence of well conserved CaM is well documented in fish , . As we observed increased intracellular Ca2+-levels in infected HKM  and CaM being a well-known Ca2+-sensor, we hypothesised a role of CaM on the pathogenicity of A. hydrophila. Indeed, results obtained from qRT-PCR, specific EIA assays, siRNA and pharmacological inhibitors conclusively demonstrated the importance of Ca2+-induced CaM expression in the initiation of A. hydrophila-induced HKM apoptosis. The kinetics of CaM expression, both at transcript and protein level demonstrated maximum expression at 2 h p.i. This suggested CaM expression at protein level closely follows pattern at transcript level, a phenomenon observed for many signalling molecules . To our knowledge, this is the first report that clearly documents the pro-apoptotic involvement of CaM in A. hydrophila infections. We had earlier reported the role of calpain-2 in A. hydrophila-induced HKM apoptosis. We questioned whether the two Ca2+-dependent molecules crosstalk to initiate HKM apoptosis. Contrary to our expectations we did not observe any crosstalk between calpain-2 and CaM activity (data not shown), which suggests two possibilities: (a) CaM is resistant to the lytic action of calpains  and (b) CaM and calpain activity are independent to each other in our model.
All the chemicals used in this study were purchased from Sigma unless otherwise stated.