Research Article: Role of Calmodulin-Calmodulin Kinase II, cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

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.

Partial Text

Aeromonas hydrophila, a Gram-negative, rod-shaped, facultatively intracellular bacterium is commonly found as part of the normal microbial flora in the aquatic environment [1]. 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 [2]. A. hydrophila is also known for its wide range of host tropism that includes amphibians, reptiles as well as mammals [2]. In humans, this bacterium is frequently associated with individuals suffering from gastroenteritis, wound infections, septicemia and immunodeficiency disorders [1].

Calmodulin plays a significant role in microbial pathogenicity. Its role has been implicated in the pathogenicity of Mycobacterium sp. [28], Clostridium perfingens[30] and for survival of Pneumocystis-infected alveolar macrophages [31]. The presence of well conserved CaM is well documented in fish [32], [33]. As we observed increased intracellular Ca2+-levels in infected HKM [19] 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 [34]. 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 [35] 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.




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