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Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella

  • H.Y. HAN (a1), S.H. ZHU (a1), L.L. JIANG (a1), Y. LI (a1), H. DONG (a1), Q.P. ZHAO (a1), C.L. KONG (a1) and B. HUANG (a1)...
Summary
SUMMARY

The calcium-dependent protein kinases (CDPKs) are unique enzymes found only in plants, green algae, ciliates and apicomplexan parasites. In this study, a novel CDPK gene of Eimeria tenella, designed EtCDPK3, was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). The entire cDNA of EtCDPK3 contained 1637 nucleotides encoding 433 amino acids and the deduced EtCDPK3 protein had canonical characteristic domains identified in other CDPKs, including a well-conserved amino-terminal kinase domain and a carboxy-terminal calmodulin-like structure with 4 EF-hand motifs for calcium binding. The expression profiles of the EtCDPK3 gene in different development stages were investigated by real-time quantitative PCR. Messenger RNA levels from the EtCDPK3 gene were higher in sporozoites than in other stages (unsporulated oocysts, sporulated oocysts and merozoites). Western blot analysis showed that rabbit antiserum against recombinant EtCDPK3 could recognize a native 49 kDa protein band of parasite. Indirect immunofluorescent antibody labelling revealed dispersed localization of EtCDPK3 during the first schizogony and intense specific staining. EtCDPK3 was located at the apical end of the sporozoites after early infection of DF-1 cells and the protein was highly expressed. Inhibition of EtCDPK3 function using specific antibodies reduced the ability of E. tenella to invade host cells. These results suggested that EtCDPK3 may be involved in invasion and survival of the parasite intracellular stages of E. tenella. Because this kinase family is absent from hosts, it represents a valid target that could be exploited for chemotherapy against Eimeria spp.

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*Corresponding author. Tel: +86(0)21 34293464. Fax: +86(0)21 54081818. E-mail address: hb@shvri.ac.cn
References
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Billker O., Tewari R., Franke-Fayard B. and Brinkman V. (2004). Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 117, 503514.
Billker O., Lourido S. and Sibley L. D. (2009). Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host and Microbe 18, 612622.
Blackman M. J. and Bannister L. H. (2001). Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation. Molecular and Biochemical Parasitology 117, 1125.
Choi K. M., Kim J. Y., Moon S. U., Lee H. W., Sattabongkot J., Na B. K., Kim D. W., Suh E. J., Kim Y. J., Cho S. H., Lee H. S., Rhie H. G. and Kim T. S. (2010). Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4. Korean Journal of Parasitology 48, 319324.
Dalloul R. A. and Lillehoj H. S. (2005). Recent advances in immunomodulation and vaccination strategies against coccidiosis. Avian Disease 49, 18.
Donald R. G. K., Zhong T., Wiersma H., Nare B., Yao D., Lee A., Allocco J. and Liberator P. A. (2006). Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase. Molecular and Biochemical Parasitology 149, 8698.
Dunn P. P., Bumstead J. M. and Tomley F. M. (1996). Sequence, expression and localization of calmodulin-domain protein kinases in Eimeria tenella and Eimeria maxima. Parasitology 113, 439448.
Dvorin J. D., Martyn D. C., Patel S. D., Grimley J. S., Collins C. R., Hopp C. S., Bright A. T., Westenberger S., Winzeler E., Blackman M. J. and Baker D. A., Wandless T. J. and Duraisingh M. T. (2010). A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes. Science 328, 910912.
Han H. Y., Lin J. J., Zhao Q. P., Dong H., Jiang L. L., Xu M. Q., Zhu S. H. and Huang B. (2010). Identification of differentially expressed genes in early stages of Eimeria tenella by suppression subtractive hybridization and cDNA microarray. Journal of Parasitology 96, 95102.
Harper J. F. and Harmon A. C. (2005). Plants, symbiosis and parasites: a calcium signaling connection. Nature Reviews Molecular Cell Biology 6, 555566.
Ishino T., Orito Y., Chinzei Y. and Yuda M. (2006). A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell. Molecular Microbiology 59, 11751184.
Jahn D., Matros A., Bakulina A. Y., Tiedemann J., Schubert U., Giersberg M., Haehnel S., Zoufal K., Mock H. and Kipriyanov S. M. (2009). Model structure of the immunodominant surface antigen of Eimeria tenella identified as a target for sporozoite-neutralizing monoclonal antibody. Parasitology Research 105, 655668.
Jiang L. L., Lin J. J., Han H. Y., Zhao Q. P., Dong H., Zhu S. H. and Huang B. (2012 a). Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella. Parasitology Research 110, 865874.
Jiang L. L., Lin J. J., Han H. Y., Dong H., Zhao Q. P., Zhu S. H. and Huang B. (2012 b). Identification and characterization of Eimeria tenella apical membrane antigen-1(AMA1). PLos One 7, e41115.
Kieschnick H., Wakefied T., Narducci C. A. and Beckers C. (2001). Toxoplasma gondii attachment to host cells is regulated by a calmodulin-like domain protein kinase. Journal of Biological Chemistry 276, 1236912377.
Kinnaird J. H., Bumstead J. M., Mann D. J., Ryan R., Shirley M. W., Shiels B. R. and Tomley F. M. (2004). EtCRK2, a cyclin-dependent kinase gene expressed during the sexual and asexual phases of the Eimeria tenella life cycle. International Journal for Parasitology 34, 683692.
Labbe M., Peroval M., Bourdieu C., Girard-Misquich F. and Pery P. (2006). Eimeria tenella enolase and pyruvate kinase: a likely role in glycolysis and in other functions. International Journal for Parasitology 36, 14431452.
Li J. L., Baker D. A. and Cox L. S. (2000). Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum. Biochimica et Biophysica Acta – Biomembranes 1491, 341349.
Lourido S., Shuman J., Zhang C., Shokat K. M., Hui R. and Sibley L. D. (2010). Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature, London 465, 359362.
Miska K. B., Fetterer R. H. and Barfield R. C. (2004). Analysis of transcripts expressed by Eimeria tenella oocysts using subtractive hybridization methods. Journal of Parasitology 90, 12451252.
Möskes C., Burghaus P. A., Wernli B., Sauder U., Durrenberger M. and Kappes B. (2004). Export of Plasmodium falciparum calcium-dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N-terminal membrane anchor motifs. Molecular Microbiology 54, 676691.
Nagamune K. and Sibley L. D. (2006). Comparative genomic and phylogenetic analysis of calcium ATPases and calcium-regulated proteins in the apicomplexa. Molecular Biology and Evolution 23, 16131627.
Ng S. T., Sanusi Jangi M., Shirley M. W., Tomley F. M. and Wan K. L. (2002). Comparative EST analyses provide insights into gene expression in two asexual developmental stages of Eimeria tenella. Experimental Parasitology 101, 168173.
Peroval M., Pery P. and Labbe M. (2006). The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth. International Journal for Parasitology 36, 12051215.
Podell S. and Gribskov M. (2004). Predicting N-terminal myristoylation sites in plant proteins. BMC Genomics 5, 37.
Ranjan R., Ahmed A., Gourinath S. and Sharma P. (2009). Dissection of mechanisms involved in the regulation of Plasmodium falciparum calcium-dependent protein kinase 4. Journal of Biological Chemistry 284, 1526715276.
Reddy V. S. and Reddy A. S. (2004). Proteomics of calcium-signaling components in plants. Phytochemistry 65, 17451776.
Shirley M. W. (1995). Eimeria species and strains of chickens. In Biotechnology – Guidelines on Techniques in Coccidiosis Research (ed. Coudert P., Eckert J., Braun R. and Shirley M. W.), pp. 910. The European Commission DGXII, Luxembourg City, Luxembourg.
Shirley M. W., Ivens A., Gruber A., Madeira A. M., Wan K. L., Dear P. H. and Tomley F. M. (2004). The Eimeria genome projects: a sequence of events. Trends in Parasitology 20, 199201.
Shirley M. W., Smith A. L. and Tomley F. M. (2005). The biology of avian Eimeria with an emphasis on their control by vaccination. Advances in Parasitology 60, 285330.
Shirley M. W., Smith A. L. and Blake D. P. (2007). Challenges in the successful control of the avian coccidia. Vaccine 25, 55405547.
Tomley F. M. (1997). Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites. Methods in Enzymology 13, 171176.
Wang B. L., Xu Y., Wu C. Q., Xu Y. M. and Wang H. H. (2005). Cloning, expression, and refolding of a secretory protein ESAT-6 of mycobacterium tuberculosis. Protein Expression and Purification 39, 184188.
Wiersma H. I., Galuska S. E., Tomley F. M., Sibley L. D., Liberator P. A. and Donald R. G. (2004). A role for coccidian cGMP-dependent protein kinase in motility and invasion. International Journal for Parasitology 34, 369380.
Zhao Y., Franklin R. M. and Kappes B. (1994 a). Plasmodium falciparum calcium-dependent protein kinase phosphorylates proteins of the host erythrocytic membrane. Molecular and Biochemical Parasitology 66, 329343.
Zhao Y., Pokutta S., Maurer P., Lindt M., Franklin R. M. and Kappes B. (1994 b). Calcium-binding properties of a calcium-dependent protein kinase from Plasmodium falciparum and the significance of individual calcium-binding sites for kinase activation. Biochemistry 33, 37143721.
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Parasitology
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