Abstract
Serine protease inhibitors (serpins) mediate many biological processes, including immune responses to pathogenic infection. In this study, a member of the serpin superfamily was identified from the common poultry parasite Eimeria tenella by expressed sequence tag analysis and the rapid amplification of cDNA ends technique. The full-length cDNA was 1,918 bp and had an open reading frame of 1,248 bp encoding a polypeptide of 415 amino acids with the theoretical isoelectric point of 5.26 and predicted molecular weight of 45.5 kDa. Real-time quantitative PCR analysis revealed that the serpin gene was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). The sequence encoding the mature protein was amplified by PCR, cloned into the pET28(a) vector, and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was prepared and used to determine invasion inhibition capacity and localization; the results suggested that the serpin may play an important role in invasion and survival of the sporoziotes in the host.
Similar content being viewed by others
References
Allen PC, Fetterer RH (2002) Recent advances in biology and immunobiology of Eimeria species and in diagnosis and control of infection with these coccidian parasites of poultry. Clin Microbiol Rev 15:58–65
Barr SC, Warner K (2003) Characterization of a serine protease activity in Sarcocystis neurona merozoites. J Parasitol 89:385–388
Bruno SV, Duschak G, Ledesma B, Ferella M, Adersson B, Guarnera EA, Angel SO (2004) Identification and characterization of serine protease inhibitors from Neospora caninum. Mol Biochem Parasitol 136:101–107
Danforth HD, Augustine PC, Clare RA (1994) Ultrastructural observations of development of Eimeria tenella in a novel established avian-derived cell line. Parasitol Res 80:588–593
Dong H, Lin JJ, Han HY, Jiang LL, Zhao QP, Zhu SH, Huang B (2010) Analysis of differentially expressed genes in the precocious line of Eimeria maxima and its parent strain using suppression subtractive hybridization and cDNA microarrays. Parasitol Res. doi:10.1007/s00436-010-2149-7
Fetterer RH, Miska KB, Lillehoj H, Barfield RC (2007a) Serine protease activity in developmental stages of Eimeria tenella. J Parasitol 93:333–340
Fetterer RH, Jenkins MC, Miska KB, Barfield RC (2007b) Characterization of the antigen SO7 during development of Eimeria tenella. J Parasitol 93:1107–1113
Fetterer RH, Miska KB, Jenkins MC, Barfield RC, Lillehoj H (2008) Identification and characterization of a serpin from Eimeria acervulina. J Parasitol 96:1269–1274
Ford L, Guiliano DB, Oksov Y, Debnath AK, Liu J, Williams SA, Blaxer ML, Lustigman S (2005) Characterization of a novel filarial serine protease inhibitor, Ov-SPI-1, from Onchocerca volvulus, with potential multifunctional roles during development of the parasite. J Biol Chem 280:40845–40856
Fuller AL, Mcdougald LR (1990) Reduction in cell entry of Eimeria tenella (Coccidia) sporozoites by protease inhibitors and partial characterization of proteolytic activity associated with intact sporozoites and merozoites. J Parasitol 76:464–467
Gent D, Sharp P, Morgan K, Noor K (2003) Serpins: structure, function and molecular evolution. Int J Biochem Cell Biol 35:1536–1547
Han HY, Lin JJ, Zhao QP, Dong H, Jiang LL, Xu MQ, Zhu SH, Huang B (2010) Identification of differentially expressed genes in early stages of Eimeria tenella by suppression subtractive hybridization and cDNA microarray. J Parasitol 96:95–102
Hwang JH, Lee WG, Na BK, Lee HW, Cho SH, Kim TS (2009) Identification and characterization of a serine protease inhibitor of Paragonimus westermani. Parasitol Res 104:495–501
Innes EA, Vermeulen AN (2006) Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora. Parasitol 133:145–168
Jahn D, Matros A, Bakulina AY, Tiedemann J, Schubert U, Giersberg M, Haehnel S, Zoufal K, Mock H, Kipriyanov SM (2009) Model structure of the immunodominant surface antigen of Eimeria tenella identified as a target for sporozoite-neutralizing monoclonal antibody. Parasitol Res 105:655–668
Kang JM, Sohn WM, Ju JW, Kim TS, Na BK (2010) Identification and characterization of a serine protease inhibitor of Clonorchis sinensis. Acta Tropica 116:134–140
Kim K (2004) Role of host protease in host cell invasion by Toxoplasma gondii and other Apicomplexa. Acta Trop 91:69–81
Klotz C, Marhofer RJ, Selzer PM, Jucius R, Pogonka T (2005) Eimeria tenella: identification of secretory and surface proteins from expressed sequence tags. Experimental Parasitol 111:14–23
Knox DP (2007) Proteinase inhibitors and helminth parasite infection. Parasite Immunol 29:57–71
Labbe M, Venevellesa PD, Girard-Misguichb F, Bourdieua C, Guillaumeb A, Pery P (2005) Eimeria tenella microneme protein EtMIC3: identification, localization and role in host cell infection. Mol Biochem Parasitol 140:43–53
Miska KB, Fetterer RH, Barfield RC (2004) Analysis of transcripts expressed by Eimeria tenella oocysts using subtractive hybridization methods. J Parasitol 90:1245–1252
Morris MT, Coppin A, Tomavo S, Carruthers VB (2002) Functional analysis of Toxoplasma gondii protease inhibitor 1. J Biol Chem 277:45259–45266
Ng ST, Jangi MS, Shirley MW, Tomley FM, Wan KL (2002) Comparative EST analyses provide insights into gene expression in two asexual developmental stages of Eimeria tenella. Experimental Parasitol 101:168–173
Patton WH (1965) Eimeria tenella: cultivation of the asexual stage in cultured animal cells. Science 150:767–769
Peroval M, Pery P, Labbe M (2006) The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth. Int J Parasitol 36:1205–1215
Pszenny V, Angel SO, Duschak VG, Paulino M, Ledesma B, Yabo MI, Guarnera E, Ruiz AM, Bontempie J (2000) Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii. Mol Biochem Parasitol 107:241–249
Shirley MW (1995) Eimeria species and strains of chickens. In: Eckert J, Braun R, Shirley MW, Coudert P (eds) Biotechnology—guidelines on techniques in coccidiosis research. The European Commission DGXII, Luxembourg City, Luxembourg, p 24
Shirley MW, Smith AL, Tomley FM (2005) The biology of avian Eimeria with an emphasis on their control by vaccination. Adv Parasitol 60:285–330
Shirley MW, Smith AL, Blake DP (2007) Challenges in the successful control of the avian coccidia. Vaccine 25:5540–5547
Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW, Pemberton PA, Remold-O'Donnell E, Salvesen GS, Travis J, Whisstock JC (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 276:33293–33296
Tomley FM (1997) Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites. Methods Enzymol 13:171–176
Tomley FM, Billington KJ, Bumstead JM (2001) EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats. Int J Parasitol 31:1303–1310
Williams RB (2006) Tracing the emergence of drug-resistance in coccidian(Eimeria spp.) of commercial broiler flocks medicated with decoquinate for the first time in the United Kingdom. Vet Parasitol 135:1–14
Xie MQ, Gilbert JM, Fuller AL, Mcdougald LR (1990) A new method for purification of Eimeria tenella merozoites. Parasitol Res 76:566–569
Zang XX, Maizels RM (2001) Serine proteinase inhibitors from nematodes and the arms race between host and pathogen. Trends Biochem Sci 26:191–197
Zang XX, Yazdanbakhsh M, Jiang HB, Kanost MR, Maizels RM (1999) A novel serpin expressed by the blood-borne microfilariae of the parasitic nematode Brugia malayi inhibits human neutrophil serine proteinases. Blood 94:1418–1428
Acknowledgments
This work was supported by the Shanghai Science Foundation of China (Grant No. 09ZR1438700). We would like to thank Prof. Chan Ding (CAAS) for generously providing the DF-1 cell line used in this study. We also thank Zhixin Zhao and Beimin Zhang for their excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Jiang, L., Lin, J., Han, H. et al. Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella . Parasitol Res 110, 865–874 (2012). https://doi.org/10.1007/s00436-011-2568-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-011-2568-0