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Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella

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Abstract

Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in birds. Cysteine protease is a major virulence factor in parasitic protozoa, and it may be a suitable chemotherapeutic target and vaccine candidate molecule. A 100 amino acid (aa.) partial sequence of cathepsin L, which is a cysteine protease, was reported by Katrib et al. (Ac. No. CDJ41293) (2012). A 219 aa. sequence was reported by Reid et al. (Ac. No. AFV92863) (2013). However, the open reading frame (ORF) was not reported. In this study, a full sequence of a cathepsin-L-like peptidase in Eimeria tenella (EtcatL) was obtained and its biochemical characterizations and expression profiles were analyzed across different stages of the parasite’s life cycle. Results showed that the EtcatL gene encodes a protein 470 aa. in length, with 47 and 49 % identity to Toxoplasma gondii and Eimeria acervulina. Considering the close phylogenetic relationship, TgcatL (PDB. ID 3F75) was selected for use as a template for homology modeling with quality factors of 90.9. Gelatin SDS-PAGE showed it to exert protease activity at ≈38 and ≈26 kDa. Further analysis showed the kinetic parameters of the recombinant peptidase to be K m  = 8.9 μM and V max = 5.7 RFU/s μM at pH 5.5 containing 10 mM dithiothreitol (DTT) in the reaction matrix, and the IC50 value of E64 was 65.32 ± 3.02 nM. The recombinant protein was active from 25 to 50 °C, with optimal activity at 42 °C. The RT-PCR and Western blot results showed it to be expressed mainly at the endogenous stages and the initial phase of the sporulation. The protective experiment showed that chickens immunized with 100 and 200 μg rEtcatL had reduction of weight loss values 48.7 and 57.9 % those of infected controls, respectively. Their reduction of lesion scores (RLS) were 25.0 and 47.2 % that of control chickens, and relative oocyst production (ROP) was 39.6 and 15.5 % that of control chickens. These results indicate that the EtcatL can be used as an effective immunogen, and further studies are needed to enhance its potential as a vaccine candidate molecule.

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Acknowledgments

This research was supported by the Joint Project of Natural Science Foundation of China and the Natural Science Foundation of Guangdong Province (no. U0831004 to J. P. Cai). The authors are grateful to the technical staff of our laboratory for their assistance during this study. We would also like to thank LetPub (www.letpub.com) for linguistic assistance during the preparation of this manuscript.

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Authors and Affiliations

  1. National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, No. 2 Yuan Ming Yuan West Road, Haidian District, Beijing, 100193, China

    Renqiang Liu, Aijun Liu & Ming Wang

  2. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institude, Chinese Academy of Agricultural Sciences, No. 1 Xujiaping, Chengguan District, Lanzhou, Gansu Province, China

    Xueting Ma & Jianping Cai

  3. Key Laboratory of Veterinary Bioproduction and Veterinary Medicine of the Ministry of Agriculture, Zhongmu Institutes of China Animal Husbandry Industry Co., Ltd, No. 156 Beiqing Road, Haidian District, Beijing, 100095, China

    Lei Zhang & Ming Wang

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  1. Renqiang Liu
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Correspondence to Jianping Cai or Ming Wang.

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Liu, R., Ma, X., Liu, A. et al. Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella . Parasitol Res 113, 4335–4348 (2014). https://doi.org/10.1007/s00436-014-4107-2

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  • DOI: https://doi.org/10.1007/s00436-014-4107-2

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