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Cloning and molecular analysis of the aspartic protease Sc-ASP110 gene transcript in Steinernema carpocapsae

Published online by Cambridge University Press:  04 June 2013

NATESAN BALASUBRAMANIAN  and
NELSON SIMÕES
NATESAN BALASUBRAMANIAN*
Affiliation:
CIRN and Department of Biology, University of Azores, 9501-855 Ponta Delgada, Portugal
NELSON SIMÕES
Affiliation:
CIRN and Department of Biology, University of Azores, 9501-855 Ponta Delgada, Portugal
*
*Corresponding author: CIRN and Department of Biology, University of Azores, 9501-855 Ponta Delgada, Portugal. E-mail: [email protected]; [email protected]
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Summary

Many protease genes have previously been shown to be involved in parasitism and in the development of Steinernema carpocapsae, including a gene predicted to encode an aspartic protease, Sc-ASP110, which was cloned and was analysed in this study. A cDNA encoding Sc-ASP110 was cloned based on an expressed sequence tag (EST) fragment from our EST library. The full-length cDNA of Sc-ASP110 consists of 1112 nucleotides with a catalytic aspartic domain (aa18–337). The putative 341 amino acid residues have a calculated molecular mass of 37·1 kDa and a theoretical pI of 4·7. BLASTp analysis of the Sc-ASP110 amino acid sequence showed 45–77% amino acid sequence identity to parasitic and non-parasitic nematode aspartic proteases. An expression analysis showed that the sc-asp110 gene was upregulated during the late parasitic stage, L4, and 24 h after induction of in vitro nematodes. A sequence comparison revealed that Sc-ASP110 was a member of an aspartic protease family; additionally, a phylogenetic analysis indicated that Sc-ASP110 was clustered with the closely related nematode Steinernema feltiae. In situ hybridization showed that sc-asp110 was expressed in the body walls of dorsal cells. The upregulated Sc-ASP110 expression revealed that this protease could play a role in the late parasitic process. In this study, we have cloned and analysed the gene transcript of Sc-ASP110 in S. carpocapsae.

Keywords

Aspartic proteasedevelopmental stagegene transcriptparasitic nematodeSteinernema carpocapsae
Type
Research Article
Information
Parasitology , Volume 140 , Issue 9 , August 2013 , pp. 1158 - 1167
Copyright
Copyright © Cambridge University Press 2013 

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References

REFERENCES

Antonov, V. K., Ginodman, L. M., Kapitannikov, Y. V., Barshcvskaya, T. N., Gurova, A. G. and Rumsh, L. D. (1978). Mechanism of pepsin catalysis general base catalysis by active site carboxylate ion. FEBS Letters 88, 87–90.CrossRefGoogle ScholarPubMed
Ausbel, F. M. (1989). Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY.Google Scholar
Balasubramanian, N., Hao, Y. J., Toubarro, D., Nascimento, G. and Simões, N. (2009). Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae. International Journal for Parasitology 39, 975–984.CrossRefGoogle ScholarPubMed
Balasubramanian, N., Toubarro, D. and Simões, N. (2010). Biochemical study and in vitro insect immune suppression by a trypsin-like secreted protease from the nematode Steinernema carpocapsae. Parasite Immunology 32, 165–175.CrossRefGoogle ScholarPubMed
Balasubramanian, N., Toubarro, D., Nascimento, G., Ferreira, R. and Simões, N. (2012 a). Purification, molecular characterization and gene expression analysis of an aspartic protease (Sc-ASP113) from the nematode Steinernema carpocapsae during the parasitic stage. Molecular and Biochemical Parasitology 182, 37–44.CrossRefGoogle ScholarPubMed
Balasubramanian, N., Nascimento, G., Ferreira, R., Martinez, M. and Simões, N. (2012 b). Pepsin-like aspartic protease (Sc-ASP155) cloning, molecular characterization and gene expression analysis in developmental stages of nematode Steinernema carpocapsae. Gene 500, 164–171.CrossRefGoogle ScholarPubMed
Becker, M. M., Harrop, S. A., Dalton, J. P., Kalinna, B. H., McManus, D. P. and Brindley, P. J. (1995). Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in haemoglobin degradation. Journal of Biological Chemistry 270, 24496–24501.CrossRefGoogle Scholar
Bellafiore, S., Shen, Z., Rosso, M. N., Abad, P., Shih, P. and Briggs, S. P. (2008). Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential. PLoS Pathogens 4, e1000192.CrossRefGoogle ScholarPubMed
Brindley, P. J., Kalinna, B. H., Wong, J. Y., Bogitsh, B. J., King, L. T., Smyth, D. J., Verity, C. K., Abbenante, G., Brinkworth, R. I., Fairlie, D. P., Smythe, M. L., Milburn, P. J., Bielefeldt-Ohmann, H., Zheng, Y. and McManus, D. P. (2001). Proteolysis of human hemoglobin by Schistosome cathepsin D. Molecular and Biochemical Parasitology 112, 103–112.CrossRefGoogle ScholarPubMed
Burman, M. (1982). Neoaplectana carpocapsae: toxin production by axenic insect parasitic nematodes. Nematologica 28, 62–70.CrossRefGoogle Scholar
Calderwood, M. S., Gannoun-Zaki, L., Wellems, T. E. and Deitsch, K. W. (2003). Plasmodium falciparum var genes are regulated by two regions with separate promoters, one upstream of the coding region and a second within the intron. Journal of Biological Chemistry 278, 34125–34132.CrossRefGoogle Scholar
de Boer, J. M., Yan, Y., Davis, E. L., Smant, G. and Baum, T. J. (1998). In-situ hybridization to messenger RNA in Heterodera glycines. Journal of Nematology 30, 309–312.Google ScholarPubMed
Dunn, B. M. (2002). Structure and mechanism of the pepsin-like family of aspartic peptidases. Chemical Review 102, 4431–4458.CrossRefGoogle ScholarPubMed
Dutky, S. R. (1959). Insect microbiology. Advances in Applied Microbiology 1, 175–200.CrossRefGoogle ScholarPubMed
Ehlers, R. U. (2001). Mass production of entomopathogenic nematodes for plant protection. Applied Microbiology and Biotechnology 56, 623–633.CrossRefGoogle ScholarPubMed
Forst, S., Dowds, B., Boemare, N. and Stackebrandt, E. (1997). Xenorhabdus and Photorhabdus spp. bugs that kill bugs. Annual Review of Microbiology 51, 47–72.CrossRefGoogle ScholarPubMed
Geier, G., Banaj, H. J., Heid, H., Bini, L., Pallini, V. and Zwilling, R. (1999). Aspartyl proteases in Caenorhabditis elegans. Isolation, identification and characterization by a combined use of affinity chromatography, two-dimensional gel electrophoresis, microsequencing and databank analysis. European Journal of Biochemistry 264, 872–879.CrossRefGoogle ScholarPubMed
Hao, Y. J., Montiel, R., Abubucker, S., Mitreva, M. and Simões, N. (2010). Transcripts analysis of the entomopathogenic nematode Steinernema carpocapsae induced in vitro with insect haemolymph. Molecular and Biochemical Parasitology 169, 79–86.CrossRefGoogle ScholarPubMed
Hao, Y. J., Montiel, R., Lucena, M. A., Costa, M. and Simões, N. (2012). Genetic diversity and comparative analysis of gene expression between Heterorhabditis bacteriophora Az29 and Az36 isolates: uncovering candidate genes involved in insect pathogenicity. Experimental Parasitology 130, 116–125.CrossRefGoogle ScholarPubMed
Hussey, R. S. (1989). Disease-inducing secretions of plant-parasitic nematodes. Annual Review of Phytopathology 27, 123–141.CrossRefGoogle Scholar
Hwang, K. P., Chang, S. H. and Wang, L. C. (2010). Alterations in the expression level of a putative aspartic protease in the development of Angiostrongylus cantonensis. Acta Tropica 113, 289–294.CrossRefGoogle ScholarPubMed
Jing, Y. J., Toubarro, D., Hao, Y. J. and Simões, N. (2010). Cloning, characterisation and heterologous expression of an astacin metalloprotease, Sc-AST, from the entomoparasitic nematode Steinernema carpocapsae. Molecular and Biochemical Parasitology 174, 101–108.CrossRefGoogle ScholarPubMed
Jolodar, A. and Miller, D. J. (1998). Identification of a novel family of non-lysosomal aspartic proteases in nematodes. Biochimica et Biophysica Acta 1382, 13–16.CrossRefGoogle ScholarPubMed
Kaya, H. K., Aguillera, M. M., Alumai, A., Choo, H. Y., de la Torre, M., Fodor, A., Ganguly, S., Hazir, S., Lakatos, T., Pye, A., Wilson, M., Yamanaka, S., Yang, H. and Ehlers, R. U. (2006). Status of entomopathogenic nematodes and their symbiotic bacteria from selected countries or regions of the world. Biological Control 38, 134–155.CrossRefGoogle Scholar
Kikuchi, T. (2008) Parasitism genes of the pine wood nematode. In Pine Wilt Disease (ed. Zhao, B. G., Futai, K., Sutherland, J. R. and Takeuchi, Y.), pp. 67–80. Springer, Tokyo, Japan.CrossRefGoogle Scholar
Laumond, C., Simões, N. and Boemare, N. (1989). Toxins of entomoparasitic nematodes. Pathogenicity of Steinernema carpocapsae prospectives of genetic engineering. Comptes rendus des séances de l'Académie d'Agriculture de France 75, 135–138.Google Scholar
Lin, S., Jian, H., Zhao, H., Yang, D., Liu, Q. (2011). Cloning and characterization of a venom allergen-like protein gene cluster from the pinewood nematode Bursaphelenchus xylophilus. Experimental Parasitology 127, 440–447.CrossRefGoogle ScholarPubMed
Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta DeltaC(T)) method. Methods 25, 402–408.CrossRefGoogle Scholar
Longbottom, D., Redmond, D. L., Russell, M., Liddell, S., Smith, W. D. and Knox, D. P. (1997). Molecular cloning and characterisation of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus. Molecular and Biochemical Parasitology 88, 63–72.CrossRefGoogle ScholarPubMed
McKerrow, J. H., Brindley, P., Brown, M., Gam, A. A., Staunton, C. and Neva, F. A. (1990). Strongyloides stercoralis: identification of a protease that facilitates penetration of skin by the infective larvae. Experimental Parasitology 70, 134–143.CrossRefGoogle ScholarPubMed
Morales, M. E., Kalinna, B. H., Heyers, O., Mann, V. H., Schulmeister, A., Copeland, C. S., Loukas, A. and Brindley, P. J. (2004). Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D. Gene 338, 99–109.CrossRefGoogle ScholarPubMed
Muhia, D. K., Swales, C. A., Eckstein-Ludwig, U., Saran, S., Polley, S. D., Kelly, J. M., Schaap, P., Krishna, S. and Baker, D. A. (2003). Multiple splice variants encode a novel adenylyl cyclase of possible plastid origin expressed in the sexual stage of the malaria parasite Plasmodium falciparum. Journal of Biological Chemistry 278, 22014–22022.CrossRefGoogle ScholarPubMed
Nickel, W. (2003). The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. European Journal of Biochemistry 270, 2109–2119.CrossRefGoogle ScholarPubMed
Rosa, J. S., Cabral, C. and Simoes, N. (2002). Differences between the pathogenic processes induced by Steinernema and Heterorhabditis Nemata: Rhabditida in Pseudaletia unipuncta Insecta: Lepidoptera. Journal of Invertebrate Pathology 80, 46–54.CrossRefGoogle ScholarPubMed
Schulmeister, A., Heyers, O., Morales, M. E., Brindley, P. J., Lucius, R., Meusel, G. and Kalinna, B. H. (2005). Organization and functional analysis of the Schistosoma mansoni cathepsin D-like aspartic protease gene promoter. Biochemical Biophysical Acta 1727, 27–34.CrossRefGoogle ScholarPubMed
Shapiro-Ilan, D. I., Lewis, E. E., Son, Y. and Tedders, W. L. (2003). Superior efficacy observed in entomopathogenic nematodes applied in infected-host cadavers compared with application in aqueous suspension. Journal of Invertebrate Pathology 83, 270–272.CrossRefGoogle ScholarPubMed
Szecsi, P. B. (1992). The aspartic proteases. Scandinavian Journal of Clinical and Laboratory Investigation. Supplementum 210, 5–22.Google Scholar
Tcherepanova, I., Bhattacharyya, L., Rubin, C. S. and Freedman, J. H. (2000). Aspartic proteases from the nematode Caenorhabditis elegans. Structural organization and developmental and cell-specific expression of asp-1. Journal of Biological Chemistry 275, 26359–26369.CrossRefGoogle ScholarPubMed
Toubarro, D., Lucena-Robles, M., Nascimento, G., Santos, R., Montiel, R., Veríssimo, P., Pires, E., Faro, C., Coelho, A. V. and Simões, N. (2010). Serine protease-mediated host invasion by the parasitic nematode Steinernema carpocapsae. Journal of Biological Chemistry 285, 30666–30675.CrossRefGoogle ScholarPubMed
Volkman, S. K., Barry, A. E., Lyons, E. J., Nielsen, K. M., Thomas, S. M., Choi, M., Thakore, S. S., Day, K. P., Wirth, D. F. and Hartl, D. L. (2001). Recent origin of Plasmodium falciparum from a single progenitor. Science 293, 482–484.CrossRefGoogle ScholarPubMed
Williamson, A. L., Brindley, P. J., Abbenante, G., Prociv, P., Berry, C., Girdwood, K., Pritchard, D. I., Fairlie, D. P., Hotez, P. J., Dalton, J. P. and Loukas, A. (2002). Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity. Federation of American Societies for Experimental Biology Journal 16, 1458–1460.CrossRefGoogle ScholarPubMed
Williamson, A. L., Brindley, P. J., Knox, D. P., Hotez, P. J. and Loukas, A. (2003). Digestive proteases of blood-feeding nematodes. Trends in Parasitology 19, 417–423.CrossRefGoogle ScholarPubMed
Yang, Y., Wei, H., Qin, W. and Zheng, J. (2009). Expression and characterization of aspartic protease gene in eggs and larvae stage of Ancylostoma caninum. Parasitology Research 104, 1327–1333.CrossRefGoogle ScholarPubMed