Acta Parasitologica

, Volume 58, Issue 1, pp 112–118 | Cite as

Cloning and molecular characterization of cDNAs encoding three Ancylostoma ceylanicum secreted proteins

  • Anna M. Siwińska
  • Piotr Bąska
  • Emilia Daniłowicz-Luebert
  • Kamil Januszkiewicz
  • Ewa Długosz
  • Halina Wędrychowicz
  • Michael Cappello
  • Marcin Wiśniewski
Original Paper
  • 240 Downloads

Abstract

Ancylostoma ceylanicum belongs to a group of soil-transmitted helminths, which infect almost 576 mln people worldwide and are a major cause of anaemia and malnutrition. Upon contact with a permissive host, third-stage larvae (L3) residing in the environment become activated larvae (ssL3), a process associated with changes in the profile of gene expression. Ancylostoma secreted proteins (ASPs) are the major proteins secreted during larvae activation and play a crucial role in hookworm adaptation to parasitism. Here we report the cloning using RACE-PCR technique of three novel ASPs from the hookworm A. ceylanicum (Ace-asp-3, Ace-asp-4, and Ace-asp-5) and computational analysis of the protein sequences. All three proteins contain SCP (Sperm Coating Protein) domain characteristic for previously described ASP proteins. Real-time PCR analysis shows significant up-regulation of Ace-asp-3 and Ace-asp-5 expression in adult worms and correlated down-regulation in ssL3 larvae. On the other hand, expression of Ace-asp-4 was increased in ssL3 stages and decreased in adult parasites.

Keywords

Ancylostoma ceylanicum ASP Ancylostoma secreted proteins 

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References

  1. Albonico M., Smith P.G., Ercole E., Hall A., Chwaya H. M., Alawi K. S., Savioli L. 1995. Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area. Transactions of the Royal Society of Tropical Medicine and Hygiene, 89, 538–541.PubMedCrossRefGoogle Scholar
  2. Albonico M. 2003. Methods to sustain drug efficacy in helminth control programmes. Acta Tropica 86, 233–242. DOI: 10.1016/S0001-706X(03)00043-3.PubMedCrossRefGoogle Scholar
  3. Albonico M., Bickle Q., Ramsan M., Montresor A., Savioli L., Taylor M. 2003. Efficiency of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bulletin of the World Health Organization, 81, 343–352.PubMedGoogle Scholar
  4. Asojo O.A., Goud G., Dhar K., Loukas A., Zhan B., Deumic V., Liu S., Borgstahl G.E.O., Hotez P.J. 2005. X-ray structure of Na-ASP-2, a pathogenesis-related-1 protein from the nematode parasite, Necator americanus, and a vaccigen for human hookworm infection. Journal for Molecular Biology, 346, 801–814. DOI:10.1016/j.jmb.2004.12.023.CrossRefGoogle Scholar
  5. Bajer A., Bednarska M., Rodo A. 2011. Risk factors and control of intestinal parasite infections in sled dogs in Poland. Veterinary Parasitology, 175, 343–350. DOI:10.1016/j.vetpar.2010. 10.029.PubMedCrossRefGoogle Scholar
  6. Benson D.A., Boguski M.S., Lipman D.J., Ostell J., Ouellette F. 1998. GeneBank. Nucleic Acids Research, 261–267. DOI: 10.1093/nar/26.1.1.Google Scholar
  7. Bethony J.M., Cole R.N., Guo X., Kamhawi S., Lightowlers M.W., Loukas A., Petri W., Reed S., Valenzuela J.G., Hotez P.J. 2011. Vaccines to combat the neglected tropical diseases. Immunological Reviews, 239, 237–270. DOI: 10.1111/j.1600-065X.2010.00976.x.PubMedCrossRefGoogle Scholar
  8. Brailsford T.J., Behnke J.M. 1992. The dynamics of trickle infections with Ancylostoma ceylanicum in inbred hamsters. Parasitology, 105, 247–253.PubMedCrossRefGoogle Scholar
  9. Cass C.L., Johanson J.R., Califf L.L. 2007. Proteomic analysis of Schistosoma mansoni egg secretions. Molecular and Biochemical Parasitology, 155, 84–93. DOI: 10.1016/j.molbiopara.2007.06.00.PubMedCrossRefGoogle Scholar
  10. Chu D., Bungiro R.D., Ibanez M., Harrison L.M., Campodonico E., Jones B.F., Mieszczanek J., Kuzmic P., Cappello M. 2004. Molecular characterization of Ancylostoma ceylanicum Kunitztype serine protease inhibitor: evidence for a role in hookworm-associated growth delay. Infection and Immunity, 72, 2214–2221. DOI: 10.1128/IAI.72.4.2214-2221.200.PubMedCrossRefGoogle Scholar
  11. Datu B.J.D., Gasser R.B., Nagaraj S.H., Ong E.K., O’Donoghue P., McInnes R., Ranganathan S., Loukas A. 2008, Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva. Neglected Tropical Diseases, 2, e130. DOI: 10.1371/journal.pntd.000 0130.CrossRefGoogle Scholar
  12. Foster J.A., Gerton G.L. 1996. Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human Tpx-1 and is a member of the cysteinrich secretory protein (CRISP) family. Molecular Reproduction and Development, 44, 221–229.PubMedCrossRefGoogle Scholar
  13. Frochman M.A. 1994. On beyond classic RACE (rapid amplification of cDNA ends). PCR Methods and Application, 4, S40–S58.CrossRefGoogle Scholar
  14. Giese A., Jude R., Kuiper H., Raudsepp T., Piumi F., Schambony A., Guérin G., Chowdhary B.P., Distl O., Töpfer-Petersen E., Leeb T. 2002. Molecular characterization of the equine testisspecific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family. Gene, 299, 101–109.PubMedCrossRefGoogle Scholar
  15. Hawdon J.M., Jones B.F., Hoffman D.R., Hotez P.J. 1996. Cloning and characterization of Ancylostoma secreted protein a novel protein associated with the transition to the parasitism by infective hookworm larvae. Journal of Biological Chemistry, 271, 6672–6678.PubMedCrossRefGoogle Scholar
  16. Hawdon J.M., Narasimhan S., Hotez P.J. 1999. Ancylostoma secreted protein 2: cloning and characterization of a second member of a family of nematode secreted proteins from Ancylostoma caninum. Molecular and Biochemical Parasitology, 99, 149–165.PubMedCrossRefGoogle Scholar
  17. Hewitson J.P., Harcus Y.M., Curwen R.S., Dowle A.A., Atmadja A.K., Ashton P.D., Wilson A., Maizels R.M. 2008. The secretome of the filaria parasite, Brugia malayi: Proteomic profile of adult excretory-secretory products. Molecular and Biochemical Parasitology, 160, 8–21. DOI: 10.1016/j.molbiopara.2008.02.00.PubMedCrossRefGoogle Scholar
  18. Hewitson J.P., Harcus Y., Murray J., van Agtmaal M., Filbey K.J., Greinger J.R., Bridgett S., Blaxter M.L., Ashton P.D., Ashford D.A., Curwen R.S., Wilson R.A., Dowe A.A., Maizels R.M. 2011. Proteomic analysis of secretory products form the model gastrointestinal nematode Heligmosomoides polygyrus reveals dominance of Venom Allergen-Like (VAL) proteins. Journal for Proteomics, 74, 1573–1594. DOI: 10.1016/j.jprot.2011.06.002.CrossRefGoogle Scholar
  19. Hotez P.J., Brooker S., Bethony J.M., Bottazzi M.E., Loukas A., Xiao S. 2004. Hookworm infection. New England Journal of Medicine, 351, 799–807.PubMedCrossRefGoogle Scholar
  20. King T.P., Sprangford M.D. 2000. Structure and biology stinging insect venom allergens. International Archives of Allergy and Immunology, 123, 99–106. DOI: 10.1159/000024440.PubMedCrossRefGoogle Scholar
  21. Keiser J., Utzinger J. 2008. Efficacy of current drugs against soiltransmitted helminth infections. Journal of the American Medical Association, 299, 1937–1947.PubMedCrossRefGoogle Scholar
  22. Livak K.J., Schmittgen T.D. 2001. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCt Method. Methods, 25, 402–408. DOI: 10.1006/meth.2001.1262.PubMedCrossRefGoogle Scholar
  23. Lu G., Villalba M., Coscia M., Hoffman D.R., King T.P. 1993. Sequence analysis and antigenic cross-reactivity of a venom allergen, antigen 5 from hornets, wasps and yellow jackets. Journal of Immunology, 150, 2823–2830.Google Scholar
  24. Mahdy M.A., Lim Y.A., Ngui R., Siti Fatimah M.R., Choy S.H., Yap N.J., Al-Mekhlafi H.M., Ibrahim J., Surin J. 2012. Prevalence and zoonotic potential of canine hookworms in Malaysia. Parasites and Vectors, 5, 88. DOI: 10.1186/1756-3305-5-88.PubMedCrossRefGoogle Scholar
  25. Moreno Y., Geary T.G. 2008. Stage- and gender-specific proteomic analysis of Brugia malayi excretory-secretory products. Neglected Tropical Diseases, 2, e326. DOI: 10.1371/journal. pntd.0000326.CrossRefGoogle Scholar
  26. Szafrańska E., Wasielewska O., Bereszyński A. 2010. A faecal analysis of helminth infections in wild and captive wolves, Canis lupus L., in Poland. Journal of Helminthology, 84, 415–419. DOI: 10.1017/S0022149X10000106.PubMedCrossRefGoogle Scholar
  27. Szyperski T., Fernandez C., Mumenthaler C., Wuthrich K. 1998. Structure comparison of human glyoma pathogenesis-related protein GliPR and the plant pathogenesis-related protein P14a indicates a functional link between the human immune system and plant defense system. Proceedings of the National Academy of Science of the United States of America, 95, 2262–2266.CrossRefGoogle Scholar
  28. World Health Organization. 2010. Working to overcome the global impact of neglected tropical diseases: first WHO report on neglected tropical diseases.Google Scholar
  29. Wu S., Zhang Y. 2007. LOMETS: A local meta-threading-server for protein structure prediction. Nucleic Acid Research, 35, 3375–3382. DOI: 10.1093/nar/gkm251.CrossRefGoogle Scholar
  30. Zhan B., Hawdon J., Qiang S., Hainan R., Huiqing Q., Wei H., Shuhua X., Liehua L., Xing G., Zheng F., Hotez P.J. 1999. Ancylostoma secreted protein 1 (ASP-1) homologues in human hookworms. Molecular and Biochemical Parasitology, 98, 143–149.CrossRefGoogle Scholar
  31. Zhan B., Liu Y., Badamchian M., Williamson A., Feng J., Loukas A., Hawdon J.M., Hotez P.J. 2003. Molecular characterisation of the Ancylostoma-secreted protein family from adult stage of Ancylostoma caninum. International Journal for Parasitology, 33, 897–907. doi:10.1016/S0020-7519(03)00111-5.PubMedCrossRefGoogle Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Wien 2013

Authors and Affiliations

  • Anna M. Siwińska
    • 1
  • Piotr Bąska
    • 1
  • Emilia Daniłowicz-Luebert
    • 1
    • 2
  • Kamil Januszkiewicz
    • 3
  • Ewa Długosz
    • 1
  • Halina Wędrychowicz
    • 1
    • 3
  • Michael Cappello
    • 4
  • Marcin Wiśniewski
    • 1
  1. 1.Division of Parasitology and Parasitic Diseases, Department Preclinical Sciences, Faculty of Veterinary MedicineWarsaw University of Life Sciences-SGGWWarsawPoland
  2. 2.Department of Molecular ParasitologyHumboldt University BerlinBerlinGermany
  3. 3.W. Stefański Institute of ParasitologyWarsawPoland
  4. 4.Program in International Child Health, Department of PediatricsYale School of MedicineNew HavenUSA

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