Advertisement

Parasitology Research

, Volume 96, Issue 5, pp 312–320 | Cite as

Cloning and expression analysis of two novel paraflagellar rod domain genes found in Trypanosoma cruzi

  • April K. Clark
  • Gennadiy Kovtunovych
  • Sachin Kandlikar
  • Shailesh Lal
  • Gabrielle A. Stryker
Original Paper

Abstract

The eukaryotic flagellum is one of the most complex macromolecular structures found in cells, containing more than 250 proteins. One unique structure in the flagella of trypanomastids is the paraflagellar rod (PFR). The PFR constitutes a lattice of cytoskeletal filaments that lies alongside the axoneme in the flagella. This unique and complex structure is critical for cell motility, though little is known about its molecular assembly or its role in the lifecycle of trypanosomatids. These proteins are of particular importance in Trypanosoma cruzi, as purified or recombinant PFR proteins have been demonstrated to be immunogenic, protecting mice from a lethal challenge with the parasite. We have searched the T. cruzi databases and discovered two novel genes containing PFR domains. Both these genes are transcribed in vivo and are significantly larger than the previously described PFR genes identified in T. cruzi (>2 Kb). Real-time PCR was used to examine the relative expression levels of six PFR genes, including the two we describe here, in all three stages of T. cruzi’s lifecycle. Database searches have further provided EST and genomic sequence support for the presence of these genes in two other pathogenic trypanosomatids, Trypanosoma brucei and Leishmania spp. One of these genes, designated PFR5 contains a carboxy terminal SH3 domain not previously seen in PFR family genes. We propose that this proline-binding SH3 domain may play an important role in the assembly of the PFR.

Keywords

Trypanosoma cruzi Chagas’ disease Paraflagellar rod Src Homology-3 Trypanomastid 

Abbreviations

PFR

Paraflagellar rod

SH3

Src Homology-3

EST

Expressed sequence tag

Notes

Acknowledgements

The authors would like to thank Bing-Bing Wang for bioinformatics support, Smriti Gupta for RNA analysis, Harvey Qu for statistical analysis, and Jill Zeilstra-Ryalls and Paul McBride for critically reading this manuscript. This study was supported by Oakland University’s Research Excellence fund and Faculty start up funds. All the experiments described comply with the current laws of the United States where the experiments were performed.

References

  1. Bastin P, Gull K (1999) Assembly and function of complex flagellar structures illustrated by the paraflagellar rod of trypanosomes. Protist 150:113–123PubMedGoogle Scholar
  2. Bastin P, Sherwin T, Gull K (1998) Paraflagellar rod is vital for trypanosome motility. Nature 391:548CrossRefPubMedGoogle Scholar
  3. Bringaud F, Robinson DR, Barradeau S, Biteau N, Baltz D, Baltz T (2000) Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition. Mol Biochem Parasitol 111:283–297CrossRefPubMedGoogle Scholar
  4. Cesareni G, Panni S, Nardelli G, Castagnoli L (2002) Can we infer peptide recognition specificity mediated by SH3 domains? FEBS Lett 513:38–44CrossRefPubMedGoogle Scholar
  5. Chagas disease in the Americas (1996) Epidemiol Bull 17:16Google Scholar
  6. Ersfeld K, Gull K (2001) Targeting of cytoskeletal proteins to the flagellum of Trypanosoma brucei. J Cell Sci 114:141–148PubMedGoogle Scholar
  7. Fitch WM, Margoliash E (1967) Construction of phylogenetic trees. Science 155:279–284PubMedGoogle Scholar
  8. Fouts DL, Stryker GA, Gorski KS, Miller MJ, Nguyen TV, Wrightsman RA, Manning JE (1998) Evidence for four distinct major protein components in the paraflagellar rod of Trypanosoma cruzi. J Biol Chem 273:21846–21855CrossRefPubMedGoogle Scholar
  9. Gadelha C, LeBowitz JH, Manning J, Seebeck T, Gull K (2004) Relationships between the major kinetoplastid paraflagellar rod proteins: a consolidating nomenclature. Mol Biochem Parasitol 136:113–115CrossRefPubMedGoogle Scholar
  10. Kohl L, Sherwin T, Gull K (1999) Assembly of the paraflagellar rod and the flagellum attachment zone complex during the Trypanosoma brucei cell cycle. J Eukaryot Microbiol 46:105–109PubMedGoogle Scholar
  11. Luhrs KA, Fouts DL, Manning JE (2003) Immunization with recombinant paraflagellar rod protein induces protective immunity against Trypanosoma cruzi infection. Vaccine 21:3058–3069CrossRefPubMedGoogle Scholar
  12. Maga JA, LeBowitz JH (1999) Unravelling the kinetoplastid paraflagellar rod. Trends Cell Biol 9:409–413CrossRefPubMedGoogle Scholar
  13. Magill AJ, Reed SG (2000) American trypanosomiasis. In: Strickland G (ed) Hunter’s tropical medicine and emerging infectious diseases. Saunders, Philadelphia, pp 653–664Google Scholar
  14. Manzullo EC, Chuit R (1999) Risk of death due to chronic chagasic cardiopathy. Mem Inst Oswaldo Cruz 94 Suppl 1:317–320CrossRefPubMedGoogle Scholar
  15. Marsden PD (1967) Trypanosoma cruzi infections in CFI mice. I. Mortality with different doses of trypanosomes. Ann Trop Med Parasitol 61:57–61PubMedGoogle Scholar
  16. Mayer BJ (2001) SH3 domains: complexity in moderation. J Cell Sci 114:1253–1263PubMedGoogle Scholar
  17. Miller MJ, Wrightsman RA, Stryker GA, Manning JE (1997) Protection of mice against Trypanosoma cruzi by immunization with paraflagellar rod proteins requires T cell, but not B cell, function. J Immunol 158:5330–5337PubMedGoogle Scholar
  18. Moncayo A (2003) Chagas’ disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the Southern Cone countries. Mem Inst Oswaldo Cruz 98:577–591CrossRefPubMedGoogle Scholar
  19. Moore LL, Santrich C, LeBowitz JH (1996) Stage-specific expression of the Leishmania mexicana paraflagellar rod protein PFR-2. Mol Biochem Parasitol 80:125–135CrossRefPubMedGoogle Scholar
  20. Morton CJ, Campbell ID (1994) SH3 domains. Molecular ‘Velcro’. Curr Biol 4:615–617CrossRefPubMedGoogle Scholar
  21. Pawson T, Schlessingert J (1993) SH2 and SH3 domains. Curr Biol 3:434–442CrossRefPubMedGoogle Scholar
  22. Saborio JL, Manuel Hernandez J, Narayanswami S, Wrightsman R, Palmer E, Manning J (1989) Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi. J Biol Chem 264:4071–4075PubMedGoogle Scholar
  23. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  24. Santrich C, Moore L, Sherwin T, Bastin P, Brokaw C, Gull K, LeBowitz JH (1997) A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts. Mol Biochem Parasitol 90:95–109CrossRefPubMedGoogle Scholar
  25. Segura EL, Paulone I, Cerisola J, Cappa SM (1976) Experimental Chagas’ disease: protective activity in relation with subcellular fractions of the parasite. J Parasitol 62:131–133PubMedGoogle Scholar
  26. Tomlinson S, Vandekerckhove F, Frevert U, Nussenzweig V (1995) The induction of Trypanosoma cruzi trypomastigote to amastigote transformation by low pH. Parasitology 110 (Pt 5):547–554PubMedGoogle Scholar
  27. Wrightsman RA, Manning JE (2000) Paraflagellar rod proteins administered with alum and IL-12 or recombinant adenovirus expressing IL-12 generates antigen-specific responses and protective immunity in mice against Trypanosoma cruzi. Vaccine 18:1419–1427CrossRefPubMedGoogle Scholar
  28. Wrightsman RA, Miller MJ, Saborio JL, Manning JE (1995) Pure paraflagellar rod protein protects mice against Trypanosoma cruzi infection. Infect Immun 63:122–125PubMedGoogle Scholar
  29. Zhang J, Madden TL (1997) PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annotation. Genome Res 7:649–656PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • April K. Clark
    • 1
  • Gennadiy Kovtunovych
    • 1
  • Sachin Kandlikar
    • 1
  • Shailesh Lal
    • 1
  • Gabrielle A. Stryker
    • 1
  1. 1.Department of Biological SciencesOakland UniversityRochesterUSA

Personalised recommendations