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Molecular characterization of calcineurin B from the non-virulent Trypanosoma rangeli kinetoplastid indicates high gene conservation

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Abstract

Calcineurin B, the regulatory subunit of calcineurin, a serine/threonine protein phosphatase, is highly conserved throughout the evolutionary scale including trypanosomatids such as Trypanosoma cruzi, and Leishmania major. Thus, in these flagellates the protein is required for mammalian host cell invasion and virulence and stress responses. With the aim of determining the presence of calcineurin B in Trypanosoma rangeli, a non-virulent trypanosome for mammals, the respective gene was amplified by PCR, cloned and sequenced. Two sequences of 531 bp in length showing a nucleotide polymorphism (314A>C) were obtained in spite of a single-copy gene was revealed by Southern blot. These sequences, probably the alleles from the gene, showed a 79 % of identity with those from T. cruzi and clustered as the sister group of this trypanosome species in a Maximum Parsimony analysis. Deduced amino acid sequence comparison with trypanosomatids and other organisms through the phylogenetic scale as well as the obtained protein structural homology model suggested the presence of the four potential EF-hand regions and the corresponding calcium binding sites of the last three of these domains. Having assessed the expression of this protein in T. rangeli epimastigotes, and taking into account the following facts: (i) calcineurin inhibitors have inhibitory effect on the in vitro replication of T. cruzi, (ii) L. major promastigote growth is inhibited by chelating agents, and (iii) T. rangeli does not seem to productively infect mammalian cells, it is hypothesized herein that the function of this protein in T. rangeli is required for epimastigote growth.

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References

  1. Rusnak F, Mertz P (2000) Calcineurin: form and function. Physiol Rev 80:1483–1521

    PubMed  CAS  Google Scholar 

  2. Stie J, Fox D (2008) Calcineurin regulation in fungi and beyond. Eukaryot Cell 7:177–186. doi:10.1128/EC.00326-07

    Article  CAS  Google Scholar 

  3. Moreno SN, Silva J, Vercesi AE, Docampo R (1994) Cytosolic-free calcium elevation in Trypanosoma cruzi is required for cell invasion. J Exp Med 180:1535–1540. doi:10.1084/jem.180.4.1535

    Article  PubMed  CAS  Google Scholar 

  4. Andrade LO, Andrews NW (2005) The Trypanosoma cruzi-host-cell interplay: location, invasion, retention. Nat Rev Microbiol 3:819–823. doi:10.1038/nrmicro1249

    Article  PubMed  CAS  Google Scholar 

  5. Moreno VR, Aguero F, Tekiel V, Sanchez DO (2007) The calcineurin A homologue from Trypanosoma cruzi lacks two important regulatory domains. Acta Trop 101:80–89. doi:10.1016/j.actatropica.2006.11.008

    Article  PubMed  CAS  Google Scholar 

  6. Araya JE, Cornejo A, Orrego PR, Cordero EM, Cortéz M, Olivares H, Neira I, Sagua H, Da Silveira JF, Yoshida N, González J (2008) Calcineurin B of the human protozoan parasite Trypanosoma cruzi is involved in cell invasion. Microbes Infect 10:892–900. doi:10.1016/j.micinf.2008.05.003

    Article  PubMed  CAS  Google Scholar 

  7. Naderer T, Dandash O, McConville MJ (2011) Calcineurin is required for Leishmania major stress response pathways and for virulence in the mammalian host. Mol Microbiol 80:471–480. doi:10.1111/j.1365-2958.2011.07584.x

    Article  PubMed  CAS  Google Scholar 

  8. Cuba CA (1999) Revisión de los aspectos biológicos y diagnósticos del Trypanosoma (Herpetosoma) rangeli. Rev Soc Bras Med Trop 31:207–220

    Google Scholar 

  9. Grisard EC, Steindel M, Guarneri AA, Eger-Mangrich I, Campbell DA, Romanha AJ (1999) Characterization of Trypanosoma rangeli strains isolated in Central and South America: an overview. Mem Inst Oswaldo Cruz 94:203–209

    Article  PubMed  CAS  Google Scholar 

  10. Guhl F, Vallejo GA (2003) Trypanosoma (Herpetosoma) rangeli Tejera, 1920: an updated review. Mem Inst Oswaldo Cruz 98:435–442

    Article  PubMed  Google Scholar 

  11. Pavia PX, Thomas MC, López MC, Puerta CJ (2012) Molecular characterization of the short interspersed repetitive element SIRE in the six discrete typing units (DTUs) of Trypanosoma cruzi. Exp Parasitol 132(2):144–150. doi:10.1016/j.exppara.2012.06.007-&gt

    Article  PubMed  CAS  Google Scholar 

  12. Puerta CJ, Sincero TC, Stoco PH, Cuervo CL, Grisard EC (2009) Comparative analysis of Trypanosoma rangeli histone H2A gene intergenic region with distinct intraspecific lineage markers. Vector Borne Zoonotic Dis 9(5):449–456. doi:10.1089/vbz.2008.0017

    Article  PubMed  Google Scholar 

  13. Puerta CJ, Urueña CP (2005) Prácticas de biología molecular. Pontificia Universidad Javeriana, Bogotá

    Google Scholar 

  14. Altschul SF, Boguski MS, Gish W, Wootton JC (1994) Issues in searching molecular sequence databases. Nat Genet 6:119–129. doi:10.1038/ng0294-119

    Article  PubMed  CAS  Google Scholar 

  15. Grisard EC, Stoco PH, Wagner G, Sincero TC, Rotava G, Rodrigues JB, Snoeijer CQ, Koerich LB, Sperandio MM, Bayer-Santos E, Fragoso SP, Goldenberg S, Triana O, Vallejo GA, Tyler KM, Dávila AM, Steindel M (2010) Transcriptomic analyses of the avirulent protozoan parasite Trypanosoma rangeli. Mol Biochem Parasitol 174:18–25. doi:10.1016/j.molbiopara.2010.06.008

    Article  PubMed  CAS  Google Scholar 

  16. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448

    Article  PubMed  CAS  Google Scholar 

  17. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113. doi:10.1186/1471-2105-5-113

    Article  PubMed  Google Scholar 

  18. Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New York

    Google Scholar 

  19. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 10:2731–2739. doi:10.1093/molbev/msr121

    Article  Google Scholar 

  20. Arnold K, Kiefer F, Kopp J, Battey JN, Podvinec M, Westbrook JD, Berman HM, Bordoli L, Schwede T (2009) The Protein Model Portal. J Struct Funct Genomics 10:1–8. doi: 10.1007/s10969-008-9048-5 http://www.proteinmodelportal.org/. Accessed 21 Feb 2012

    Google Scholar 

  21. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738. doi:10.1038/nprot.2010.5

    Article  PubMed  CAS  Google Scholar 

  22. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40. doi:10.1186/1471-2105-9-40

    Article  PubMed  Google Scholar 

  23. Schultz J, Milpetz F, Bork P, Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA 95:5857–5864

    Article  PubMed  CAS  Google Scholar 

  24. Letunic I, Doerks T, Bork P (2012) SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res 40 (Database issue):D302-5. Simple modular architecture research tool. doi: 10.1093/nar/gkr931 http://smart.embl-heidelberg.de/. Accessed 21 Feb 2012

  25. Bordoli L, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc 4:1–13. ExPASy bioinformatics resource portal doi:10.1038/nprot.2008.197 http://swissmodel.expasy.org/workspace/. Accessed 21 Feb 2012

    Google Scholar 

  26. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. doi:10.1093/bioinformatics/bti770

    Article  PubMed  CAS  Google Scholar 

  27. Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35(Web Server issue):W407-10. Protein structure analysis ProSA-Web. doi: 10.1093/nar/gkm290 http://prosa.services.came.sbg.ac.at/prosa.php. Accessed 21 Feb 2012

  28. Sippl MJ (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17:355–362

    Article  PubMed  CAS  Google Scholar 

  29. McGuffin LJ, Roche DB (2010) Rapid model quality assessment for protein structure predictions using the comparison of multiple models without structural alignments. Bioinformatics 26:182–188. doi:10.1093/bioinformatics/btp629

    Article  PubMed  CAS  Google Scholar 

  30. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723. ExPASy bioinformatics resource portal web. http://www.expasy.org/spdbv/. Accessed 21 Feb 2012

    Google Scholar 

  31. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14: 33–38. Theoretical and computational biophysics group web. http://www.ks.uiuc.edu/Research/vmd/. Accessed 21 Feb 2012

    Google Scholar 

  32. Steinbach WJ, Reedy JL, Cramer RA Jr, Perfect JR, Heitman J (2007) Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections. Nat Rev Microbiol 5:418–430. doi:10.1038/nrmicro1680

    Article  PubMed  CAS  Google Scholar 

  33. Cuervo C, López MC, Puerta C (2006) The Trypanosoma rangeli histone H2A gene sequence serves as a differential marker for KP1 strains. Infect Genet Evol 6:401–409. doi:10.1016/j.meegid.2006.01.005

    Article  PubMed  CAS  Google Scholar 

  34. Diez H, Thomas MC, Urueña CP, Santander SP, Cuervo CL, López MC, Puerta CJ (2005) Molecular characterization of the kinetoplastid membrane protein-11 genes from the parasite Trypanosoma rangeli. Parasitology 130(Pt 6):643–651. doi:10.1017/S0031182004006936

    Article  PubMed  CAS  Google Scholar 

  35. Urrea DA, Carranza JC, Cuba Cuba CA, Gurgel-Gonc Alves R, Guhl F, Schofield CJ, Triana O, Vallejo GA (2005) Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panamá, supports a co-evolutionary association between parasites and vectors. Infect Genet Evol 5:123–129. doi:10.1016/j.meegid.2004.07.005

    Article  PubMed  CAS  Google Scholar 

  36. Urrea DA, Guhl F, Herrera CP, Falla A, Carranza JC, Cuba-Cuba C, Triana-Chávez O, Grisard EC, Vallejo GA (2011) Sequence analysis of the spliced-leader intergenic region (SL-IR) and random amplified polymorphic DNA (RAPD) of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis, R. colombiensis, R. pallescens and R. prolixus suggests a degree of co-evolution between parasites and vectors. Acta Trop 120:59–66. doi:10.1016/j.actatropica.2011.05.016

    Article  PubMed  CAS  Google Scholar 

  37. Deschamps P, Lara E, Marande W, López-García P, Ekelund F, Moreira D (2011) Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids. Mol Biol Evol 28:53–58. doi:10.1093/molbev/msq289

    Article  PubMed  CAS  Google Scholar 

  38. Nocua P, Gómez C, Cuervo C, Puerta C (2009) Cl gene cluster encoding several small nucleolar RNAs: a comparison amongst trypanosomatids. Mem Inst Oswaldo Cruz 104:473–480. doi:10.1590/S0074-02762009000300013

    Article  CAS  Google Scholar 

  39. Barton N, Briggs D, Eisen J, Goldstein D, Patel N (2007) Species and speciation. In: Evolution, Cold Spring Harbor Laboratory Press, New York, pp 619–656

  40. Maurer-Stroh S, Eisenhaber B, Eisenhaber F (2002) N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence. J Mol Biol 317:541–557. doi:10.1006/jmbi.2002.5426

    Article  PubMed  CAS  Google Scholar 

  41. Price HP, Menon MR, Panethymitaki C, Goulding D, McKean PG, Smith DF (2003) Myristoyl-CoA:protein N-myristoyltransferase, an essential enzyme and potential drug target in kinetoplastid parasites. J Biol Chem 278:7206–7214. doi:10.1074/jbc.M211391200

    Article  PubMed  CAS  Google Scholar 

  42. Búa J, Ruiz AM, Potenza M, Fichera LE (2004) In vitro anti-parasitic activity of cyclosporin A analogs on Trypanosoma cruzi. Bioorg Med Chem Lett 14:4633–4637. doi:10.1016/j.bmcl.2004.07.003

    Article  PubMed  Google Scholar 

  43. Eger-Mangrich I, de Oliveira MA, Grisard EC, De Souza W, Steindel M (2001) Interaction of Trypanosoma rangeli Tejera, 1920 with different cell lines in vitro. Parasitol Res 87:505–509. doi:10.1007/s004360000356

    Article  CAS  Google Scholar 

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Acknowledgments

This work was financially supported by Colciencias Research project No. RC-595-2009. CAB and MM, were supported by the program Jóvenes Investigadores e Innovadores 2010 and 2011 from Colciencias. MC López and MC Thomas, were supported by Grants P08-CVI-04037 from PAI (Junta de Andalucía), BFU2010-1670 and SAF2012-35777 from Plan Nacional I+D+i (MICINN) and RD12/008/0021 from ISCIII-RETIC (MICINN), Spain and FEDER.

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Authors and Affiliations

  1. Laboratorio de Parasitología Molecular, Departamento de Microbiología, Facultad Ciencias, Pontificia Universidad Javeriana, Carrera 7 No. 43–82, Bogotá, Colombia

    M. Montenegro, C. Cuervo, C. Bernal & C. J. Puerta

  2. Núcleo de Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330, Valparaíso, Chile

    C. Cardenas

  3. Laboratórios de Protozoologia e de Bioinformática, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Caixa Postal 476, Florianópolis, SC, 88040-900, Brazil

    E. C. Grisard

  4. Instituto de Parasitología y Biomedicina López Neyra, Consejo Superior de Investigaciones Científicas (IPBLN-CSIC), Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento, s/n, 18100, Granada, Spain

    M. C. Thomas & M. C. Lopez

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  1. M. Montenegro
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Correspondence to C. J. Puerta.

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Montenegro, M., Cardenas, C., Cuervo, C. et al. Molecular characterization of calcineurin B from the non-virulent Trypanosoma rangeli kinetoplastid indicates high gene conservation. Mol Biol Rep 40, 4901–4912 (2013). https://doi.org/10.1007/s11033-013-2590-7

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