Skip to main content

Advertisement

Log in

Molecular characterization of cytosolic and mitochondrial tryparedoxin peroxidase in Trypanosoma cruzi populations susceptible and resistant to benznidazole

Parasitology Research Aims and scope Submit manuscript

Abstract

Antioxidant defense in Trypanosomatids has been indicated as a potential target for chemotherapy. Tryparedoxin peroxidase (TXNPx) participates in this defense by metabolizing hydrogen peroxide to water molecules. In this work, genes encoding both cytosolic (cTcTXNPx) and mitochondrial (mTcTXNPx) TXNPx were characterized in 15 benznidazole-susceptible and resistant Trypanosoma cruzi strains. Northern blot and real-time RT-PCR analyses revealed that the levels of cTcTXNPx and mTcTXNPx mRNA were two-fold higher in the in-vitro-induced resistant 17 LER T. cruzi population than its drug-susceptible counterpart 17 WTS. The mRNA levels for both genes were similar among the other T. cruzi samples studied. No amplification of these genes was observed in the parasite genome. In silico analyses indicated that cTcTXNPx and mTcTXNPx genes present eight and two copies, respectively, dispersed in the parasite genome. By western blot analysis, anti-cTcTXNPx and anti-mTcTXNPx polyclonal antibodies recognized a 23- and 25-kDa peptide, respectively, in all T. cruzi samples analyzed. The expression levels of these native proteins were similar for all samples except 17 LER, which displayed two-fold greater expression. In addition, the oxidized mTcTXNPx protein (50 kDa) demonstrated 5.5-fold greater expression in the 17 LER population than 17 WTS. Our findings demonstrate increased expression of the cytosolic and mitochondrial TcTXNPx in the T. cruzi population with in-vitro-induced resistance to benznidazole.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

cTcTXNPx:

cytosolic tryparedoxin peroxidase T. cruzi

mTcTXNPx:

mitochondrial tryparedoxin peroxidase T. cruzi

TXNPx:

tryparedoxin peroxidase

BZ:

benznidazole

ROS:

reactive oxygen species

References

  • Allen TE, Ullman B (1994) Molecular characterization and overexpression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma cruzi. Mol Biochem Parasitol 65:233–245

    Article  PubMed  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  • Andrade HM, Murta SM, Chapeaurouge A, Perales J, Nirde P, Romanha AJ (2008) Proteomic analysis of Trypanosoma cruzi resistance to benznidazole. J Proteome Res 7:2357–2367

    Article  PubMed  Google Scholar 

  • Bannister JV, Bannister WH, Rotilio G (1987) Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit Rev Biochem 22:111–180

    Article  PubMed  CAS  Google Scholar 

  • Castro H, Sousa C, Santos M, Cordeiro-da-Silva A, Flohe L, Tomas AM (2002) Complementary antioxidant defense by cytoplasmic and mitochondrial peroxiredoxins in Leishmania infantum. Free Radic Biol Med 33:1552–1562

    Article  PubMed  CAS  Google Scholar 

  • Centers for Disease Control and Prevention (2007) Available from: www.cdc.gov/chagas. Reviewed August 8.

  • Docampo R (1990) Sensitivity of parasites to free radical damage by antiparasitic drugs. Chem Biol Interact 73:1–27

    Article  PubMed  CAS  Google Scholar 

  • El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN et al (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 309:409–415

    Article  PubMed  CAS  Google Scholar 

  • Filardi LS, Brener Z (1987) Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas Disease. Trans R Soc Trop Med Hyg 81:755–759

    Article  PubMed  CAS  Google Scholar 

  • Finzi JK, Chiavegatto CW, Corat KF, Lopez JA, Cabrera OG, Mielniczki-Pereira AA, Colli W, Alves MJ, Gadelha FR (2004) Trypanosoma cruzi response to the oxidative stress generated by hydrogen peroxide. Mol Biochem Parasitol 133:37–43

    Article  PubMed  CAS  Google Scholar 

  • Galtier N, Gouy M, Gautier C (1996) SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12:543–548

    PubMed  CAS  Google Scholar 

  • Guerrero SA, Lopez JA, Steinert P, Montemartini M, Kalisz HM, Colli W, Singh M, Alves MJ, Flohe L (2000) His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening. Appl Microbiol Biotechnol 53:410–414

    Article  PubMed  CAS  Google Scholar 

  • Guimond C, Trudel N, Brochu C, Marquis N, El Fadili A, Peytavi R, Briand G, Richard D, Messier N, Papadopoulou B, Corbeil J, Bergeron MG, Legare D, Ouellette M (2003) Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays. Nucleic Acids Res 31:5886–5896

    Article  PubMed  CAS  Google Scholar 

  • Hofmann B, Hecht HJ, Flohe L (2002) Peroxiredoxins. Biol Chem 383:347–364

    Article  PubMed  CAS  Google Scholar 

  • Levick MP, Tetaud E, Fairlamb AH, Blackwell JM (1998) Identification and characterisation of a functional peroxidoxin from Leishmania major. Mol Biochem Parasitol 96:125–137

    Article  PubMed  CAS  Google Scholar 

  • Lin YC, Hsu JY, Chiang CS, Lee ST (2005) Distinct overexpression of cytosolic and mitochondrial tryparedoxin peroxidases results in preferential detoxification of different oxidants in arsenite-resistant Leishmania amazonensis with and without DNA amplification. Mol Biochem Parasitol 142:66–75

    Article  PubMed  CAS  Google Scholar 

  • Lopez JA, Carvalho TU, de Souza W, Flohe L, Guerrero SA, Montemartini M, Kalisz HM, Nogoceke E, Singh M, Alves MJ, Colli W (2000) Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi. Free Radic Biol Med 28:767–772

    Article  PubMed  CAS  Google Scholar 

  • Lyer JP, Kaprakkaden A, Choudhary ML, Shaha C (2008) Crucial role of cytosolic tryparedoxin peroxidase in Leishmania donovani survival, drug response and virulence. Mol Microbiol 68:372–391

    Article  Google Scholar 

  • Maya JD, Repetto Y, Agosin M, Ojeda JM, Tellez R, Gaule C, Morello A (1997) Effects of nifurtimox and benznidazole upon glutathione and trypanothione content in epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi. Mol Biochem Parasitol 86:101–106

    PubMed  CAS  Google Scholar 

  • Montemartini M, Nogoceke E, Singh M, Steinert P, Flohe L, Kalisz HM (1998) Sequence analysis of the tryparedoxin peroxidase gene from Crithidia fasciculata and its functional expression in Escherichia coli. J Biol Chem 273:4864–4871

    Article  PubMed  CAS  Google Scholar 

  • Moser DR, Kirchhoff LV, Donelson JE (1989) Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction. J Clin Microbiol 27:1477–1482

    PubMed  CAS  Google Scholar 

  • Murta SM, Romanha AJ (1998) In vivo selection of a population of Trypanosoma cruzi and clones resistant to benznidazole. Parasitology 116:165–171

    Article  PubMed  CAS  Google Scholar 

  • Murta SM, Gazzinelli RT, Brener Z, Romanha AJ (1998) Molecular characterization of susceptible and naturally resistant strains of Trypanosoma cruzi to benznidazole and nifurtimox. Mol Biochem Parasitol 93:203–214

    Article  PubMed  CAS  Google Scholar 

  • Murta SM, Krieger MA, Montenegro LR, Campos FF, Probst CM, Avila AR, Muto NH, de Oliveira RC, Nunes LR, Nirde P, Bruna-Romero O, Goldenberg S, Romanha AJ (2006) Deletion of copies of the gene encoding old yellow enzyme (TcOYE), a NAD(P)H flavin oxidoreductase, associates with in vitro-induced benznidazole resistance in Trypanosoma cruzi. Mol Biochem Parasitol 146:151–162

    Article  PubMed  CAS  Google Scholar 

  • Murta SM, Nogueira FB, dos Santos PF, Campos FF, Volpe C, Liarte DB, Nirdé P, Probst CM, Krieger MA, Goldenberg S, Romanha AJ (2008) Differential gene expression in Trypanosoma cruzi populations susceptible and resistant to benznidazole. Acta Trop 107:59–65

    Article  PubMed  CAS  Google Scholar 

  • Neal RA, Van Bueren J (1988) Comparative studies of drug susceptibility of five strains of Trypanosoma cruzi in vivo and in vitro. Trans R Soc Trop Med Hyg 82:709–714

    Article  PubMed  CAS  Google Scholar 

  • Nirde P, Larroque C, Barnabe C (1995) Drug-resistant epimastigotes of Trypanosoma cruzi and persistence of this phenotype after differentiation into amastigotes. C R Acad Sci III 318:1239–1244

    PubMed  CAS  Google Scholar 

  • Nogueira FB, Krieger MA, Nirdé P, Goldenberg S, Romanha AJ, Murta SM (2006) Increased expression of iron-containing superoxide dismutase-A (TcFeSOD-A) enzyme in Trypanosoma cruzi population with in vitro-induced resistance to benznidazole. Acta Trop 100:119–132

    Article  PubMed  CAS  Google Scholar 

  • Nozaki T, Engel JC, Dvorak JA (1996) Cellular and molecular biological analyses of nifurtimox resistance in Trypanosoma cruzi. Am J Trop Med Hyg 55:111–117

    PubMed  CAS  Google Scholar 

  • Piñeyro MD, Pizarro JC, Lema F, Pritsch O, Cayota A, Bentley GA, Robello C (2005) Crystal structure of the tryparedoxin peroxidase from the human parasite Trypanosoma cruzi. J Sruct Biol 150:11–22

    Article  Google Scholar 

  • Piñeyro MD, Parodi-Talice A, Arcari T, Robello C (2008) Peroxiredoxins from Trypanosoma cruzi: virulence factors and drug targets for treatment of Chagas disease? Gene 408:45–50

    Article  PubMed  Google Scholar 

  • Steenkamp DJ (2002) Thiol metabolism of the trypanosomatids as potential drug targets. IUBMB life 53:243–248

    Article  PubMed  CAS  Google Scholar 

  • Teixeira SMR, DaRocha W (2003) Control of gene and genetic manipulation in the Trypanosomatidae. Genet Mol Res 2:148–158

    PubMed  Google Scholar 

  • Tetaud E, Giroud C, Prescott AR, Parkin DW, Baltz D, Biteau N, Baltz T, Fairlamb AH (2001) Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei. Mol Biochem Parasitol 116:171–183

    Article  PubMed  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  • Toledo M, Tafuri W, Bahia MT, Tibayrenc M, Lana M (2004) Genetic diversity and drug resistance in Trypanosoma cruzi, the agent of Chagas disease. Antimicrob Agents Chemother 4:11–22

    Google Scholar 

  • Trujillo M, Budde H, Pineyro MD, Stehr M, Robello C, Flohe L, Radi R (2004) Trypanosoma brucei and Trypanosoma cruzi tryparedoxin peroxidases catalytically detoxify peroxynitrite via oxidation of fast reacting thiols. J Biol Chem 279:34175–34182

    Article  PubMed  CAS  Google Scholar 

  • Turrens JF (2004) Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa. Mol Aspects Med 25:211–220

    Article  PubMed  CAS  Google Scholar 

  • Urbina JA, Docampo R (2003) Specific chemotherapy of Chagas disease: controversies and advances. Trends Parasitol 19:495–501

    Article  PubMed  CAS  Google Scholar 

  • Wassmann C, Hellberg A, Tannich E, Bruchhaus I (1999) Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase. J Biol Chem 274:26051–26056

    Article  PubMed  CAS  Google Scholar 

  • Wilkinson SR, Temperton NJ, Mondragon A, Kelly JM (2000) Distinct mitochondrial and cytosolic enzymes mediate trypanothione-dependent peroxide metabolism in Trypanosoma cruzi. J Biol Chem 275:8220–8225

    Article  PubMed  CAS  Google Scholar 

  • Wilkinson SR, Taylor MC, Horn D, Kelly JM, Cheeseman I (2008) A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes. Proc Natl Acad Sci USA 105:5022–5027

    Article  PubMed  CAS  Google Scholar 

  • Wyllie S, Vickers TJ, Fairlamb AH (2008) Roles of trypanothione S-transferase and tryparedoxin peroxidase in resistance to antimonials. Antimicrob Agents Chemother 52:1359–1365

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Centro de Pesquisas René Rachou (CPqRR-FIOCRUZ), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Programa de Desenvolvimento Tecnológico em Insumos para Saúde (PDTIS-FIOCRUZ).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Silvane M. F. Murta.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nogueira, F.B., Ruiz, J.C., Robello, C. et al. Molecular characterization of cytosolic and mitochondrial tryparedoxin peroxidase in Trypanosoma cruzi populations susceptible and resistant to benznidazole. Parasitol Res 104, 835–844 (2009). https://doi.org/10.1007/s00436-008-1264-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-008-1264-1

Keywords

Navigation