Skip to main content
SpringerLink
Log in

PTR1-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite Leishmania major

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1 − null mutants were 18-fold more sensitive to H2O2 than PTR1-overproducing lines, and significant three- to fivefold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H2O2 susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H4B metabolism specifically. Neither H2O2 consumption nor the level of intracellular oxidative stress was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols, these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.

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

Access this article

Log in via an institution

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
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

PTR1:

Pteridine reductase 1

DHFR-TS:

Dihydrofolate reductase-thymidylate synthase

ROS:

Reactive oxygen species

RNS:

Reactive nitrogen species

DCFH-DA:

2′7′-Dichlorofluorescein diacetate

DCF:

Dichlorofluorescin

GSH:

Glutathione

T[SH]2 :

Trypanothione

SIN-1:

3-Morpholino-sydnonimine

H2B:

Dihydrobiopterin

H4B:

Tetrahydrobiopterin

WT:

Wild-type

References

  • Ariyanayagam MR, Fairlamb AH (2001) Ovothiol and trypanothione as antioxidants in trypanosomatids. Mol Biochem Parasitol 115:189–198

    Article  PubMed  CAS  Google Scholar 

  • Ashutosh, Sundar S, Goyal N (2007) Molecular mechanisms of antimony resistance in Leishmania. J Med Microbiol 56:143–153

    Article  PubMed  CAS  Google Scholar 

  • Augusto O, Alves MJM, Colli W, Filardi LS, Brener S (1986) Primaquine can mediate hydroxyl radical generation by Trypanosoma cruzi extracts. Biochem Biophys Res Commun 135:1029–1034

    Article  PubMed  CAS  Google Scholar 

  • Bello AR, Nare B, Freedman D, Hardy L, Beverley SM (1994) PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc Natl Acad Sci 91:11442–11446

    Article  PubMed  CAS  Google Scholar 

  • Beverley SM (1991) Gene Amplification in Leishmania. Annu Rev Microbiol 45:417–444

    Article  PubMed  CAS  Google Scholar 

  • Beverley SM (2003) Genetic and genomic approaches to the analysis of Leishmania virulence. In: Marr JM, Nilsen T, Komuniecki R (eds) Molecular and medical parasitology. Academic Press, New York

    Google Scholar 

  • Beverley SM, Coderre JA, Santi DV, Schimke RT (1984) Unstable DNA amplifications in methotrexate resistant Leishmania consist of extra-chromosomal circles which relocate during stabilization. Cell 38:431–439

    Article  PubMed  CAS  Google Scholar 

  • Bishai WR, Howard NS, Winkelstein JA, Smith HO (1994) Characterization and virulence analysis of catalase mutants of Haemophilus influenzae. Infect Immun 62:4855–4860

    PubMed  CAS  Google Scholar 

  • Brudler R, Hitomi K, Daiyasu H, Toh H, Kucho K, Ishiura M, Kanehisa M, Roberts VA, Todo T, Tainer JA, Getzoff ED (2003) Identification of a new cryptochrome class. Structure, function, and evolution. Mol Cell 11:59–67

    Article  PubMed  CAS  Google Scholar 

  • Callahan HL, Beverley SM (1991) Heavy metal resistance: a new role for P-glycoproteins in Leishmania. J Biol Chem 266:18427–18430

    PubMed  CAS  Google Scholar 

  • Clos J, Choudhury K (2006) Functional cloning as a means to identify Leishmania genes involved in drug resistance. Mini Rev Med Chem 6:123–129

    Article  PubMed  CAS  Google Scholar 

  • Cotrim PC, Garrity LK, Beverley SM (1999) Isolation of genes mediating resistance to inhibitors of nucleoside and ergosterol metabolism in Leishmania by overexpression/selection. J Biol Chem 274:37723–37730

    Article  PubMed  CAS  Google Scholar 

  • Cruz A, Beverley SM (1990) Gene replacement in parasitic protozoa. Nature 348:171–173

    Article  PubMed  CAS  Google Scholar 

  • Cruz A, Coburn C, Beverley SM (1991) Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci 88:7170–7174

    Article  PubMed  CAS  Google Scholar 

  • Cruz AK, Titus R, Beverley SM (1993) Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. Proc Natl Acad Sci USA 90:1599–1603

    Article  PubMed  CAS  Google Scholar 

  • Cunningham ML, Beverley SM (2001) Pteridine salvage throughout the Leishmania infectious cycle: implications for antifolate chemotherapy. Mol Biochem Parasitol 113:199–213

    Article  PubMed  CAS  Google Scholar 

  • Cunningham ML, Titus RG, Turco SJ, Beverley SM (2001) Regulation of differentiation to the infective stage of the protozoan parasite Leishmania major by tetrahydrobiopterin. Science 292:285–287

    Article  PubMed  CAS  Google Scholar 

  • Davies KJ (1995) Oxidative stress: the paradox of aerobic life. Biochem Soc Symp 61:1–31

    PubMed  CAS  Google Scholar 

  • Denicola A, Rubbo H, Rodriguez D, Radi R (1993) Peroxynitrite-mediated cytotoxicity to Trypanosoma cruzi. Arch Biochem Biophys 304:279–286

    Article  PubMed  CAS  Google Scholar 

  • Denkers EY, Butcher BA (2005) Sabotage and exploitation in macrophages parasitized by intracellular protozoans. Trends Parasitol 21:35–41

    Article  PubMed  CAS  Google Scholar 

  • Desjeux P (2004) Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27:305–318

    Article  PubMed  CAS  Google Scholar 

  • Devine SE, Boeke JD (1994) Efficient intergration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis. Nucleic acid Res 22:3765–3772

    Article  PubMed  CAS  Google Scholar 

  • Dumas C, Ouellette M, Tovar J, Cunningham ML, Fairlamb AH, Tamar S, Olivier M, Papadopoulou B (1997) Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages. EMBO J 16:2590–2598

    Article  PubMed  CAS  Google Scholar 

  • Ellenberger TE, Beverley SM (1989) Multiple drug resistance and conservative amplification of the H region in Leishmania major. J Biol Chem 264:15094–150103

    PubMed  CAS  Google Scholar 

  • Fairlamb AH, Cerami A (1992) Metabolism and functions of trypanothione in the kinetoplastida. Annu Rev Microbiol 46:695–729

    Article  PubMed  CAS  Google Scholar 

  • Fang FC (2004) Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev 2:820–832

    Article  CAS  Google Scholar 

  • Feelisch M, Ostrowski J, Noack E (1989) On the mechanism of NO release from sydnonimines. J Cardiovasc Pharmacol 14:S13–S22

    PubMed  CAS  Google Scholar 

  • Garraway LA, Tosi LRO, Wang Y, Moore JB, Dobson DE, Beverley SM (1997) Insertional mutagenesis using a modified in vitro Ty1 transposition system. Gene 198:27–36

    Article  PubMed  CAS  Google Scholar 

  • Gatti RM, Augusto O, Kwee JK, Giorgio S (1995) Leishmanicidal activity of peroxynitrite. Redox Rep 1:261–265

    CAS  Google Scholar 

  • Homan-Muller JWT, Weening RT, Roos D (1975) Production of hydrogen peroxide by phagocytizing human granulocytes. J Lab Clin Med 85:198–207

    PubMed  CAS  Google Scholar 

  • Huang HM, Chen HL, Xu H, Gibson GE (2005) Modification of endoplasmic reticulum Ca2+ stores by select oxidants produces changes reminiscent of those in cells from patients with Alzheimer disease. Free Radic Biol Med 39:979–989

    Article  PubMed  CAS  Google Scholar 

  • Iyer 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  PubMed  CAS  Google Scholar 

  • Jaeger T, Flohe L (2006) The thiol-based redox networks of pathogens: unexploited targets in the search for new drugs. Biofactors 27:109–120

    Article  PubMed  Google Scholar 

  • Kapler GM, Coburn CM, Beverley SM (1990) Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol 10:1084–1094

    PubMed  CAS  Google Scholar 

  • Kaufman S (1963) The structure of phenylalanine hydroxylation cofactor. Proc Natl Acad Sci 50:1085–1093

    Article  PubMed  CAS  Google Scholar 

  • Kelly JM, Taylor MC, Smith K, Hunter KJ, Fairlamb AH (1993) Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which over-express trypanothione reductase. Sensitivity towards agents that are thought to induce oxidative stress. Eur J Biochem 218:29–37

    Article  PubMed  CAS  Google Scholar 

  • Keston AS, Brandt R (1965) The fluorimetric analysis of ultramicroquantities of hydrogen peroxide. Anal Biochem 11:1–5

    Article  PubMed  CAS  Google Scholar 

  • Kirsch M, Korth HG, Stenert V, Sustmann R, de Groot H (2003) The autoxidation of tetrahydrobiopterin revisited. Proof of superoxide formation from reaction of tetrahydrobiopterin with molecular oxygen. J Biol Chem 278:24481–24490

    Article  PubMed  CAS  Google Scholar 

  • Kosar-Hashemi B, Armarego WLF (1993) A convenient spectrophotometric method for measuring the kinetic parameters of glyceryl-ether monooxygenase (EC1.14.16.5). Biol Chem Hoppe-Seyler 374:9–25

    PubMed  CAS  Google Scholar 

  • Krauth-Siegel RL, Ludemann H (1996) Reduction of dehydroascorbate by trypanothione. Mol Biochem Parasitol 80:203–208

    Article  PubMed  CAS  Google Scholar 

  • Krauth-Siegel RL, Meiering SK, Schmidt H (2003) The parasite-specific trypanothione metabolism of Trypanosoma and Leishmania. Biol Chem 384:539–549

    Article  PubMed  CAS  Google Scholar 

  • Krauth-Siegel LR, Comini MA, Schlecker T (2007) The trypanothione system. Subcell Biochem 44:231–251

    Article  PubMed  Google Scholar 

  • Liew FY, Millott S, Parkinson C, Palmer RMJ, Moncada S (1990) Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from l-arginine. J Immunol 144:4794–4797

    PubMed  CAS  Google Scholar 

  • Ma D, Beverley SM, Turco SJ (1996) Leishmania donovani possess a NADPH-dependent alkylglyceryl cleavage enzyme. Biochem Biophys Res Commun 227:885–889

    Article  PubMed  CAS  Google Scholar 

  • Marchini JF, Cruz AK, Beverley SM, Tosi LR (2003) The H region HTBF gene mediates terbinafine resistance in Leishmania major. Mol Biochem Parasitol 131:77–81

    Article  PubMed  CAS  Google Scholar 

  • Milstien S, Katusic Z (1999) Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun 263:681–684

    Article  PubMed  CAS  Google Scholar 

  • Moreira W, Leblanc E, Ouellette M (2009) The role of reduced pterins in resistance to reactive oxygen and nitrogen intermediates in the protozoan parasite Leishmania. Free Radic Biol Med 46:367–375

    Article  PubMed  CAS  Google Scholar 

  • Moutiez M, Meziane-Cherif D, Aumercier M, Sergheraert C, Tartar A (1994) Compared reactivities of trypanothione and glutathione in conjugation reactions. Chem Pharmacol Bull 42:2641–2644

    CAS  Google Scholar 

  • Nare B, Hardy L, Beverley SM (1997a) The roles of pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHFR-TS) in pteridine metabolism in the protozoan parasite Leishmania major. J Biol Chem 272:13883–13891

    Article  PubMed  CAS  Google Scholar 

  • Nare B, Luba J, Hardy LW, Beverley S (1997b) New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity. Parasitology 114:S101–S110

    PubMed  Google Scholar 

  • Oettl K, Reibnegger G (2002) Pteridine derivatives as modulators of oxidative stress. Curr Drug Metab 3:203–209

    Article  PubMed  CAS  Google Scholar 

  • Ouellette M, Legare D, Papadopoulou B (1994) Microbial multidrug-resistance ABC transporters. Trends Microbiol 2:407–411

    Article  PubMed  CAS  Google Scholar 

  • Ouellette M, Drummelsmith J, El-Fadili A, Kundig C, Richard D, Roy G (2002) Pterin transport and metabolism in Leishmania and related trypanosomatid parasites. Int J Parasitol 32:385–398

    Article  PubMed  CAS  Google Scholar 

  • Ouellette M, Drummelsmith J, Papadopoulou B (2004) Leishmaniasis: drugs in the clinic, resistance and new developments. Drug Resist Updat 7:257–266

    Article  PubMed  CAS  Google Scholar 

  • Penketh PG, Klein RA (1986) Hydrogen peroxide metabolism in Trypanosoma brucei. Mol Biochem Parasitol 20:111–121

    Article  PubMed  CAS  Google Scholar 

  • Pick E, Keisari Y (1980) A simple calorimetric assay for the measurement of hydrogen peroxide produced by cells in culture. J Immunol Methods 38:161–170

    Article  PubMed  CAS  Google Scholar 

  • Plewes KA, Barr SD, Gedamu L (2003) Iron superoxide dismutases targeted to the glycosomes of Leishmania chagasi are important for survival. Infect Immun 71:5910–5920

    Article  PubMed  CAS  Google Scholar 

  • Robinson JP, Bruner LH, Bassoe CF, Hudson JL, Ward PA, Phan SH (1988) Measurement of intracellular fluorescence of human monoctytes relative to oxidative metabolism. J Leucoc Biol 43:304–310

    CAS  Google Scholar 

  • Roy G, Kundig C, Olivier M, Papadopoulou B, Ouellette M (2001) Adaptation of Leishmania cells to in vitro culture results in a more efficient reduction and transport of biopterin. Exp Parasitol 97:161–168

    Article  PubMed  CAS  Google Scholar 

  • Ryan KA, Dasgupta S, Beverley SM (1993) Shuttle cosmid vectors for the trypanosomatid parasite Leishmania. Gene 131:145–150

    Article  PubMed  CAS  Google Scholar 

  • Sancar A (2004) Photolyase and cryptochrome blue-light photoreceptors. Adv Protein Chem 69:73–100

    Article  PubMed  CAS  Google Scholar 

  • Spath GF, Garraway LA, Turco SJ, Beverley SM (2003) The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts. Proc Natl Acad Sci USA 100:9536–9541

    Article  PubMed  Google Scholar 

  • Stamler JS (1994) Redox signalling: nitrosylation and related target interactions of nitric oxide. Cell 78:931–936

    Article  PubMed  CAS  Google Scholar 

  • Tayeh MA, Marletta MA (1989) Macrophage oxidation of l-argenine to nitric oxide, nitrite and nitrate. J Biol Chem 264:19654–19658

    PubMed  CAS  Google Scholar 

  • Titus RG, Gueiros-Filho FJ, de Freitas LA, Beverley SM (1995) Development of a safe live Leishmania vaccine line by gene replacement. Proc Natl Acad Sci USA 92:10267–10271

    Article  PubMed  CAS  Google Scholar 

  • Vergauwen B, Herbert M, Van Beeumen JJ (2006) Hydrogen peroxide scavenging is not a virulence determinant in the pathogenesis of Haemophilus influenzae type b strain Eagan. BMC microbiology 6:3

    Article  PubMed  Google Scholar 

  • Vogt RN, Steenkamp DJ (2003) The metabolism of S-nitrosothiols in the trypanosomatids: the role of ovothiol A and trypanothione. Biochem J 371:49–59

    Article  PubMed  CAS  Google Scholar 

  • Wanasen N, Soong L (2008) l-Arginine metabolism and its impact on host immunity against Leishmania infection. Immunol Res 41:15–25

    Article  PubMed  CAS  Google Scholar 

  • Wang J, Leblanc E, Chang CF, Papadopoulou B, Bray T, Whiteley JM, Lin SX, Ouellette M (1997) Pterin and folate reduction by the Leishmania tarentolae H locus short- chain dehydrogenase/reductase PTR1. Arch Biochem Biophys 342:197–202

    Article  PubMed  CAS  Google Scholar 

  • Werner-Felmayer G, Golderer G, Werner ER (2002) Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects. Curr Drug Metab 3:159–173

    Article  PubMed  CAS  Google Scholar 

  • Wyllie S, Cunningham ML, Fairlamb AH (2004) Dual action of antimonial drugs on thiol redox metabolism in the human pathogen Leishmania donovani. J Biol Chem 279:39925–39932

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank James Schwarz for help with flow cytometry, and Dr. David Scott for providing the FV1 ptr1 −/SSU::PTR1 line. We thank Alan Fairlamb and CC Wang for their discussions and comments on this work, and are grateful to the members of the lab for critical comments on the manuscript, including M. Cunningham, D. Dobson, L. Epstein, F. Gueiros-Filho, L. Garrity, J. Moore, K. R. Nagar-Anthal, and D. Scott. Supported by NIH grant AI21903.

Author information

Author notes

  1. Bakela Nare

    Present address: SCYNEXIS, Inc., P.O. Box 12878, Research Triangle Park, NC, 27709, USA

  2. Levi A. Garraway

    Present address: Department of Medical Oncology, Dana Farber Cancer Institute, 44 Binney, St. Boston, MA, 02115, USA

Authors and Affiliations

  1. Department of Molecular Microbiology, Washington University School of Medicine, Campus Box 8230, 660 S. Euclid Ave., St. Louis, MO, 63110, USA

    Tim J. Vickers & Stephen M. Beverley

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA

    Bakela Nare, Levi A. Garraway & Stephen M. Beverley

Authors
  1. Bakela Nare
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Levi A. Garraway
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tim J. Vickers
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen M. Beverley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stephen M. Beverley.

Additional information

Communicated by G. Kapler.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nare, B., Garraway, L.A., Vickers, T.J. et al. PTR1-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite Leishmania major . Curr Genet 55, 287–299 (2009). https://doi.org/10.1007/s00294-009-0244-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-009-0244-z

Keywords

Search

Navigation