PTR1-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite Leishmania major.
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 H(2)O(2) 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. H(2)O(2) susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H(4)B metabolism specifically. Neither H(2)O(2) 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.
Nare
Bakela
B
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Garraway
Levi A
LA
Vickers
Tim J
TJ
Beverley
Stephen M
SM
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018320:Leishmania major / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011621:Pteridines / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism
D009097:Multienzyme Complexes; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D016877:Oxidants; D015800:Protozoan Proteins; D011621:Pteridines; C056930:thymidylate synthase-dihydrofolate reductase; D008769:Methylnitronitrosoguanidine; D001708:Biopterins; C002385:linsidomine; C024942:S-nitrosopenicillamine; D005492:Folic Acid; D006861:Hydrogen Peroxide; D008981:Molsidomine; D010088:Oxidoreductases; C085592:pteridine reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; C003402:sapropterin