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

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 H 2 O 2 than PTR1-overproducing lines, and significant 3-5 fold 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 reductasedependent 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.

State of the Art in African Trypanosome Drug Discovery

“…Unlike humans and most other organisms that have monofunctional proteins, trypanosomatids express a bifunctional DHFR-TS in which a single polypeptide chain contains both catalytic activities [80]. DHFR-TS and PTR1, have been proposed as key drug targets in trypanosomatids because folates are essential for synthesis of thymidylate while pterins are implicated in parasites growth and oxidant resistance [81, 82]. …”

Selection of Molecular Targets for Drug Development Against Trypanosomatids

“…2.5 ). These two enzymes have been proposed as drug targets in trypanosomatids, since they participate in essential pathways for the parasite metabolism, like thymidylate production by folates and oxidant resistance by the reduction of pterins (Moreira et al 2009 ;Nare et al 2009 ). Drugs that have been used to inhibit DHFR have proven ineffective in Leishmania (Neal and Croft 1984 ).…”

Proteomic analysis of metacyclogenesis in Leishmania infantum wild-type and PTR1 null mutant

“…One of the earliest known cellular functions of THB was as a growth factor for the parasite Crithidia fasciculata [12], and many studies have indicated that THB is indeed an essential growth factor for Leishmania [13][14][15][16], although its exact role is still to be discovered (reviewed in [17]). THB was also shown to react readily with oxygen, superoxide, H 2 O 2 , and peroxynitrite and it was demonstrated that it protects cells against oxidative damages [18,19] (and reviewed in [20,21]). PTR1 and reduced pterins thus play important roles in Leishmania differentiation and life cycle in which the infective metacyclic form encounters an impressive oxidative stress as it meets with the host immune system.…”