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Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies., Competing Interests: The authors declare no competing financial interests.

The Indian subcontinent is the only region where arsenic contamination of drinking water coexists with widespread resistance to antimonial drugs that are used to treat the parasitic disease visceral leishmaniasis. We have previously proposed that selection for parasite resistance within visceral leishmaniasis patients who have been exposed to trivalent arsenic results in cross-resistance to the related metalloid antimony, present in the pentavalent state as a complex in drugs such as sodium stibogluconate (Pentostam) and meglumine antimonate (Glucantime). To test this hypothesis, Leishmania donovani was serially passaged in mice exposed to arsenic in drinking water at environmentally relevant levels (10 or 100 ppm). Arsenic accumulation in organs and other tissues was proportional to the level of exposure and similar to that previously reported in human liver biopsies. After five monthly passages in mice exposed to arsenic, isolated parasites were found to be completely refractory to 500 μg · mL(-1) Pentostam compared with the control passage group (38.5 μg · mL(-1)) cultured in vitro in mouse peritoneal macrophages. Reassessment of resistant parasites following further passage for 4 mo in mice without arsenic exposure showed that resistance was stable. Treatment of infected mice with Pentostam confirmed that resistance observed in vitro also occurred in vivo. We conclude that arsenic contamination may have played a significant role in the development of Leishmania antimonial resistance in Bihar because inadequate treatment with antimonial drugs is not exclusive to India, whereas widespread antimonial resistance is.

Methylglyoxal is a toxic by-product of glycolysis and other metabolic pathways. In mammalian cells, the principal route for detoxification of this reactive metabolite is via the glutathione-dependent glyoxalase pathway forming d-lactate, involving lactoylglutathione lyase (GLO1; EC 4.4.1.5) and hydroxyacylglutathione hydrolase (GLO2; EC 3.2.1.6). In contrast, the equivalent enzymes in the trypanosomatid parasites Trypanosoma cruzi and Leishmania spp. show >200-fold selectivity for glutathionylspermidine and trypanothione over glutathione and are therefore sensu stricto lactoylglutathionylspermidine lyases (EC 4.4.1.-) and hydroxyacylglutathionylspermidine hydrolases (EC 3.2.1.-). The unique substrate specificity of the parasite glyoxalase enzymes can be directly attributed to their unusual active site architecture. The African trypanosome differs from these parasites in that it lacks GLO1 and converts methylglyoxal to l-lactate rather than d-lactate. Since Trypanosoma brucei is the most sensitive of the trypanosomatids to methylglyoxal toxicity, the absence of a complete and functional glyoxalase pathway in these parasites is perplexing. Alternative routes of methylglyoxal detoxification in T. brucei are discussed along with the potential of exploiting trypanosomatid glyoxalase enzymes as targets for anti-parasitic chemotherapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The clinical value of antimonial drugs, the mainstay therapy for leishmaniasis, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is required. Using the model organism Leishmania tarentolae, we have examined the role of trypanothione S-transferase (TST) in trivalent antimony [Sb(III)] resistance. TST has S-transferase activity with substrates such as chlorodinitrobenzene as well as peroxidase activity with alkyl and aryl hydroperoxides but not with hydrogen peroxide. Although S-transferase activity and TST protein levels were unchanged in Sb(III)-sensitive and -resistant lines, rates of metabolism of hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide were significantly increased. Elevated peroxidase activities were shown to be both trypanothione and tryparedoxin dependent and were associated with the overexpression of classical tryparedoxin peroxidase (TryP) in the cytosol of L. tarentolae. The role of TryP in Sb(III) resistance was verified by overexpression of the recombinant Leishmania major protein in Sb(III)-sensitive promastigotes. An approximate twofold increase in the level of TryP activity in this transgenic cell line was accompanied by a significant decrease in sensitivity to Sb(III) (twofold; P < 0.001). Overexpression of an enzymatically inactive TryP failed to result in Sb(III) resistance. This indicates that TryP-dependent resistance is not due to sequestration of Sb(III) and suggests that enhanced antioxidant defenses may well be a key feature of mechanisms of clinical resistance to antimonial drugs.

Leishmaniasis is a complex disease, with visceral and cutaneous manifestations, and is caused by over 15 different species of the protozoan parasite genus Leishmania. There are significant differences in the sensitivity of these species both to the standard drugs, for example, pentavalent antimonials and miltefosine, and those on clinical trial, for example, paromomycin. Over 60% of patients with visceral leishmaniasis in Bihar State, India, do not respond to treatment with pentavalent antimonials. This is now considered to be due to acquired resistance. Although this class of drugs has been used for over 60 years for leishmaniasis treatment, it is only in the past 2 years that the mechanisms of action and resistance have been identified, related to drug metabolism, thiol metabolism, and drug efflux. With the introduction of new therapies, including miltefosine in 2002 and paromomycin in 2005-2006, it is essential that there be a strategy to prevent the emergence of resistance to new drugs; combination therapy, monitoring of therapy, and improved diagnostics could play an essential role in this strategy.

Crithidia fasciculata is a monogenetic parasite of insects. It grows in fully defined media without requiring serum, which facilitates biochemical analysis. We have constructed a series of expression systems that allows expression of transfected genes in the kinetoplastid protozoa Crithidia and Leishmania. These cells can be readily transfected with plasmid DNA by electroporation and transformants selected with various antibiotic resistance markers. 5'-Trans-splicing signals and poorly defined regions within the 3'-untranslated regions of genes are required for optimal expression of genes in trypanosomatids. We, therefore, inserted the intergenic region of the C. fasciculata phosphoglycerate kinase (PGK) genes A and B, which allows polyadenylation of the target gene and spliced leader addition to the selectable marker gene. Part of the intergenic region of the PGK locus was added upstream of the target gene to permit its trans-splicing. A 3'-untranslated sequence from the Crithidia glutathionylspermidine synthetase (GSPS) was also added to allow the polyadenylation of the selectable marker gene. Genes can be readily inserted using a multiple cloning site and can be expressed as a fusion protein with a poly-histidine sequence at either the N or C-terminus or fused with green fluorescent protein. Biologically active proteins can be expressed in C. fasciculata or L. amazonensis promastigotes and purified by affinity chromatography using a metal chelating column.

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

Drug discovery pipelines for the “neglected diseases” are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic nitro-compounds (R)-PA-824, DNDI-VL-2098 and delamanid have been identified as potential candidates for the treatment of visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative NAD(P)H oxidase as the activating nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic nitro-compounds, but only marginally to the monocyclic nitro-drugs, nifurtimox and fexinidazole sulfone, known to be activated by a mitochondrial oxygen-insensitive nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic nitro-compounds and only marginally resistant to nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series. [ABSTRACT FROM AUTHOR]

Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis. [ABSTRACT FROM AUTHOR]

The genome of Leishmania major, the causative agent of cutaneous leishmaniasis, contains three almost identical genes encoding putative glutathione peroxidases, which differ only at their N- and C-termini. Because the gene homologues are essential in trypanosomes, they may also represent potential drug targets in Leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as the electron donor indicating that it is not a bone fide glutathione peroxidase. By contrast, high peroxidase activity was obtained with tryparedoxin, indicating that these proteins belong to a new class of monomeric tryparedoxin -dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in the formation of an intramolecular disulfide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity, whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping-pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than towards t-butyl hydroperoxide, whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 × 104 m−1·s−1, whereas TryP1 exhibits higher rate constants ∼ 106 m−1·s−1. All three TDPX proteins together constitute ∼ 0.05% of the L. major promastigote protein content, whereas the TryPs are ∼ 40 times more abundant. Possible specific functions of TDPXs are discussed. [ABSTRACT FROM AUTHOR]

Trypanothione replaces glutathione in defence against cellular damage caused by oxidants, xenobiotics and methylglyoxal in the trypanosomatid parasites, which cause trypanosomiasis and leishmaniasis. In Leishmania major, the first step in methylglyoxal detoxification is performed by a trypanothione-dependent glyoxalase I (GLO1) containing a nickel cofactor; all other characterized eukaryotic glyoxalases use zinc. In kinetic studies L. major and human enzymes were active with methylglyoxal derivatives of several thiols, but showed opposite substrate selectivities: N 1-glutathionylspermidine hemithioacetal is 40-fold better with L. major GLO1, whereas glutathione hemithioacetal is 300-fold better with human GLO1. Similarly, S-4-bromobenzylglutathionylspermidine is a 24-fold more potent linear competitive inhibitor of L. major than human GLO1 ( Kis of 0.54 µM and 12.6 µM, respectively), whereas S-4-bromobenzylglutathione is > 4000-fold more active against human than L. major GLO1 ( Kis of 0.13 µM and > 500 µM respectively). The crystal structure of L. major GLO1 reveals differences in active site architecture to both human GLO1 and the nickel-dependent Escherichia coli GLO1, including increased negative charge and hydrophobic character and truncation of a loop that may regulate catalysis in the human enzyme. These differences correlate with the differential binding of glutathione and trypanothione-based substrates, and thus offer a route to the rational design of L. major-specific GLO1 inhibitors. [ABSTRACT FROM AUTHOR]

Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way. [ABSTRACT FROM AUTHOR]

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX-LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis-Menten saturation kinetics with Km values of 36 μM and 9 μM, respectively, the former yielding a kcat/Km of 5.0×106 M-1 s-1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti-leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time-dependent manner, with the pseudo-first-order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme-drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge-transfer complex in the two-electron-reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2.3-dimercaptopropanol, 2,3-dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid-unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed. [ABSTRACT FROM AUTHOR]

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol-redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine-activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti-leishmanial agents for chemotherapeutic use. [ABSTRACT FROM AUTHOR]

Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR-knockout mutants by gene disruption in L.donovani and L.major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L.donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR-containing region to a larger chromosome. Mutants of L.donovani and L.major possessing only one wild-type TR allele express less TR mRNA and have lower TR activity compared with wild-type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates. [ABSTRACT FROM AUTHOR]

Trypanothione reductase is thought to be important in maintaining an intracellular reducing environment in trypanosomatids. To investigate the role of trypanotione reductase we transfected Leishmania donovani and Trypanosoma cruzi with an expression vector containing the L. donovani trypanothione reductase gene and achieved over-expression of enzyme activity (10–14-fold) in transformed cells. Following treatment of L. donovani cells with the thiol-oxidizing agent diamide, the ability to regenerate dihydrotrypanothione from trypanothione disulphide was considerably enhanced in cells which over-expressed trypanothione reductase. However, the growth of transformed and control cells was equally sensitive to inhibition by nifurtimox, nitrofurazone and gentian violet, drugs that are thought to act by inducing oxidative damage. Likewise, growth of transformed and control cells were equally susceptible to inhibition by hydrogen peroxide, and control and transformed L. donovani promastigotes metabolized hydrogen peroxide at comparable rates. Thus, these experiments suggest that the ability to regenerate dihydrotrypanothione from trypanothione disulphide is not a rate-limiting step in the metabolism of hydrogen peroxide. [ABSTRACT FROM AUTHOR]

The enzyme trypanothione reductase has been identified as a prime target for the rational design of inhibitors which may have clinical use in the treatment of tropical diseases caused by the genera Trypanosoma and Leishmania. To aid the design or identification of new inhibitors of this enzyme we have elucidated the structural detail of a trypanothione reductase complexed with one of the naturally occurring substrates, N1-glutathionylspermidine disulphide, by single-crystal X-ray diffraction methods at 0.28-nm resolution. The model for the Crithidia fasciculata enzyme-substrate complex has an R-factor of 14.8% and root-mean-square deviations of 0.0015 nm and 3.3 degrees on bond lengths and angles respectively. Hydrogen bonding and van der Waals interactions between the enzyme and substrate are dominated by the amino acid side chains. The substrate binds in a rigid active site such that one glutathione moiety is in a V-shape, the other in an extended conformation. One spermidine moiety binds closely to a hydrophobic patch in the active site formed by a tryptophan and a methionine. Distances between the methionine S δ and the terminal N of this spermidine suggest that a hydrogen bond may supplement the hydrophobic interactions in this part of the active site. [ABSTRACT FROM AUTHOR]

Provides information on a study using a trans-dominant mutational strategy to down-regulate trypanothione reductase (TR) activity levels in Leishmani donovani. Methodology and materials used to conduct the study; Results of the study.

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania. [ABSTRACT FROM AUTHOR]

In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified α, β, and γ subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg-1). Kinetic studies suggest a ternary complex mechanism, with Km values of 140 μM for trypanothione and 7.4 mM for cumene hydroperoxide and kcat = 25 s-1. Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation. [ABSTRACT FROM AUTHOR]

Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH- dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H4B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity associated with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiological pH and ionic strength and compared with LmQDPR. Specificity constants (kcat/Km) for LmPTR1 are similar with dihydrobiopterin (H2B) and quinon-old dihydrobiopterin (qH2B) as substrates and about 20-fold lower than LmQDPR with qH2B. In contrast, TbPTR1 shows a 10-fold higher kcat/Km for H2B over qH2B. Analysis of Trypanosoma brucei isolated from infected rats revealed that H4B (430 nM, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H4B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained from lines overexpressing LrnQDPR with H4B as a medium supplement. These cells grew normally with H4B, which spontaneously oxidizes to qH2B, but were unable to survive in the absence of pterin or with either biopterin or H2B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metabolism in African trypanosomes and underline its potential as a drug target. [ABSTRACT FROM AUTHOR]

Abstract: Enhancement of the anti-oxidant metabolism of Leishmania parasites, dependent upon the unique dithiol trypanothione, has been implicated in laboratory-generated antimony resistance. Here, the role of the trypanothione-dependent anti-oxidant pathway is studied in antimony-resistant clinical isolates. Elevated levels of tryparedoxin and tryparedoxin peroxidase, key enzymes in hydroperoxide detoxification, were observed in antimonial resistant parasites resulting in an increased metabolism of peroxides. These data suggest that enhanced anti-oxidant defences may play a significant role in clinical resistance to antimonials. [Copyright &y& Elsevier]