Comparative metabolism of conjugated and unconjugated pterins in Crithidia, Leishmania and African trypanosomes
Han B. Ong, Susan Wyllie, Alan H. Fairlamb, Martin Craig Taylor, Martin Craig Taylor, Martin Craig Taylor
TL;DR
The study compares how three types of parasites use different forms of pterins for growth, revealing that they convert pterins to tetrahydro forms and prefer specific structures.
Contribution
The study identifies structural requirements of pterins for growth in kinetoplastid parasites and reveals that pterins are reduced to tetrahydro forms without further conversion.
Findings
T. brucei and C. fasciculata prefer 6-biopterin, while L. major strongly prefers reduced pterins.
Pterins supporting growth are metabolized to tetrahydro forms with no evidence of further interconversion.
Folate or dihydrofolate can partially replace unconjugated pterins in some cases.
Abstract
Mammalian cells synthesise tetrahydrobiopterin de novo, an essential cofactor for hydroxylation of aromatic amino acids, cleavage of ether lipids and the synthesis of nitric oxide. In contrast, kinetoplastid parasites are pterin auxotrophs and none of the above metabolic functions can account for the essential requirement of an unconjugated pterin for growth. Here we investigate the pterin requirements for growth and survival of two medically important parasites (T. brucei and L. major) in comparison with the model insect parasite, Crithidia fasciculata. The pterin concentration required to support 50% of maximum growth of each parasite was determined in defined pterin-free media for a variety of naturally occurring pterins. T. brucei and C. fasciculata showed an identical order of preference with the most active being 6-biopterin, followed by dihydrobiopterin > tetrahydrobiopterin >…
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Fig 7- —http://dx.doi.org/10.13039/100010269Wellcome Trust
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Taxonomy
TopicsTrypanosoma species research and implications · Biochemical and Molecular Research · Research on Leishmaniasis Studies
Introduction
Pterins are a class of nitrogen-containing heterocyclic compounds that share a characteristic 2-amino-4-oxopteridine moiety and can be further classified as conjugated or unconjugated [1,2]. These compounds can be reduced at the 5, 6, 7 and 8 positions of the pteridine ring to exist in different oxidation states (Fig 1A). Conjugated pterins contain a p-aminobenzoate substituent at the 6-position of the pteridine moiety coupled a single glutamate residue and can be further polyglutamated to facilitate retention within cells (Fig 1B). Unconjugated pterins contain neither of these substitutions and instead have varying lengths of hydrocarbon side chains on the 6 or 7-position of the moiety (Fig 1C). Despite their structural similarities, both classes of pterins are required for many important metabolic processes. Conjugated pterins are more commonly referred to as folates whose reduced derivatives are essential cofactors for several enzymes catalysing one-carbon transfer reactions [3–5]. The term “pterin” is used primarily to describe unconjugated pterins. Oxidised pterins, such as xanthopterin and leucopterin, are major components of butterfly wings [6], while the fully reduced tetrahydro-form of biopterin (tetrahydrobiopterin, H_4_B) is an essential cofactor for various one-electron transfer enzymatic reactions [7,8]. Pterins are also essential metabolic intermediates in the biosynthesis of a 7-deazaguanine derivative, queuosine, in eubacteria. Queuosine is incorporated into the wobble position 34 of the anticodon loop of tyrosyl, asparaginyl, aspartyl and histidinyl tRNAs not only in eubacteria, but also by salvage in plants, animals and fungi [9]. This modification is important for translational fidelity [10,11]. Like other eukaryotes, trypanosomes do not appear to be able to synthesise queuine de novo and obtain it from their environment [12,13]. Although levels of queuosine in tRNA can change in response to availability of certain amino acids, these changes have negligible effect on growth rate or global protein synthesis, eliminating this as an essential role for pterins [12,13].
Chemical structure of unconjugated and conjugated pterins.Panel A: Basic pterin moiety and the numbering system in the pteridine ring. Panel B: Chemical structures of different unconjugated pterins used in the present study. Panel C: Chemical structures of pteroic acid and folic acid which are pterins conjugated to an aminobenzoate group with and without glutamylation, respectively.
H_4_B is the best studied pterin due to its important biological functions [14]. In mammalian cells, H_4_B is synthesised de novo from guanosine triphosphate (GTP) and acts as an essential cofactor for several enzymatic reactions such as the hydroxylation of aromatic amino acids (phenylalanine, tyrosine and tryptophan) by their respective hydroxylases [15–17]. H_4_B is also required by nitric oxide synthases (NOS) to produce nitric oxide, both an important signalling molecule and a powerful antimicrobial agent [18–20]. In lipid metabolism, the enzyme glyceryl-ether monooxygenase also utilises H_4_B for the hydroxylation of glyceryl-ethers, resulting in their subsequent cleavage [21,22].
Leishmania spp. and Trypanosoma ssp. are protozoan parasites that cause some of the most neglected tropical diseases known as the Leishmaniases, and the South American and African trypanosomiases. Crithidia spp. are parasites of insects that have been used as convenient models for the study of these fastidious vertebrate parasites [23,24]. Unlike mammalian cells, trypanosomatids such as Leishmania spp. and Crithidia spp. are unable to synthesise pterins de novo and are therefore pteridine auxotrophs. The importance of pterins was first described in the 1950s when biopterin was found to be an important growth factor for the insect parasite, Crithidia fasciculata [25,26]. The term “Crithidia growth factor” originated from these studies and is used to describe pterins, including biopterin, that are able promote the growth of C. fasciculata. A fully defined media (KD media) was formulated for culturing of C. fasciculata where high levels of folate supplementation were determined to be required for cell growth [27]. This requirement for high folate was significantly lowered by the addition of trace amounts of biopterin [13] and this observation has been used in the biological assay of unconjugated pteridines in biological fluids and tissues [27,28]. Despite the extensive knowledge of the roles of pterins in other organisms, the function(s) of pterins within the Kinetoplastida is still not yet understood.
Pterin metabolism in Leishmania spp. was studied extensively following the discovery of an enzyme, pteridine reductase 1 (PTR1), which plays a role in parasite resistance to the classical antifolate drug methotrexate [29,30]. This enzyme plays a pivotal role in the generation of H_4_B from salvaged biopterin in trypanosomatids [31,32]. PTR1 from both Leishmania major and Trypanosoma brucei has also been shown to be capable of regenerating H_4_B from quinonoid dihydrobiopterin (qH_2_B), an intermediary product of pterin-dependent hydroxylation reactions [2]. This reaction is normally catalysed by a dedicated NADH-dependent quinonoid dihydropteridine reductase (qDPR) that is also present in L. major but absent in T. brucei [2,33]. The essentiality of PTR1 in T. brucei was confirmed by the knockdown of PTR1 by RNAi, which, unlike L. major PTR1 knockout mutants, could not be rescued by any pterin supplementation [34]. These findings made PTR1 an attractive novel target for drug discovery initiatives against T. brucei for the treatment of Human African Trypanosomiasis (HAT). However, inhibitors designed against this enzyme have been met by a lack of cellular potency [35]. This study is aimed at better understanding the requirement for pterins in the Kinetoplastida, particularly T. brucei. Specifically, we investigate the growth requirements and metabolism of pterins in T. brucei in a comparison to L. major and C. fasciculata. Our findings should facilitate future drug development against this area of parasite metabolism.
Methods
Organisms and reagents – All pterins were purchased from Schircks laboratories. Other chemicals and reagents used in this study were of the highest grade and purity available. C. fasciculata clone HS6 [36] was cultured at 28 °C with shaking at 200 rpm in medium D plus 2.27 nM folate [27]. T. brucei bloodstream-form S427 was cultured at 37 °C in a modified HMI-9 medium containing 31 nM folate (FDM [37]) supplemented with 10% dextran/charcoal-treated foetal calf serum (FCS, Hyclone) and 15 µg ml^-1^ gentamycin sulphate (G418, Invitrogen). L. major promastigotes (Friedlin strain; WHO designation MHOM/JL/81/Friedlin) were cultured at 28 °C with shaking at 150 rpm in M199 medium (Invitrogen, folate content 22.7 nM) containing 0.5% FCS (PAA). L-Biopterin (10 µM) was added for routine culturing of all parasites.
Concentration of pterins required for half maximal growth – parasites were depleted of pterins by subculturing (1 x 10^4^ cells ml^-1^) in pterin-free media for 72 h. The concentration of pterins required for growth were determined in 96-well microtiter plates in a final culture volume of 200 µl per well and an initial parasite seeding density of 1 x 10^4^ cells ml^-1^. The pH of the assay media was adjusted to correct for HCl (1 mM, H_4_B) or NaOH (1 mM, all other pterins) used for the preparation of stock pterin solutions. Cells were incubated at their normal culture media as above. Cell densities were determined daily using a CASY cell-counter. To assay cell viability of parasites grown in the absence of pterins, cells were subcultured in fresh medium for 72 h before growth was determined using a resazurin-based assay [38,39]. The ability of each pterin to support 50% maximum cell growth (GC_50_) was determined by comparison with cultures supplemented with 10 µM L-biopterin (positive control for maximum growth, 100%). GC_50_ values were determined by plotting cell growth with respect to pterin concentration and analysed by non-linear regression with a 4-parameter logistic equation in GraFit.
Analysis of intracellular pterins – Intracellular pterins of parasites were determined by HPLC as previously described [2]. Intracellular pterin concentrations were calculated based upon previously published cell volumes of 10.5, 5.8 and 4.3 µl per 10^8^ cells for C. fasciculata [40], T. brucei [41], and L. major [42], respectively.
Enzymatic assays – Cell lysates were prepared as previously described [43]. Trypanothione reductase and PTR1 activities in these lysates were determined as previously described [44,45].
Results and discussion
Effect of extracellular L-biopterin concentrations on cellular pterin levels in C. fasciculata – The insect parasite, C. fasciculata was used as a model organism to develop methods for studying pterin metabolism in other trypanosomatids. Unlike T. brucei, these parasites can be cultured in a fully defined medium that does not require serum, thus enabling controlled modulation of pterin structure and concentration. Initially, C. fasciculata was grown in medium D supplemented with 4 nM L-biopterin, as recommended by Dewey and Kidder [27]. However, under these conditions, intracellular pterin content was below the limits of detection in our HPLC assay. This problem was resolved by increasing the biopterin concentration to 50 nM in the culture medium. Under these conditions, biopterin was readily detected in log-phase cells (harvested by centrifugation at ~1 x 10^7^ cells ml^-1^) such that total biopterin was proportional to cell numbers between 1 × 10^7^ and 2.5 × 10^8^ per assay (Fig 2). All subsequent HPLC analyses were carried out on ~5 x 10^7^ cells. Biopterin was found to be the predominant intracellular pterin in these log-phase parasites, with the fully reduced H_4_B constituting more than 95% of the total (Fig 3). The total intracellular content of biopterin in mid-log phase C. fasciculata cultured in 50 nM biopterin (Fig 2) was determined as 62.2 ± 0.4 pmol/ 10^8^ cells, from which an intracellular concentration of 5.9 ± 0.04 µM can be calculated.
Linearity of HPLC based assay for intracellular pterin analysis.C. fasciculata were treated with iodine in 0.1 M HCl to oxidise 7,8-dihydrobiopterin and tetrahydrobiopterin to biopterin [46], protein removed by centrifugation and the supernatant analysed by HPLC [2]. Total biopterin was proportional to cell numbers up to 2.5 × 108 cells (linear regression coefficient of 1.00).
Intracellular pterin content of C. fasciculata during growth.C. fasciculata was cultured in KD medium supplemented with either 100 nM (pink) or 10 µM (blue) of L-biopterin. Cells were cultured at an initial seeding density of 5 x 104 cells ml-1 and harvested during different growth stages namely early log (1 x 106 cells ml-1), mid-log (1 x 107 cells ml-1), early stationary (5 x 107 cells ml-1) and late stationary (5 x 107 cells ml-1) phases, oxidised with iodine and analysed by HPLC for H4B (light bars) and biopterin plus H2B (dark bars). Results are presented as means ± S.D. of triplicate measurements.
Biopterin is essential for growth of L. major with decreases in H_4_B levels regulating parasite differentiation from the sand fly promastigote stage to the infective mammalian metacyclic stage [47]. A similar decrease in H_4_B concentration was also found with C. fasciculata when cultured in 0.1 µM biopterin (Fig 3, pink). Mid-log phase cells (harvested at ~1 x 10^7^ cells ml^-1^) were found to maintain the highest concentrations of biopterin (9.6 ± 0.03 µM), with levels decreasing 6-fold as cells entered stationary phase. The degradation of PTR1 during stationary phase has been proposed to be directly responsible for the decrease in H_4_B levels in Leishmania [48]. However, this was found not to be the case with C. fasciculata, where PTR1 activity in clarified lysates of these parasites was maintained at 55.9 ± 2.4 pmol min^-1^ mg^-1^ (~0.02% of the trypanothione reductase activity, 330 ± 10.3 nmol min^-1^ mg^-1^, used as a control for cell breakage) throughout the different growth phases. Instead, HPLC analyses of media supernatants from stationary-phase cultures indicated that the complete exhaustion of L-biopterin was a more credible reason for the decrease in pterin levels. To test this hypothesis, pterin levels were measured in cells cultured in the presence of saturating amounts of L-biopterin (10 µM) during different growth stages (Fig 3, blue). Consistent with the previous observations, H_4_B (>95%) was again the predominant form of intracellular biopterin in these cells. However, total biopterin levels (21.8 ± 0.13 µM) were considerably higher compared to cells cultured in 0.1 µM L-biopterin, across the different growth stages. Of note, H_4_B levels remained high even in stationary phase parasites. These results suggest the intracellular pterin levels in C. fasciculata are directly dependent upon the availability of pterin in the media and that these parasites can accumulate pterins against a concentration gradient.
Biopterin requirement in the trypanosomatids – The doubling time of C. fasciculata cultured in medium D supplemented with 10 µM L-biopterin was determined to be 6.57 ± 0.03 h (Fig 4A, open symbols). Cells sub-cultured in biopterin-free medium grew normally for 72 h before growth ceased by 120 h (Fig 4A, closed symbols). Biopterin-starved parasites were present for up to 14 days in subcultures, although the parasites progressively lost viability commencing on day 4 (Fig 4A, inset). These observations are consistent with pterin levels in cells falling below the limit of detection after 72 h subculturing in biopterin-free medium D.
Growth analyses of C. fasciculata, T. brucei and L. major cultured in media with and without 10µM L-biopterin.Cumulative growth curves of C. fasciculata (panel A), T. brucei (panel B) or L. major (panel C) cultured in media supplemented with 10 µM L-biopterin (open circles) or without L-biopterin (closed circles). Cell densities were determined daily using a CASY cell-counter. Cells were harvested and resuspended in fresh media with or without L-biopterin every third day. The inset shows the viability of biopterin-free cultures. Viability of biopterin-free cultures was assessed by daily resuspension at a final density of 1 x 104 cells ml-1 in fresh media containing 10 µM L-biopterin. Culture density was measured after a further 72 h incubation and the viability of these cells expressed as a percentage of maximum growth from day 3 cultures. Results are the mean of triplicate measurements.
The importance of nutritional supplementation of biopterin was further demonstrated in T. brucei. In the first instance, bloodstream trypanosomes were conditioned to grow in minimal medium where growth is only permissive when L-biopterin is included. T. brucei was cultured in a previously described folate-deficient medium [34], with the “10% FCS and 0.5% Serum-plus” replaced by 10% charcoal/dextran-treated FCS (FDM^dex^). The medium was supplemented with 10 µM L-biopterin and the doubling times for these parasites were determined to be 8.4 ± 0.08 h (Fig 4B). Consistent with observations made with C. fasciculata, these parasites continued to grow normally for about 3 days when cultured in the absence of biopterin before growth ceased (Fig 4B). At this point the intracellular pterin levels were below the limit of detection. However, unlike C. fasciculata, despite supplementation with fresh biopterin-free media, these cells were unable to survive in culture beyond day 4, with prominent loss of viability evident by day 5 (Fig 4B, inset).
Growth of L. major promastigotes in biopterin-replete medium replicated that observed with C. fasciculata under the same conditions (Fig 4C). Linear growth was observed when subcultured in replete media with a doubling time of 10.4 ± 0.08 h. In biopterin-free medium, growth ceased entirely by day 3. Progressive loss of viability over time was also observed.
Comparing the ability of different pterins to support growth - C. fasciculata can utilise folic acid, in place of L-biopterin for optimal growth in medium D (Fig 5A). However, the difference in pterin concentration required to support 50% of maximum cell growth (GC_50)_ spans 4 orders of magnitude (0.2 ± 0.01 nM versus 3,680 ± 190 nM for biopterin and folate, respectively). T. brucei has a similar GC_50_ of 0.83 ± 0.11 nM for biopterin to that determined for C. fasciculata. However, only 30% of maximum growth could be achieved with folate up to 5 μM (Fig 5B). L. major required considerably more biopterin to support growth (GC_50_ = 567 ± 31 nM) and folate was incapable of supporting growth in the absence of biopterin up to the maximum concentration tested (500 μM) (Fig 5C).
Growth of parasites cultured in varying concentrations of folic acid and biopterin.Panels A, B and C respectively show the GC50 curves of C. fasciculata, T. brucei and L. major cultured in varying concentrations of biopterin (open circles) and folic acid (closed circles). Pterin-depleted cells were seeded at densities as specified in methods and cell growth was quantified after 72 h using a resazurin-based cell-viability assay. Results were analysed by 4-parameter non-linear regression using GraFit and presented as means ± S.E.M of triplicate measurements.
C. fasciculata can use a range of pterins in place of biopterin to support growth [27]. To determine whether this is a generic feature amongst the trypanosomatids, a selection of pterins were tested for their ability to restore growth in pterin-depleted parasites. Several pterins were found to support growth at optimal concentrations (Table 1), while becoming inhibitory at higher concentrations (Table 2). L-Biopterin and its reduced forms were the most adept in supporting the growth of C. fasciculata, with GC_50_ values in the sub-nanomolar range. This was followed by L-neopterin and L-sepiapterin with GC_50_ values in the low nanomolar range. D-neopterin and 7-biopterin were considerably less efficient in supporting growth with GC_50_ values that were >800-fold and >8,000-fold higher than their respective isoforms, L-neopterin and L-biopterin. Conjugated pterins (pteroic acid and folic acid) were intermediary in supporting growth with GC_50_ values in the low micromolar range, with the parasites found to prefer H_2_F over the fully oxidised folic acid. The sub-nanomolar GC_50_-values for L-biopterin and L-neopterin are in reasonable agreement (within 2- to 9-fold) with the previous work of Dewey and Kidder [27] and Rembold [49]. The general ranking of different pterins’ ability to support growth is also in good agreement. L-Sepiapterin, an intermediate in the salvage and de novo synthetic pathways of tetrahydrobiopterin in humans [50], was only moderately active in our assay compared to Dewey and Kidder (58.3 versus 0.34 nM). This discrepancy may be due to an impurity of the latter material, which was isolated from a Drosophila melanogaster mutant in the earlier study [27]. Pteridoxamine, 6-carboxypterin, and xanthopterin were reported as weakly active (GC_50_ > 50 μM) in previous studies [27,49], but were unable to support growth in our experiments. The pterins that support maximal growth at lower concentrations but inhibit growth at higher concentrations (Table 2) are reduced pterins or the unnatural isomers of biopterin or neopterin. The toxicity associated with high concentrations of reduced pterins could be due to autoxidation in the media causing oxidative stress [51] and/or high substrate inhibition of PTR1 [2,32].
Table 1: Pterin requirement for growth in C. fasciculata, T. brucei and L. major. Concentration required for half maximum growth (GC50) were determined as described in the methods. Values are the mean ± S.E.M of triplicate measurements.
Table 2: Pterins supporting maximum growth at low concentrations that inhibited growth at higher concentrations.
T. brucei was found to have a similar growth requirement for unconjugated pterins compared to C. fasciculata, with an identical ranking order for the top six pterins listed in Table 1, albeit with higher GC_50_ values (4.2- to 12.8- fold higher). In contrast, their ability to utilise conjugated pterins was markedly different. Based upon the GC_50_ values, folic acid would appear to be the most efficient in supporting growth of T. brucei. However, the maximum growth of these cells (cultures supplemented with between 10 nM and 500 µM folic acid) was only ~30% of the positive controls (Fig 5B). Likewise, H_2_F was only able to support the growth of T. brucei up to 80% of the maximum growth with growth inhibition evident above 50 µM (Table 2). Unlike, C. fasciculata pteroic acid was unable to support growth.
The pterin requirements for growth of L. major are in excellent agreement with the earlier work of Nare et al. [32] who reported L-biopterin, H_2_B, L-neopterin and sepiapterin to be “good pterin nutrients” when cultures were supplemented with 5 μg ml^-1^ (~20 μM) of these pterins. Consistent with our findings in Table 1, D-neopterin,7-biopterin, pteroic acid, pteridoxamine, xanthopterin, isoxanthopterin and 6-carboxypterin were reported as “inactive pterin nutrients” in wild-type promastigotes. Unexpectedly, we note that reduced pterins (H_2_B and H_4_B) had GC_50_-values for L. major that were orders of magnitude lower than that for biopterin or neopterin, a feature that was not observed with T. brucei or C. fasciculata (Table 1). The reason for this striking preference in L. major for a reduced pterin is not clear. Perhaps uptake of biopterin is less efficient than uptake of H_2_B or H_4_B (or its oxidation product qH_2_B) in L. major, compared to T. brucei or C. fasciculata. Leishmania has fourteen members of the Folate Biopterin Transporter (FBT) family [52], some of which have been characterised [53,54]. In contrast, T. brucei has only seven [55], whereas C. fasciculata Cf C1 [56] has 51 putative FBT genes listed in TriTrypDB. Another explanation might be that biopterin (or neopterin) is less efficiently converted to their respective tetrahydro-forms in L. major. However, the comprehensive enzymatic studies on PTR1 in L. major [32] and T. brucei [2] would not support this. Moreover, it is difficult to envisage an explanation based on the presence or absence of qDPR activity, since this enzyme is present in L. major [33] and C. fasciculata [57], but absent in T. brucei [2].
Further metabolism of pterins in C. fasciculata - To determine whether pterins that support growth undergo further metabolism, parasites were cultured in the presence of a variety of pterins and/or folates (10 µM) and their intracellular pterins then analysed during log phase (Fig 6 and Table 3). No difference in intracellular pterin levels was observed in cells cultured with either H_4_B or L-biopterin, with H_4_B constituting over 96% of the total. The trace amount of pteridoxamine (0.6%) observed in H_4_B cultures is likely to be an extracellular breakdown product of this highly unstable pterin [51]. Interestingly, L-neopterin was found to be principally converted to tetrahydroneopterin (H_4_N) in cells, but not further metabolised. As observed with cells cultured in L-biopterin or H_4_B, over 98% of intracellular neopterin was present in the tetrahydro-form (H_4_N), although total pterin levels within these cells were ~3.5-fold higher (59 ± 3 µM).
Table 3: Pterin content and interconversion in C. fasciculata. Parasites were cultured supplemented with 10 µM pterins and analysed for intracellular pterin content as described in the methods. Values are the means (μM) plus standard deviation of three biological replicates.
Chromatograms illustrating HPLC-mediated separation of pterins.
Pterins were separated on a C_18_ reverse phase column using a mobile phase of 20 mM sodium phosphate plus 4% methanol, pH 6.5. Fully oxidised pterins were detected fluorometrically using excitation wavelengths of 360 and 440 nm, respectively. Panel A: Representative chromatogram of extracts of C. fasciculata cultured in L-biopterin (red line), H_4_B (pink line), and neopterin (blue line), Panel B: folic acid (green line) and H_2_F (purple line), respectively. Note difference in scale in A and B. Quantification is provided in Table 3. Samples were prepared by oxidising cell suspension by iodine under acidic conditions. HPLC analyses were carried out on 10 µl of injected samples (equivalent ~1 x 10^6^ cells).
High concentrations of folate can support growth of C. fasciculata in the absence of pterins (Table 1). Analysis of C. fasciculata cultured under these conditions revealed that folate is recovered as pterins that co-elute with pteridoxamine and 6-hydroxymethylpterin (HMP) (Fig 6 and Table 3). Pteridoxamine is likely to be derived from intracellular tetrahydrofolates following acidic oxidation with iodine required for our analytical method [46], whereas HMP (predominantly present in the tetrahydro-form, H_4_HMP) is a known metabolic product of folate [58]. Pteridoxamine and HMP were also present in C. fasciculata cells cultured in H_2_F with markedly higher levels of H_4_HMP plus HMP (~10-fold) compared to folate cultures. Unlike folic acid, reduced folates are highly unstable (t^½^ ~ 5h) undergoing autooxidation at neutral pH in the absence of ant-oxidants to 6-formylpterin [59]. This might be taken up and reduced by PTR1 to dihydro-6-formylpterin which could then undergo reduction to dihydro-HMP by the action of a broad specificity aldehyde reductase [60]. Radiotracer experiments of Kidder and colleagues [58] identified biopterin, as well as HMP, as a metabolite of 2-[^14^C]folate. Biopterin was not detected in our analyses, but it is worth noting that the amount of radiolabelled biopterin in the experiment by Kidder et al was only 0.25% of that of HMP, well below the limit of detection in our assay.
Conclusion and future prospects. This investigation has demonstrated that pterins are essential for growth in African trypanosomes as well as confirming and extending knowledge on the essential pterin requirements in Crithidia and Leishmania. A common feature of the pterins that supported growth in these organisms is the presence of at least one or more linear carbon substituents at position 6 of the pteridine ring with at least one hydroxyl group, ideally in the 1S configuration – cf. L-biopterin with, L- and D-neopterin (Table 1 and Fig 1). Further metabolism of oxidised pterins appears to be restricted to reduction to the tetrahydro-forms via PTR1, a potential drug target in Leishmania spp. [31,32,61] and T. brucei [2,34]. Which of these chemical features are critical for uptake, reduction to their active form or their vital biological function(s) requires further investigation. The metabolic functions of biopterin in other organisms are well established: principally involving hydroxylation of aromatic amino acids (phenylalanine, tyrosine and tryptophan) by their respective hydroxylases or hydroxylation and cleavage of glyceryl ethers by glyceryl ether monooxygenase. Although Beverley and colleagues have provided biochemical and genetic evidence for phenylalanine hydroxylase activity in leishmania [62], Leishmania spp. are auxotrophic for both phenylalanine and tyrosine [63–65], as are C. fasciculata [26] and T. brucei [66] that also lacks genes for amino acid hydroxylases [67]. These findings eliminate aromatic amino acid hydroxylation as the essential role for pterins in these trypanosomatids. All these kinetoplastids can synthesise and hydrolyse ether lipids [67–71]. However, the alkylglycerol monooxygenase activity in L. major uses NADPH rather than H_4_B as the cofactor used in other organisms [63]. Moreover, abolition of all ether lipid biosynthesis is not lethal in leishmania [72] or T. brucei [67].. A role as an anti-oxidant has been reported in leishmania [73,74], but it is difficult to envisage this as a specific and unique essential function given the plethora of other anti-oxidant defences in these organisms [75–77]. An overall summary of the metabolic pathways in these trypanosomatids (Fig 7) underlines the fact that, of the known biopterin-dependent pathways involving oxygen (phenylalanine, tyrosine, tryptophan hydroxylases, glyceryl ether monooxygenase and arginine-dependent nitric oxide synthase), only the non-essential phenylalanine hydroxylase is present in Leishmania and Crithidia spp. However, the fact that this metabolic cycle is retained suggests that another unidentified essential metabolite or essential biological process is involved.
Biopterin metabolic pathways in trypanosomatids.Transporters and enzymes common to L. major, T. brucei and C. fasciculata are coloured in green; enzymes common to L. major and C. fasciculata are shown in pink. An unknown oxygen dependent essential metabolite “Y” is proposed to account for retention of the cyclical pathway. Abbreviations: FBT, folate biopterin transporter; PTR1, pterdine reductase; qDPR, quinonoid dihydropteridine reductase; PAH, phenylalanine hydroxylase; PCD, pterin carbinolamine dehydratase.
One area for future research that has received little attention since the pioneering work of Kidder and colleagues is a potential role for pterins in the synthesis of lipids, specifically, unsaturated fatty acids and sterols. Growth requirement for pterin can be spared by the addition of crude lipid extracts from C. fasciculata [78,79]. Moreover, perturbations in lipid and sterol metabolism of [^14^C]-acetate have been reported in pterin-deficient parasites [80]. Our preliminary experiments with pterin-free subcellular fractions of C. fasciculata would support these earlier reports of growth sparing, suggesting this subject merits further investigation.
Seventy-five years have elapsed since the discovery of “Crithidia factor”, yet we still do not understand the essential role(s) pterins in kinetoplastids. Further work in this area using the modern tools of metabolomics and lipidomics should resolve this unanswered question and could lead to the identification of novel targets for drug development.
Supporting information
S1 TableRaw data used to build graphs in Figs 2A, 3, 4, 5A, 5B and 5C.(XLSX)
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