Biopterin conversion to reduced folates byLeishmania donovani promastigotes

Beck, Joanne T.; Ullman, Buddy

doi:10.1016/0166-6851(91)90126-q

Cited by 30 publications

(18 citation statements)

References 31 publications

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“…Accumulation ofether-linked lysophospholipids is known to be toxic to Leishmania (23,24), and perhaps failure to cleave these lipids is toxic in virulent but not attenuated parasites. Other possibilities may arise from unique aspects of folate and pterin metabolism in Leishmania, which differ significantly from other organisms (27)(28)(29) (1). If the gene is essential, homozygous knockouts will be lethal; however, the remarkable ability of Leishmania to undergo changes in chromosome number will permit the recovery of cells simultaneously bearing wild-type and both planned neo and hyg replacement chromosomes.…”

Section: Discussionmentioning

confidence: 99%

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Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.

Cruz

Titus

Beverley

1993

Proc. Natl. Acad. Sci. U.S.A.

181

138

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We It is now possible by DNA transfection to engineer trypanosomatid parasites that overexpress or lack a given gene product. Since trypanosomatids are generally diploid and it is currently either impossible (Leishmania) or difficult (Trypanosoma brucei) to perform sexual crosses, homozygous gene knockouts are usually obtained by the process ofdouble gene replacement with independent selectable markers (1). This approach is limited to genes whose function is not essential during in vitro culture; however, many interesting parasite-specific molecules are known or likely to be nonessential for growth in vitro (2, 3). One approach to the study of essential genes is to use situations in which knockouts are conditionally lethal. We have shown that homozygous knockouts of the Leishmania major dhfr-ts (dihydrofolate reductase-thymidylate synthese) locus can be obtained if parasites are provided with thymidine (1, 4), in keeping with the expected role of DHFR-TS in intermediary metabolism. The dhfr-ts knockout will facilitate studies of folate metabolism and antifolate chemotherapy, and conditionally lethal mutants like these thy-(thymidine negative) auxotrophs may have important applications in the development of safe, avirulent lines that could be used as potential vaccination strains, as could parasites lacking molecules essential for infectivity in vivo but nonessential for growth in vitro (1). Since virulent Leishmania may provide superior vaccination potential relative to attenuated lines, we attempted to develop homozygous knockouts of dhfr-ts in virulent lines. To our surprise, we were unable to obtain the desired mutants, despite the ease with which such knockouts were obtained with attenuated lines (1). Instead, virulent Leishmania parasites possess a remarkable ability to undergo changes in chromosome number, permitting retention of one or more chromosomes containing dhfr-ts despite the success of the double targeting.MATERIALS AND METHODS Virulent L. major were LV39 clone 5 (Rho/SU/59/P) and Friedlin (MHOM/JL/80/Friedlin); attenuated lines were LT252 clone CC-1 (MHOM/IR/83/IR; ref. 5) and CB rev3 (6, 7). Cells were grown in M199 medium and transfected by electroporation and plating as described (1,4,5). KS supplements were added in transfections expected to yield dhfr-ts-lines (4). Virulent parasites recovered from lesions were cultured in NNN medium (7), transformed into promastigotes, and grown <2 weeks prior to transfection. Stationary-phase parasites (5 x 106) were injected subcutaneously into the foot of BALB/c mice in triplicate, and development of lesions followed (7). General molecular methods were performed as described (1,4,5,8). Gene-specific hybridization probes were as follows: dhfr-ts, 0.8-kb Xho I fragment (nt 658-1483; ref. 8); neo (neomycin phosphotransferase), 0.9-kb Spe I fragment from pSpeNEOA (5); hyg (hygromycin B phosphotransferase), a PCR-derived cassette (1); probe P, 1.6-kb EcoRI/EcoRV fragment (8). DNA content was measured by flow cytometry using ethanol-fixed cells (9). He...

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Section: Discussionmentioning

confidence: 99%

“…We propose that this is a diagnostic criterion for essentiality of gene function, as one is scoring a positive result and not just failure to obtain knockouts. Since Leishmania appear to be generally tolerant of chromosome number changes (28) …”

Section: Discussionmentioning

confidence: 99%

Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.

Cruz

Titus

Beverley

1993

Proc. Natl. Acad. Sci. U.S.A.

181

138

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“…Although growth studies can be compromised by the presence of trace contaminants, incorporation of radiolabeled biopterin into folates has been shown in L. donovani (9), suggesting the occurrence of a de novo synthetic pathway. In contrast, another study failed to find incorporation of radiolabeled para-aminobenzoic acid into folate in L. major (54), which would be expected assuming that folates are synthesized by the classic route of dihydropteroate synthase.…”

Section: Interconversions Of Pterins and Folates-mentioning

confidence: 99%

“…A number of studies have demonstrated that the trypanosomatid growth requirement for folate can be reduced or even eliminated by inclusion of pterins such as biopterin (5)(6)(7)(8)(9)(10)(11) (Table IV). Although growth studies can be compromised by the presence of trace contaminants, incorporation of radiolabeled biopterin into folates has been shown in L. donovani (9), suggesting the occurrence of a de novo synthetic pathway.…”

Section: Interconversions Of Pterins and Folates-mentioning

confidence: 99%

“…This feature led historically to an appreciation of the pterin requirement of eukaryotes, where pterins are now known to participate as essential cofactors in hydroxylations, ether-lipid cleavage, and NO synthase (12)(13)(14)(15). However, the pathways involved in the salvage and metabolism of pterins, and their function in Leishmania, are only beginning to emerge (9,10).…”

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confidence: 99%

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The Roles of Pteridine Reductase 1 and Dihydrofolate Reductase-Thymidylate Synthase in Pteridine Metabolism in the Protozoan Parasite Leishmania major

Nare

Hardy

Beverley

1997

Journal of Biological Chemistry

174

159

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Trypanosomatid protozoans depend upon exogenous sources of pteridines (pterins or folates) for growth. A broad spectrum pteridine reductase (PTR1) was recently identified in Leishmania major, whose sequence places it in the short chain alcohol dehydrogenase protein family although its enzymatic activities resemble dihydrofolate reductases. The properties of PTR1 suggested a role in essential pteridine salvage as well as in antifolate resistance. To prove this, we have characterized further the properties and relative roles of PTR1 and dihydrofolate reductase-thymidylate synthase in Leishmania pteridine metabolism, using purified enzymes and knockout mutants. Recombinant L. major and Leishmania tarentolae, and native L. major PTR1s, were tetramers of 30-kDa subunits and showed similar catalytic properties with pterins and folates (pH dependence, substrate inhibition with H 2 pteridines). Unlike PTR1, dihydrofolate reductase-thymidylate synthase showed weak activity with folate and no activity with pterins. Correspondingly, studies of ptr1 ؊ and dhfr-ts ؊ mutants implicated only PTR1 in the ability of L. major to grow on a wide array of pterins. PTR1 exhibited 2000-fold less sensitivity to inhibition by methotrexate than dihydrofolate reductase-thymidylate synthase, suggesting several mechanisms by which PTR1 may compromise antifolate inhibition in wild-type Leishmania and lines bearing PTR1 amplifications. We incorporate these results into a comprehensive model of pteridine metabolism and discuss its implications in chemotherapy of this important human pathogen.

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Pterin and Folate Reduction by theLeishmania tarentolaeH Locus Short-Chain Dehydrogenase/Reductase PTR1

Wang

Leblanc

Chang

et al. 1997

Archives of Biochemistry and Biophysics

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Biopterin conversion to reduced folates byLeishmania donovani promastigotes

Cited by 30 publications

References 31 publications

Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.

Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.

The Roles of Pteridine Reductase 1 and Dihydrofolate Reductase-Thymidylate Synthase in Pteridine Metabolism in the Protozoan Parasite Leishmania major

Pterin and Folate Reduction by theLeishmania tarentolaeH Locus Short-Chain Dehydrogenase/Reductase PTR1

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