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• W1990493428 abstract "Leishmania donovani cannot synthesize purines de novo and express a multiplicity of enzymes that enable them to salvage purines from their hosts. Previous efforts to generate an L. donovani strain deficient in both hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) using gene replacement approaches were not successful, lending indirect support to the hypothesis that either HGPRT or XPRT is crucial for purine salvage by the parasite. We now report the genetic confirmation of this hypothesis through the construction of a conditional Δhgprt/Δxprt mutant strain that exhibits an absolute requirement for 2′-deoxycoformycin, an inhibitor of the leishmanial adenine aminohydrolase enzyme, and either adenine or adenosine as a source of purine. Unlike wild type parasites, the Δhgprt/Δxprt strain cannot proliferate indefinitely without 2′-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient. The Δhgprt/Δxprt mutant infects murine bone marrow-derived macrophages <5% as effectively as wild type parasites and cannot sustain an infection. These data establish genetically that either HGPRT or XPRT is absolutely essential for purine acquisition, parasite viability, and parasite infectivity of mouse macrophages, that all exogenous purines are funneled to hypoxanthine and/or xanthine by L. donovani, and that the purine sources within the macrophage to which the parasites have access are HGPRT or XPRT substrates. Leishmania donovani cannot synthesize purines de novo and express a multiplicity of enzymes that enable them to salvage purines from their hosts. Previous efforts to generate an L. donovani strain deficient in both hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) using gene replacement approaches were not successful, lending indirect support to the hypothesis that either HGPRT or XPRT is crucial for purine salvage by the parasite. We now report the genetic confirmation of this hypothesis through the construction of a conditional Δhgprt/Δxprt mutant strain that exhibits an absolute requirement for 2′-deoxycoformycin, an inhibitor of the leishmanial adenine aminohydrolase enzyme, and either adenine or adenosine as a source of purine. Unlike wild type parasites, the Δhgprt/Δxprt strain cannot proliferate indefinitely without 2′-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient. The Δhgprt/Δxprt mutant infects murine bone marrow-derived macrophages <5% as effectively as wild type parasites and cannot sustain an infection. These data establish genetically that either HGPRT or XPRT is absolutely essential for purine acquisition, parasite viability, and parasite infectivity of mouse macrophages, that all exogenous purines are funneled to hypoxanthine and/or xanthine by L. donovani, and that the purine sources within the macrophage to which the parasites have access are HGPRT or XPRT substrates. Leishmania donovani is a protozoan parasite that is the etiologic agent of visceral leishmaniasis, a devastating and often fatal disease in humans. Leishmania spp. are digenetic, existing in both insect vector and mammalian forms. The flagellated, motile, extracellular promastigote proliferates in the midgut of phlebotomine sandfly family members, whereas the nonflagellated, nonmotile, intracellular amastigote resides in phagolysosomes of macrophages and other reticuloendothelial cells within the vertebrate host. Because of the absence of effective vaccines, chemotherapy has offered the only avenue of defense for the treatment and prevention of leishmaniasis and other parasitic diseases. Unfortunately, drug therapy for leishmaniasis is compromised by toxicity, expense, prolonged and invasive routes of administration, and resistance. Thus, the need for new and more efficacious drugs is acute. The institution of an effective parasite-specific therapeutic regimen for the treatment of leishmaniasis, or for that matter any parasitic disease, depends upon exploiting fundamental biochemical or metabolic differences between parasite and host. Perhaps the most striking metabolic disparity between parasites and their mammalian hosts is the avenue by which they synthesize purine nucleotides. Whereas mammalian cells can generate the purine ring de novo, all of the protozoan parasites studied to date are incapable of synthesizing the purine ring (1Marr J.J. Berens R.L. Nelson D.J. Biochim. Biophys. Acta. 1978; 544: 360-371Crossref PubMed Scopus (148) Google Scholar). As a consequence, each genus of parasite has evolved a unique complement of purine salvage enzymes that enables it to scavenge host purines (1Marr J.J. Berens R.L. Nelson D.J. Biochim. Biophys. Acta. 1978; 544: 360-371Crossref PubMed Scopus (148) Google Scholar, 2Looker D.L. Berens R.L. Marr J.J. Mol. Biochem. Parasitol. 1983; 9: 15-28Crossref PubMed Scopus (82) Google Scholar). Leishmania expresses a number of purine salvage enzymes. These enzymes include hypoxanthine-guanine phosphoribosyltransferase (HGPRT), 3The abbreviations used are: HGPRT, hypoxanthine-guanine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase; XPRT, xanthine phosphoribosyltransferase; AK, adenosine kinase; dCF, 2′-deoxycoformycin; AAH, adenine aminohydrolase; UTR, untranslated region; DME-L, Dulbecco's Modified Eagle-Leishmania; PBS, phosphate-buffered saline. adenine phosphoribosyltransferase (APRT), xanthine phosphoribosyltransferase (XPRT), and adenosine kinase (AK), and the genes encoding all four L. donovani proteins have been isolated and sequenced (3Allen T. Hwang H.Y. Wilson K. Hanson S. Jardim A. Ullman B. Mol Biochem. Parasitol. 1995; 74: 99-103Crossref PubMed Scopus (24) Google Scholar, 4Allen T.E. Hwang H.Y. Jardim A. Olafson R. Ullman B. Mol. Biochem. Parasitol. 1995; 73: 133-143Crossref PubMed Scopus (38) Google Scholar, 5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 6Sinha K.M. Ghosh M. Das I. Datta A.K. Biochem. J. 1999; 339: 667-673Crossref PubMed Scopus (23) Google Scholar). Interestingly, both HGPRT and XPRT are targeted to the glycosome (7Shih S. Hwang H.Y. Carter D. Stenberg P. Ullman B. J. Biol. Chem. 1998; 273: 1534-1541Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Zarella-Boitz J.M. Rager N. Jardim A. Ullman B. Mol Biochem. Parasitol. 2004; 134: 43-51Crossref PubMed Scopus (27) Google Scholar), a subcellular organelle unique to Leishmania and related parasites (9Parsons M. Furuya T. Pal S. Kessler P. Mol. Biochem. Parasitol. 2001; 115: 19-28Crossref PubMed Scopus (83) Google Scholar), and this glycosomal localization is mediated by a COOH-terminal tripeptide referred to as the peroxisomal targeting signal 1 (9Parsons M. Furuya T. Pal S. Kessler P. Mol. Biochem. Parasitol. 2001; 115: 19-28Crossref PubMed Scopus (83) Google Scholar). The parasite also accommodates a variety of purine interconversion enzymes including nucleosidases (1Marr J.J. Berens R.L. Nelson D.J. Biochim. Biophys. Acta. 1978; 544: 360-371Crossref PubMed Scopus (148) Google Scholar, 10Koszalka G.W. Krenitsky T.A. J. Biol. Chem. 1979; 254: 8185-8193Abstract Full Text PDF PubMed Google Scholar), IMP branchpoint enzymes, phosphorylases, and deaminases (1Marr J.J. Berens R.L. Nelson D.J. Biochim. Biophys. Acta. 1978; 544: 360-371Crossref PubMed Scopus (148) Google Scholar, 11Kidder G.W. Nolan L.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3670-3672Crossref PubMed Scopus (41) Google Scholar). Thus, the purine salvage pathway of Leishmania is divagating and redundant, and this metabolic complexity, as well as the diploid nature of the parasite, has hindered a thorough characterization of the pathway. The ability of Leishmania to carry out efficient homologous gene replacement (12Kapler G.M. Coburn C.M. Beverley S.M. Mol. Cell. Biol. 1990; 10: 1084-1094Crossref PubMed Scopus (361) Google Scholar, 13LeBowitz J.H. Coburn C.M. McMahon-Pratt D. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9736-9740Crossref PubMed Scopus (191) Google Scholar) and take up foreign DNAs (14Cruz A. Beverley S.M. Nature. 1990; 348: 171-173Crossref PubMed Scopus (193) Google Scholar), however, can overcome these impediments and enables the genetic dissection of complex metabolic pathways such as that for purine acquisition. Implementing targeted gene replacement strategies, L. donovani promastigotes deficient in HGPRT, APRT, XPRT, and/or AK were created in almost every conceivable combination (5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Hwang H.Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 16Hwang H.Y. Ullman B. J. Biol. Chem. 1997; 272: 19488-19496Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), although it was not possible to create a Δhgprt/Δxprt double mutant in any genetic background. These genetic studies underscored our central hypothesis governing purine metabolism that either HGPRT or XPRT is both necessary and sufficient for all of purine acquisition by L. donovani. The inability to create the Δhgprt/Δxprt double knock-out, however, was negative evidence that did not confirm the premise. We have now isolated and characterized a conditional Δhgprt/Δxprt line of L. donovani that was selected by targeted gene replacement in the presence of 2′-deoxycoformycin (dCF), an inhibitor of the leishmanial adenine aminohydrolase (AAH) enzyme (11Kidder G.W. Nolan L.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3670-3672Crossref PubMed Scopus (41) Google Scholar), and adenine as a source of purine. The creation and characterization of this Δhgprt/Δxprt mutant confirms our main supposition that either HGPRT or XPRT is absolutely essential for purine acquisition and parasite viability. Materials—[8-14C]Adenine (50 mCi/mmol), [8-14C]adenosine (53 mCi/mmol), [8-14C]guanine (55 mCi/mmol), [8-14C]guanosine (50 mCi/mmol), [8-14C]hypoxanthine (51 mCi/mmol), [8-14C]inosine (52 mCi/mmol), and [8-14C]xanthine (53 mCi/mmol) were all purchased from Moravek Biochemicals (Brea, CA). dCF was obtained from the National Cancer Institute (Bethesda, MD). All restriction and DNA-modifying enzymes and all other chemicals and reagents were of the highest quality commercially available. Axenic Parasite Cell Culture—The L. donovani strain 1S2D originated with Dr. Dennis Dwyer (NIH) and was adapted for growth as axenic amastigotes as described (17Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar). A clonal derivative of this strain, LdBob (17Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar), was provided by Dr. Stephen Beverley (Washington University, St. Louis, MO). LdBob promastigotes were cultured at 26 °C in purine-replete M199-based medium as detailed (17Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar) or in a modified Dulbecco's Modified Eagle-Leishmania (DME-L) medium (18Iovannisci D.M. Ullman B. J. Parasitol. 1983; 69: 633-636Crossref PubMed Scopus (123) Google Scholar) that lacks bovine serum albumin and is supplemented with 10% dialyzed fetal bovine serum, 1 mm glutamine, 1× RPMI 1640 vitamin mix, 10 μm folate, 100 μm adenine, and 20 μm dCF. Axenic amastigotes were cultured at 37 °C as described (17Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar). The characterization of LdBob strains harboring single Δhgprt, Δaprt, or Δxprt lesions is currently under review. Targeting and Episomal Constructs—The flanking regions from the HGPRT, APRT, and XPRT loci and the oligonucleotides used for their amplification by the PCR have been described (5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Hwang H.Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The construction and authentication of the pX63-HYG-Δhgprt, pX63-NEO-Δxprt, and pX63-HYG-Δxprt targeting vectors employed in the previous allelic replacements of the HGPRT and XPRT loci have also been described (5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Hwang H.Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The pX63-NEO-Δhgprt/Δxprt construct was created by replacing the 5′-untranslated region (UTR) of XPRT in the pX63-NEO-Δxprt vector with the 5′-UTR of the HGPRT. The pX63-PHLEO-Δxprt replacement plasmid was generated by excising the 5′- and 3′-UTRs of XPRT from pX63-HYG-Δxprt and inserting them into the appropriate sites within pX63-PHLEO (19Freedman D.J. Beverley S.M. Mol. Biochem. Parasitol. 1993; 62: 37-44Crossref PubMed Scopus (58) Google Scholar). To generate episomal constructs of HGPRT and XPRT, the two genes were amplified by PCR and inserted into the SmaI-BamHI restriction sites within the pXG-BSD blasticidin resistance expression plasmid generously provided by Dr. Stephen Beverley. The complementation vectors were designated pXG-BSD-HGPRT and pXG-BSD-XPRT, respectively. The other two episomal constructs, pSNBR-HYG-hgprtΔ(209–211) and pXG-BSD-xprtΔAKL, which lack a peroxisomal targeting signal 1 and therefore produce cytosolic hgprt and xprt, respectively, were generated as described (7Shih S. Hwang H.Y. Carter D. Stenberg P. Ullman B. J. Biol. Chem. 1998; 273: 1534-1541Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Zarella-Boitz J.M. Rager N. Jardim A. Ullman B. Mol Biochem. Parasitol. 2004; 134: 43-51Crossref PubMed Scopus (27) Google Scholar). pSNBR-HYG-hgprtΔ(209–211) will now be referred to as pSNBR-HYG-hgprtΔSKV. Gene Replacements and Complemented Lines—All genetic manipulations were conducted on LdBob promastigotes. Single Δhgprt, Δaprt, and Δxprt knock-outs were created in LdBob using the same targeting constructs, protocols, and gene replacement strategies employed previously for the generation of these mutants within an avirulent L. donovani background (5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Hwang H.Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The Δhgprt/Δxprt double knock-out was generated after three sequential rounds of targeted gene replacement. The pX63-NEO-Δhgprt/Δxprt, pX63-HYG-Δhgprt, and pX63-PHLEO-Δxprt plasmids were linearized with HindIII and BglII and transfected into 5 × 107 parasites using reported electroporation conditions (17Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar). Homologous integrations were selected by plating parasites on semisolid medium containing selective concentrations of either Geneticin, hygromycin, or phleomycin (19Freedman D.J. Beverley S.M. Mol. Biochem. Parasitol. 1993; 62: 37-44Crossref PubMed Scopus (58) Google Scholar), as appropriate for the drug resistance marker of the targeting cassette. The HGPRT/hgprt/XPRT/xprt and Δhgprt/XPRT/xprt lines were generated and maintained in the M199-based medium, whereas the Δhgprt/Δxprt null mutant was selected and maintained in the modified DME-L medium described above. The HGPRT/hgprt/XPRT/xprt, Δhgprt/XPRT/xprt, and Δhgprt/Δxprt lines were all maintained continuously under selective pressure in the drugs for which they contained resistance markers. The Δhgprt/Δxprt line was also transfected separately with pXG-BSD-HGPRT, pXG-BSD-XPRT, or pXG-BSD-xprtΔAKL, and transfectants selected in 20 μg/ml blasticidin and either 100 μm hypoxanthine or 100 μm xanthine to generate the complemented lines Δhgprt/Δxprt-[pXG-BSD-HGPRT], Δhgprt/Δxprt[pXG-BSD-XPRT], and Δhgprt/Δxprt[pXG-BSD-xprtΔAKL], respectively. These cell lines are designated Δhgprt/Δxprt[pXPRT], Δhgprt/Δxprt[pHGPRT], and Δhgprt/Δxprt[pxprtΔAKL]. The Δhgprt/Δxprt cell line that was transfected with pSNBR-HYG-hgprtΔSKV was selected in 50 μg/ml hygromycin and 100 μm hypoxanthine and is designated Δhgprt/Δxprt [phgprtΔSKV]. DNA Manipulations and Western Blotting—Isolation of genomic DNA and Southern blotting were performed by conventional protocols (20Sambrook J. Maniatis T. Fritsch E.F. Molecular Cloning: a Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.1989: 9.31-9.57Google Scholar). Monospecific antibodies to purified recombinant L. donovani HGPRT, APRT, and XPRT proteins have been described (3Allen T. Hwang H.Y. Wilson K. Hanson S. Jardim A. Ullman B. Mol Biochem. Parasitol. 1995; 74: 99-103Crossref PubMed Scopus (24) Google Scholar, 4Allen T.E. Hwang H.Y. Jardim A. Olafson R. Ullman B. Mol. Biochem. Parasitol. 1995; 73: 133-143Crossref PubMed Scopus (38) Google Scholar, 5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), and Western blotting protocols were carried out as conveyed (20Sambrook J. Maniatis T. Fritsch E.F. Molecular Cloning: a Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.1989: 9.31-9.57Google Scholar). Mouse monoclonal antibody to the amastigote-specific A2 protein (21Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (120) Google Scholar) was generously provided by Dr. Greg Matlashewski of McGill University Faculty of Medicine, Montreal, Quebec, Canada. Enzyme Assays—2 × 109 parasites were washed two times in phosphate-buffered saline (PBS), resuspended in 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 1 mm phosphoribosylpyrophosphate, and protease inhibitor mixture, lysed by sonication, and fractionated by centrifugation at 9,740 × g at 4 °C. Radiolabel incorporation assays were performed on cell-free lysates of promastigote extracts as described (22Iovannisci D.M. Goebel D. Allen K. Kaur K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar), whereas amastigote enzymatic assays were conducted with lysates that were not fractionated by centrifugation. These assays measure the rates of radiolabeled preformed purine nucleoside and nucleobases into phosphorylated, anionic metabolites, i.e. nucleotides and nucleic acids. Purine Metabolism in Live Cells—The rates of conversion of radiolabeled purine into nucleotides were measured using the earlier described DE-81 filter disk method (22Iovannisci D.M. Goebel D. Allen K. Kaur K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar). Cells were washed with PBS and resuspended at a density of 1 × 108 cells/ml in either promastigote or amastigote medium containing 2 μm radiolabeled purine but lacking bovine serum albumin, FBS, and hemin. At each time point 1 × 107 cells were removed, washed once in PBS, lysed in 1% Triton X-100, and spotted onto DE-81 filter disks. Parasite metabolism of various radiolabeled purines was quantified by liquid scintillation. Growth Phenotypes—To assess the abilities of genetically manipulated strains to grow in various different purine sources, all parasites were washed several times with PBS, resuspended in modified DME-L medium lacking purine, and incubated at 26 °C for 4 h before they were seeded at a density of 5 × 104 cells/ml in 1.0-ml aliquots of modified DME-L containing 100 μm purine and 5% dialyzed FBS. Amastigotes were seeded at a density of 5 × 103 cells/ml into amastigote medium containing 20% FBS and 100 μm purine, incubated for 7–10 days, and counted by hemacytometer. Macrophage Infections—Stationary phase promastigotes were washed two times in purine-free promastigote medium and resuspended in Dulbecco's modified Eagle's medium supplemented with 4 mm l-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, and 10% FBS. 2 × 105 bone marrow-derived mouse macrophages, from Balb/c mice, and 2 × 106 promastigotes were placed in 4-well Lab-TekII CHAMBER SLIDES (Nalge Nunc International Corp., Naperville, IL) containing 1.0 ml of macrophage growth medium and incubated at 37 °C in a humidified 5% CO2 incubator. After 16 h, adherent macrophages were washed 10 times in PBS to eliminate residual extracellular promastigotes after which fresh growth medium was added and then again 24 h later. After an additional 24-h incubation, the chambers were washed three times with PBS, and macrophages were stained using the Diff-Quik kit (International Medical Equipment Inc., San Marcos, CA). Parasites were visualized on a Zeiss Axiovert 200 M scope (Carl Zeiss Microimaging, Thornwood, NY) using 60× oil immersion light and photographed with an AxioCam MRm camera (Zeiss), and parasites were enumerated. Color photographs of parasitized macrophages were visualized on a Zeiss Axiophot microscope using 40× oil immersion and photographed with a Leica DC 300 camera (Leica Camera AG, Solms, Germany). The Δhgprt/Δxprt knock-out was created after three rounds of transfection with drug resistance cassettes carrying 5′- and 3′-UTRs of HGPRT and XPRT (Fig. 1). Because HGPRT and XPRT are colocalized within a 4359-bp EcoRI fragment in the L. donovani genome (5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the first copy of both genes was displaced with X63-NEO-Δhgprt/Δxprt (linearized pX63-NEO-Δhgprt/Δxprt), a construct containing the 5′-UTR of HGPRT and the 3′-UTR of XPRT, to create the HGPRT/hgprt/XPRT/xprt double heterozygote. The heterozygote was then transfected with X63-HYG-Δhgprt to generate the Δhgprt/XPRT/xprt line, and the latter was transfected with X63-PHLEO-Δxprt to create the Δhgprt/Δxprt double knock-out (Fig. 1). The last round of transfection was performed in medium containing 20 μm dCF and 100 μm adenine, whereas the HGPRT/hgprt/XPRT/xprt and Δhgprt/XPRT/xprt progenitors of the double knock-out were isolated in medium lacking dCF and containing 100 μm adenine as a purine source. 50–100 drug-resistant colonies were obtained within days after the first two cycles of transfections, but surprisingly only two barely visible colonies were obtained after 4 weeks following the last round of transfection in the adenine-dCF medium. Both colonies were picked and expanded in liquid culture medium containing adenine and dCF. Southern blot analysis of the HGPRT/hgprt/XPRT/xprt, Δhgprt/XPRT/xprt, and Δhgprt/Δxprt strains divulged the new alleles that had been created by the homologous gene replacement events (Fig. 2). The digestion of genomic DNA with EcoRI and hybridization to the HGPRT and XPRT open reading frames revealed the presence of the common 4359-bp restriction fragment in the wild type and HGPRT/hgprt/XPRT/xprt lines and its absence in the Δhgprt/Δxprt null mutant. The Δhgprt/XPRT/xprt strain, as expected, lacks a band for HGPRT but exhibits a hybridization signal at 5939 bp when probed with the XPRT open reading frame that reflects the appropriate integration of the hygromycin resistance marker within the X63-HYG-Δhgprt cassette into the HGPRT locus. For comparison, a Southern blot analysis of previously isolated single knock-outs of HGPRT, APRT, and XPRT are also depicted, and the signals are appropriate for the expected homologous integrations (Fig. 2). Western blot analysis of wild type, HGPRT/hgprt/XPRT/xprt, Δhgprt/XPRT/xprt, and Δhgprt/Δxprt extracts confirmed the absence of HGPRT and XPRT protein in strains in which the corresponding gene had been eliminated (Fig. 3). The single Δhgprt, Δaprt, and Δxprt null mutants for which Southern blot data are shown in Fig. 2 also lacked the proteins corresponding to the deleted genes. The abilities of wild type and Δhgprt/Δxprt promastigote lysates to incorporate individual radiolabeled purines into nucleotides were assessed over a 15-min time interval (Fig. 4). Whereas wild type promastigotes were capable of incorporating all purine bases and nucleosides tested, the Δhgprt/Δxprt could not convert guanine, guanosine, hypoxanthine, or inosine to nucleotides. The double knock-out could, however, incorporate radiolabeled adenine and adenosine into phosphorylated products during the 15 min assay interval, whether or not dCF was added to the extracts (Fig. 4). [14C]Xanthine conversion to nucleotides could not be measured in promastigote extracts for technical reasons. The ability of live promastigotes to metabolize various [14C] radiolabeled purine bases and nucleosides was also measured. These results corroborated the results from the enzymatic assays described above. Additionally, the metabolism of [14C]xanthine could be measured in intact wild type cells but not in the double knock-out (Fig. 5). [14C]Adenine and [14C]adenosine metabolism could be measured in both wild type and mutant cells during the 2-h time course in the presence or absence of dCF. The ability of Δhgprt/Δxprt promastigotes to proliferate in various purine sources was assessed (Fig. 6). Whereas wild type parasites could grow in modified DME-L medium with any of the added purine nucleobases or nucleosides as a purine source, continuous and robust growth of Δhgprt/Δxprt promastigotes could be maintained only in adenine or adenosine in the presence of 20 μm dCF. A small number of mutant parasites were observed at the end of the experiment in adenine or adenosine alone (when the knock-out parasites in dCF had reached stationary phase growth), but these parasites were not capable of further proliferation in the absence of dCF and soon died (data not shown). The double mutant could not grow in xanthine, xanthosine, guanine, guanosine, inosine, or hypoxanthine, although a limited amount of growth was observed when inosine was added as the purine in the presence of dCF. The Δhgprt/Δxprt parasites incubated in inosine and dCF were also not capable of long term survival after several additional weeks of incubation. The limited proliferation in inosine was further investigated with immucillin H, an iminoribitol analog of inosine that is a potent inhibitor of the leishmanial inosine-uridine nucleoside hydrolase activity (23Shi W. Schramm V.L. Almo S.C. J. Biol. Chem. 1999; 274: 21114-21120Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Although immucillin H at 20 μm allowed the Δhgprt/Δxprt double knock-out to replicate for several cell cycles in 100 μm inosine, indefinite growth in the inosine-immucillin H combination could also not be sustained (data not shown). The Δhgprt/Δxprt transfectants Δhgprt/Δxprt[pHGPRT] and Δhgprt/Δxprt[pXPRT] exhibited the same growth phenotypes as the Δxprt and Δhgprt single mutants, respectively; Δhgprt/Δxprt[pXPRT] parasites could grow on all purines, whereas Δhgprt/Δxprt[pHGPRT] could grow on all purines except guanine, xanthine, and xanthosine (data not shown). dCF was not necessary for growth with adenine or adenosine in the complemented lines. Complementation of the Δhgprt/Δxprt line could also be achieved with episomes expressing cytosolic versions of hgprt and xprt that lacked their glycosomal targeting signals (7Shih S. Hwang H.Y. Carter D. Stenberg P. Ullman B. J. Biol. Chem. 1998; 273: 1534-1541Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Zarella-Boitz J.M. Rager N. Jardim A. Ullman B. Mol Biochem. Parasitol. 2004; 134: 43-51Crossref PubMed Scopus (27) Google Scholar). The Δhgprt/Δxprt[phgprtΔSKV] and Δhgprt/Δxprt[pxprtΔAKL] cell lines grew robustly in either hypoxanthine or xanthine thus confirming that proper localization of either enzyme is not necessary for enzyme function in the promastigote stage of the parasite (7Shih S. Hwang H.Y. Carter D. Stenberg P. Ullman B. J. Biol. Chem. 1998; 273: 1534-1541Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Zarella-Boitz J.M. Rager N. Jardim A. Ullman B. Mol Biochem. Parasitol. 2004; 134: 43-51Crossref PubMed Scopus (27) Google Scholar). Both wild type and Δhgprt/Δxprt parasites were capable of transformation to axenic amastigotes, as assessed by their expression of A2 proteins, a family of amastigote-specific markers (24Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (137) Google Scholar). No A2 was observed in the promastigotes. The growth phenotypes of both wild type and knock-out axenic amastigotes were identical to their promastigote counterparts (data not shown). The metabolic capacities of the axenic amastigotes toward sundry purines were also indistinguishable from the promastigote equivalent. Wild type L. donovani promastigotes were capable of sustaining a robust infection of bone marrow-derived murine macrophages, whereas the Δhgprt/Δxprt knock-out could not (Fig. 7). Parasitemia of the wild type strain was ∼21 parasites/macrophage, whereas the double knock-out infectivity was ∼1 parasite/macrophage, a 20-fold difference. The inability of Δhgprt/Δxprt cells to proliferate inside macrophages could not be imputed to a failure to infect the mammalian cells, because similar numbers of intracellular parasites were observed for both wild type and knock-out parasites 4 h postinfection (data not shown). Supplementation of the macrophage medium with either 100 μm adenine or a combination of 100 μm adenine plus 20 μm dCF allowed the Δhgprt/Δxprt mutant to reach a parasite load of 7.5 parasites/macrophage and 12 parasites/macrophage, respectively, indicating that it is possible for the Δhgprt/Δxprt mutant to proliferate within macrophages if vital nutrients are provided to its host cells (Fig. 7D). Both complemented lines, Δhgprt/Δxprt[pHGPRT] and Δhgprt/Δxprt[pXPRT], also sustained robust infections in the macrophages, although the infectivity was lower than that of wild type parasites. Parasite loads of ∼8 parasites/macrophage and ∼13 parasites/macrophage were observed for Δhgprt/Δxprt[pHGPRT] and Δhgprt/Δxprt[pXPRT], respectively (data not shown). The purine salvage pathway of Leishmania is myriad and complex, although determining which of the many routes of purine salvage are functional has defied genetic dissection (1Marr J.J. Berens R.L. Nelson D.J. Biochim. Biophys. Acta. 1978; 544: 360-371Crossref PubMed Scopus (148) Google Scholar, 25Berens R.L. Krug E.C. Marr J.J. Marr J.J. Muller M. Biochemistry and Molecular Biology of Parasites. Academic Press Ltd., London, San Diego1995: 89-117Crossref Google Scholar). Mutational and gene replacement schemes in L. donovani have demonstrated that none of the four known enzymes capable of converting host purine nucleobases or nucleosides to the nucleotide level, HGPRT, APRT, XPRT, or AK, is essential by itself (5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Hwang H.Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 16Hwang H.Y. Ullman B. J. Biol. Chem. 1997; 272: 19488-19496Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 26Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar). Furthermore, the ability to generate viable Δhgprt/Δaprt/ak– (16Hwang H.Y. Ullman B. J. Biol. Chem. 1997; 272: 19488-19496Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and Δxprt/Δaprt/ak–4 L. donovani promastigotes reveals that the parasite can rely on either a functional XPRT or HGPRT activity for all of its purine nutritional requirements. Indeed, it has been possible to generate mutant parasites by targeted gene replacement in every conceivable combination except mutants accommodating a combined Δhgprt and Δxprt genotype. These results were the basis for our fundamental hypothesis that either HGPRT or XPRT is necessary and sufficient for Leishmania parasites to salvage purines, maintain viability, and sustain proliferation. The hypothesis was sustained by the inability to generate a Δhgprt/Δxprt double knock-out even within a genetic background complemented with an episomal copy of either HGPRT or XPRT. 4J. M. Boitz and B. Ullman, unpublished observation. We now report genetic proof of our principal hypothesis by the creation and characterization of a conditional Δhgprt/Δxprt double knockout. Taking advantage of the colocalization of the HGPRT and XPRT genes in the leishmanial genome (5Jardim A. Bergeson S.E. Shih S. Carter N. Lucas R.W. Merlin G. Myler P.J. Stuart K. Ullman B. J. Biol. Chem. 1999; 274: 34403-34410Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), a Δhgprt/Δxprt mutant was selected after three rounds of targeted gene replacement, with the final transfection being achieved in the presence of 100 μm adenine and 20 μm dCF, an inhibitor of the L. donovani AAH (11Kidder G.W. Nolan L.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3670-3672Crossref PubMed Scopus (41) Google Scholar). The Δhgprt/Δxprt mutant is absolutely reliant on the presence of dCF and either adenine or adenosine as a purine source for lasting survival and growth. No other naturally occurring purine tested could enable indefinite, long term proliferation of either stage of the parasite. However, several rounds of replication by the double knock-out could be sustained with adenine or adenosine in the absence of dCF, but this proliferation could not be perpetuated interminably (Fig. 6). The ability of the Δhgprt/Δxprt to undergo several cell divisions in adenine or adenosine can be ascribed to the time interval required for AAH to convert the 6-aminopurine source to hypoxanthine. This contention is buttressed by short term radiolabel incorporation experiments demonstrating that Δhgprt/Δxprt lysates (Fig. 4), as well as intact parasites (Fig. 5), were perfectly capable of converting adenine or adenosine to the nucleotide level via APRT. Eventually, however, the actions of AAH convert adenine or adenosine, which is cleaved to adenine by L. donovani promastigotes (11Kidder G.W. Nolan L.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3670-3672Crossref PubMed Scopus (41) Google Scholar, 22Iovannisci D.M. Goebel D. Allen K. Kaur K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar, 26Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar), to hypoxanthine, a dead end nutrient for Δhgprt/Δxprt cells. The limited ability of the mutant promastigotes to grow sparingly in inosine plus dCF or immucillin H suggests that L. donovani express an activity capable of salvaging the nucleoside. Although inosine kinase activity has not been detected in Leishmania (1Marr J.J. Berens R.L. Nelson D.J. Biochim. Biophys. Acta. 1978; 544: 360-371Crossref PubMed Scopus (148) Google Scholar), a nucleoside phosphotransferase activity capable of phosphorylating the pyrazolopyrimidine nucleoside analogs allopurinol riboside and formycin B, has been observed in L. donovani promastigotes (27Nelson D.J. Bugge C.J. Elion G.B. Berens R.L. Marr J.J. J. Biol. Chem. 1979; 254: 3959-3964Abstract Full Text PDF PubMed Google Scholar, 28Rainey P. Santi D.V. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 288-292Crossref PubMed Scopus (54) Google Scholar). Whether this phosphotransferase is also capable of recognizing inosine is not known. To date, there are no effective vaccines to protect against visceral leishmaniasis (29Breton M. Tremblay M.J. Ouellette M. Papadopoulou B. Infect. Immun. 2005; 73: 6372-6382Crossref PubMed Scopus (109) Google Scholar). Generating a strain with intrinsic attenuating mutation(s) could theoretically be exploited as a live attenuated vaccine for immunizing against the disease is a valid alternative strategy to control leishmaniasis rather than the conventional paradigm of treatment with toxic drugs. Previous studies using noninfectious Leishmania tarentolae or attenuated strains of Leishmania major have demonstrated that these lines are capable of triggering a protective immune response against further challenge by virulent Leishmania in susceptible rodents (29Breton M. Tremblay M.J. Ouellette M. Papadopoulou B. Infect. Immun. 2005; 73: 6372-6382Crossref PubMed Scopus (109) Google Scholar, 30Uzonna J.E. Spath G.F. Beverley S.M. Scott P. J. Immunol. 2004; 172: 3793-3797Crossref PubMed Scopus (116) Google Scholar). The inability of the Δhgprt/Δxprt knock-out to sustain an infection of murine macrophages bolsters this mutant strain as a candidate for such a live vaccine strategy against visceral leishmaniasis. The Δhgprt/Δxprt mutant infects macrophages at a level <5% of that of wild type parasites. Preliminary results indicate that the overall infection rate of the Δhgprt/Δxprt mutant in macrophages can be increased by the addition of either adenine alone or, a combination of adenine and dCF, to the growth medium. Thus, it should be theoretically feasible to sustain an infection of the Δhgprt/Δxprt strain in susceptible mammals by dietary supplementation with adenine and/or dCF until a protective immune response has been established. The strain could then be eliminated by withdrawal of the dietary additions. It is important, of course, to determine the stability of the mutant genotype and phenotype. Genetic studies are currently underway to determine whether the Δhgprt/Δxprt double mutant is stable and does not revert by down-regulating its AAH activity thereby allowing the parasite to presumably grow on adenine or adenosine alone. We thank Dr. Stephen Beverley for generously providing the LdBob strain and Dr. Archie Bouwer, Dr. Michael Riscoe, and Dr. Jane Kelly for supplying the bone-derived macrophages and for their technical assistance. We also thank Dr. Greg Matlashewski and Dr. Wen-Wei Zhang for providing us with the A2 antibody and technical assistance regarding its use. Immucillin H was a generous gift from Dr. Vern L. Schramm of the Albert Einstein College of Medicine." @default.
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• W1990493428 title "A Conditional Mutant Deficient in Hypoxanthine-guanine Phosphoribosyltransferase and Xanthine Phosphoribosyltransferase Validates the Purine Salvage Pathway of Leishmania donovani" @default.
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