Title: Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania
Abstract: The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and Δarg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The Δarg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the Δarg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the Δarg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis. The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and Δarg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The Δarg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the Δarg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the Δarg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis. Leishmania is a genus of protozoan parasite that is the causative agent of leishmaniasis, a spectrum of devastating and potentially deadly diseases that affects ∼12 million people worldwide. The parasite exhibits a digenetic life cycle in which the extracellular promastigote form resides in the phlebotomine sandfly vector, whereas the intracellular amastigote inhabits the phagolysosomes of macrophages from the infected mammalian host. Because there are no vaccines available to prevent leishmaniasis, chemotherapy offers the only avenue to combat the disease. Unfortunately, the current arsenal of drugs used to treat leishmaniasis is far from ideal, mainly because of toxicity and therapeutic unresponsiveness. Thus, the identification, characterization, and validation of novel therapeutic targets are urgently needed. One biochemical pathway that has been successfully exploited for the treatment of parasitic disease is that for polyamine biosynthesis. Polyamines are ubiquitous organic cations found in virtually every eukaryotic cell and play critical roles in key cellular processes such as growth, differentiation, and macromolecular biosynthesis (1Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3235) Google Scholar, 2Pegg A. Williams-Ashman H.G. Morris D. Marton L.J. Polyamines in Biology and Medicine. Marcel Dekker, New York1981: 3-42Google Scholar). dl-α-Difluoromethylornithine (DFMO), 1The abbreviations used are: DFMO, dl-α-difluoromethylornithine; ODC, ornithine decarboxylase; AdoMet, S-adenosylmethionine; dAdoMet, decarboxy-S-adenosylmethionine; ADOMETDC, S-adenosylmethionine decarboxylase; SPDSYN, spermidine synthase; ARG, arginase; G418, Geneticin; PTS-1, peroxisomal targeting signal-1; GFP, green fluorescent protein; ORF, open reading frame; PBS, phosphate-buffered saline; AGM, agmatinase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; DME-L, Dulbecco's modified Eagle-based medium specifically developed for growing Leishmania promastigotes; CS, chicken serum. an irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is capable of eradicating Trypanosoma brucei infections in mice (3Bacchi C.J. Nathan H.C. Hutner S.H. McCann P.P. Sjoerdsma A. Science. 1980; 210: 332-334Crossref PubMed Scopus (310) Google Scholar) and patients with late stage African sleeping sickness (4Bacchi C. McCann P. McCann P. Pegg A.E. Sjoerdsma A. Inhibition of Polyamine Metabolism: Biolgogical Significance and Basis for New Therapies. Academic Press, Orlando, FL1987: 317-344Crossref Google Scholar, 5Schechter P. Barlow J.L.R. Sjoerdsma A. McCann P. Pegg A.E. Sjoerdsma A. Inhibition of Polyamine Metabolism: Biological Significance and Basis for New Therapies. Academic Press, Orlando, FL1987: 345-364Crossref Google Scholar). DFMO is also active against many other parasites (6Bitonti A.J. McCann P.P. Sjoerdsma A. Exp. Parasitol. 1987; 64: 237-243Crossref PubMed Scopus (49) Google Scholar, 7Gillin F.D. Reiner D.S. McCann P.P. J. Protozool. 1984; 31: 161-163Crossref PubMed Scopus (63) Google Scholar), including the promastigotes of Leishmania donovani (8Kaur K. Emmett K. McCann P.P. Sjoerdsma A. Ullman B. J. Protozool. 1986; 33: 518-521Crossref PubMed Scopus (67) Google Scholar). Interestingly, the selectivity of DFMO for the metabolic machinery of trypanosomes is not based on dissimilar DFMO binding affinities but rather to differences in ODC turnover rates between parasites and mammals (9Ghoda L. Phillips M.A. Bass K.E. Wang C.C. Coffino P. J. Biol. Chem. 1990; 265: 11823-11826Abstract Full Text PDF PubMed Google Scholar, 10Ghoda L. van Daalen Wetters T. Macrae M. Ascherman D. Coffino P. Science. 1989; 243: 1493-1495Crossref PubMed Scopus (196) Google Scholar, 11Phillips M.A. Coffino P. Wang C.C. J. Biol. Chem. 1987; 262: 8721-8727Abstract Full Text PDF PubMed Google Scholar). In addition, inhibitors of S-adenosylmethionine decarboxylase (ADOMETDC), the enzyme that provides aminopropyl moieties for spermidine and spermine synthesis, are also effective anti-trypanosomal agents (12Bacchi C.J. Nathan H.C. Yarlett N. Goldberg B. McCann P.P. Bitonti A.J. Sjoerdsma A. Antimicrob. Agents Chemother. 1992; 36: 2736-2740Crossref PubMed Scopus (64) Google Scholar, 13Bitonti A.J. Byers T.L. Bush T.L. Casara P.J. Bacchi C.J. Clarkson Jr., A.B. McCann P.P. Sjoerdsma A. Antimicrob. Agents Chemother. 1990; 34: 1485-1490Crossref PubMed Scopus (88) Google Scholar). Leishmania sp. have served as particularly valuable model systems for dissecting metabolic pathways in protozoan parasites because of their ability to proliferate axenically in defined growth medium and the facility by which their genome is genetically tractable (14Cruz A. Beverley S.M. Nature. 1990; 348: 171-173Crossref PubMed Scopus (193) Google Scholar, 15Cruz A. Coburn C.M. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7170-7174Crossref PubMed Scopus (280) Google Scholar, 16Coburn C.M. Otteman K.M. McNeely T. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 1991; 46: 169-179Crossref PubMed Scopus (67) Google Scholar, 17LeBowitz 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). All the genes of the polyamine pathway, ODC, ADOMETDC, and spermidine synthase (SPDSYN), have been cloned from L. donovani, and the creation of Δodc, Δadometdc, and Δspdsyn knockouts by targeted gene replacement has demonstrated the essential role of each of these enzymes in L. donovani promastigote proliferation and revealed significant dissimilarities between the polyamine biosynthetic pathways of this genus of protozoan parasite and the mammalian host (18Jiang Y. Roberts S.C. Jardim A. Carter N.S. Shih S. Ariyanayagam M. Fairlamb A.H. Ullman B. J. Biol. Chem. 1999; 274: 3781-3788Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Roberts S.C. Jiang Y. Jardim A. Carter N.S. Heby O. Ullman B. Mol. Biochem. Parasitol. 2001; 115: 217-226Crossref PubMed Scopus (75) Google Scholar, 20Roberts S.C. Scott J. Gasteier J.E. Jiang Y. Brooks B. Jardim A. Carter N.S. Heby O. Ullman B. J. Biol. Chem. 2002; 277: 5902-5909Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Despite the plethora of molecular and biochemical studies on the polyamine biosynthetic pathway of T. brucei, L. donovani, and other parasites (21Muller S. Coombs G.H. Walter R.D. Trends Parasitol. 2001; 17: 242-249Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 22Muller S. Wittich R.M. Walter R.D. Adv. Exp. Med. Biol. 1988; 250: 737-743Crossref PubMed Scopus (11) Google Scholar, 23Bacchi C.J. Yarlett N. Mini. Rev. Med. Chem. 2002; 2: 553-563Crossref PubMed Scopus (53) Google Scholar, 24Bacchi C.J. Yarlett N. Acta Trop. 1993; 54: 225-236Crossref PubMed Scopus (35) Google Scholar, 25Heby O. Roberts S.C. Ullman B. Biochem. Soc. Trans. 2003; 31: 415-419Crossref PubMed Scopus (78) Google Scholar), little is known about the metabolic avenues by which polyamine precursors are produced. Ornithine, the amino acid from which polyamines are generated, is produced from arginine in mammalian cells by two genetically and biochemically distinct arginase (ARG) enzymes. An ARG activity has also been detected in Leishmania (26Coons T. Hanson S. Bitonti A.J. McCann P.P. Ullman B. Mol. Biochem. Parasitol. 1990; 39: 77-89Crossref PubMed Scopus (30) Google Scholar), and an arginase gene sequence from Leishmania amazonensis has been reported, although neither the gene nor protein was functionally characterized (27da Silva E.R. Castilho T.M. Pioker F.C. Tomich de Paula Silva C.H. Floeter-Winter L.M. Int. J. Parasitol. 2002; 32: 727-737Crossref PubMed Scopus (46) Google Scholar). ARG has also been touted as a potential antileishmanial drug target, because n-hydroxyarginine, an inhibitor of ARG that is produced by macrophages as an intermediate during the formation of nitric oxide, can reduce polyamine levels in Leishmania amastigotes and lowers parasite loads (28Iniesta V. Gomez-Nieto L.C. Corraliza I. J. Exp. Med. 2001; 193: 777-784Crossref PubMed Scopus (235) Google Scholar). To initiate an investigation into the pathways by which polyamine precursors are synthesized and to begin a validation of ARG as a potential therapeutic target, we have cloned arginase from L. mexicana and generated Δarg knockouts by double targeted gene replacement. The characterization of these gene deletion mutants reveals that the arginase is essential for promastigote viability and that the lethality of the null mutation could be conditionally bypassed by either low concentrations of putrescine, high concentrations of ornithine or spermidine, or episomal complementation. Furthermore, we have established that the L. mexicana ARG is localized to the glycosome, a unique organelle found exclusively in Leishmania and close relatives (29Hart D.T. Opperdoes F.R. Mol. Biochem. Parasitol. 1984; 13: 159-172Crossref PubMed Scopus (101) Google Scholar, 30Opperdoes F.R. Borst P. FEBS Lett. 1977; 80: 360-364Crossref PubMed Scopus (485) Google Scholar), and that this localization is mediated by the peroxisomal targeting signal type 1 (PTS-1). However, genetic complementation demonstrated that this glycosomal venue is not essential for ARG function, at least in promastigotes. Materials, Chemicals, and Reagents—[14C]Glutamate (219 mCi/mmol), [14C]putrescine (110 mCi/mmol), and [14C]ornithine (53 mCi/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA), whereas l-[guanidino-14C]arginine (55 mCi/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). All restriction enzymes were bought from either Invitrogen Corp. or New England Biolabs, Inc. (Beverly, MA). Synthetic oligonucleotides were acquired from Invitrogen. Advantage HF 2 DNA polymerase was purchased from BD Bioscience, Pfu Turbo DNA polymerase was acquired from Stratagene (La Jolla, CA), and TaqDNA polymerase was from Promega Corp. The transfection vectors pX63-HYG (15Cruz A. Coburn C.M. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7170-7174Crossref PubMed Scopus (280) Google Scholar), pX63-PHLEO (31Freedman D.J. Beverley S.M. Mol. Biochem. Parasitol. 1993; 62: 37-44Crossref PubMed Scopus (58) Google Scholar), and pXG-GFP+2′ (32Ha D.S. Schwarz J.K. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 1996; 77: 57-64Crossref PubMed Scopus (222) Google Scholar) were constructed by and obtained from Dr. Stephen M. Beverley (Washington University, St. Louis, MO). Hygromycin was procured from Roche Applied Science, phleomycin from Research Products International (Mt. Prospect, IL), and Geneticin (G418) from BioWhittaker (Walkersville, MD). Ornithine, putrescine, spermidine, glutamate, and proline were purchased from Sigma-Aldrich Corp. All other chemicals and reagents were of the highest quality commercially available. Cell Culture—Wild type L. mexicana parasites (MNYC/BZ/62/M379) were generously provided by Dr. Scott M. Landfear (Oregon Health & Science University, Portland, OR). Parasites were cultivated in DME-L, a completely defined Dulbecco's modified Eagle-based medium that was specifically developed for growing Leishmania promastigotes (33Iovannisci D.M. Ullman B. J. Parasitol. 1983; 69: 633-636Crossref PubMed Scopus (123) Google Scholar). Transfected parasites were maintained in a modified DME-L medium, DME-L-CS, in which the bovine serum albumin component of DME-L was replaced with 10% chicken serum to avert polyamine oxidase-mediated polyamine toxicity (8Kaur K. Emmett K. McCann P.P. Sjoerdsma A. Ullman B. J. Protozool. 1986; 33: 518-521Crossref PubMed Scopus (67) Google Scholar). arginase/arg heterozygotes were maintained continually in 50 μg/ml hygromycin, whereas Δarg homozygotes were grown in DME-L-CS supplemented with 50 μg/ml phleomycin, 50 μg/ml hygromycin, and either 200 μm ornithine and/or 200-500 μm putrescine. Parasite growth experiments were initiated at 1.0 × 105 parasites/ml, and parasites were enumerated either every 24 or 48 h by hemacytometer or using the vital dye alamarBlue™ (BioSource International, Camarillo, CA) technology (34Mikus J. Steverding D. Parasitol. Int. 2000; 48: 265-269Crossref PubMed Scopus (322) Google Scholar). Reduction of alamarBlue™ was measured at 570 and 600 nm on a Multiskan Ascent plate reader (Thermo Labsystems, Vantaa, Finland). The percent reduction of dye was calculated according to a formula published in the manufacturer's brochure. The greatest reduction was expressed as maximal proliferation, and growth was then plotted as a function of nutrient concentration. Cloning of L. mexicana ARG—The arginase sequence from L. amazonensis (GenBank™ accession number AF038409) was used to design primers to amplify the ARG from L. mexicana genomic DNA using the PCR. Genomic DNA was isolated by standard protocols. The sense primer, 5′-GGATCCATGGAGCACGTGCAGTACAAGTTC-3′, encompassed the initiation methionine codon (boldface type) and was preceded by a BamHI restriction site (underlined), whereas the antisense primer, 5′-TCTAGACTACAGCTTGGAGCTCGTATGCGGAGT-3′, encoded the termination codon (boldface type) to which a BglII site (underlined) was attached at the 5′-end. The arginase open reading frame (ORF) was amplified from 20 ng of L. mexicana DNA using a high fidelity polymerase (Advantage HF 2 DNA polymerase, BD Bioscience, Palo Alto, CA) and standard PCR conditions for amplifying sequences from genomic DNA (95 °C for 5 min followed by 32 cycles of 95 °C for 1 min, 55 °C for 1 min, and 68 °C for 2 min). The ∼1.0-kb-amplified DNA fragment was subcloned using the pCR® 2.1-TOPO® vector of the TOPO TA Cloning™ kit (Invitrogen), and limited nucleotide sequencing confirmed the identity of the putative arginase fragment. The resulting plasmid was designated TOPO TA-arginase. The subcloned arginase fragment was then used to screen an L. mexicana cosmid library constructed by Dr. Scott M. Landfear (Oregon Health & Science University, Portland, OR) using high stringency conditions. Positive cosmids were subjected to two rounds of purification, and the cosmid DNA isolated by standard methods. The arginase locus within a positive cosmid was mapped by Southern blotting, and a ∼7-kb BamHI fragment encompassing the arginase ORF was subcloned into pBluescript (Stratagene, La Jolla, CA). The resulting plasmid was designated pBluescript-arginase and submitted for nucleotide sequencing. The entire nucleotide sequences of the arginase ORF (both directions), and ∼1 kb of each of the flanks was obtained. Construction of pXG-GFP+2′-ARG—To localize ARG, the gene was inserted into pXG-GFP+2′, a leishmanial expression vector that confers resistance to G418 and synthesizes foreign protein as an NH2-terminal fusion with green fluorescent protein (GFP). The arginase ORF was excised from the TOPO TA-arginase vector with BamHI and ligated into the BamHI cut pXG-GFP+2′ vector. Sequence analysis verified the correct orientation and reading frame of the arginase coding region. The resulting expression plasmid was designated pXG-GFP+2′- arginase. This plasmid was then transfected into wild type L. mexicana using standard electroporation conditions (17LeBowitz 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, 18Jiang Y. Roberts S.C. Jardim A. Carter N.S. Shih S. Ariyanayagam M. Fairlamb A.H. Ullman B. J. Biol. Chem. 1999; 274: 3781-3788Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Transfected parasites were selected in 20 μg/ml G418 and used for localization studies. Fluorescence Microscopy—Lab-Tek® II Chambered Coverglass slides (Fisher Scientific) were coated with a 1:10 dilution of poly-l-lysine (Sigma-Aldrich Corp.) for 15 min. The chambers were rinsed with double-deionized water to remove excess poly-l-lysine and allowed to dry at room temperature. 1 × 107 Leishmania promastigotes were resuspended in 1.0 ml of phosphate-buffered saline (PBS), pipetted into the chamber, and allowed to attach for 15 min. Chambers were rinsed once and then overlaid with 500 μl of PBS. Images were taken on a Zeiss Axiovert 200 inverted microscope and deconvolution performed using the constrained iterative method by Axiovision 3.1 software (Carl Zeiss Optical, Chesterfield, VA). Immunofluorescence Microscopy—L. donovani promastigotes at a density of ∼5 × 106/ml were pelleted by centrifugation at 3000 × g for 10 min, rinsed with PBS, and affixed to 4-well chamber slides (Nalge Nunc. International, Rochester, NY) that had been pretreated with poly-l-lysine. Cells were fixed in a PBS solution containing 4% paraformaldehyde and 0.1% glutaraldehyde or PBS containing only 4% paraformaldehyde for 30 min at room temperature. Chamber slides were rinsed once with PBS to remove fixative and incubated in a blocking solution consisting of PBS supplemented with 2% goat serum, 0.1% Tween 20, and 0.1% Triton X-100, which also permeabilized the parasites, for 1 h at room temperature. Antibodies against the L. donovani ODC, SPDSYN, and ADOMETDC proteins (18Jiang Y. Roberts S.C. Jardim A. Carter N.S. Shih S. Ariyanayagam M. Fairlamb A.H. Ullman B. J. Biol. Chem. 1999; 274: 3781-3788Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Roberts S.C. Jiang Y. Jardim A. Carter N.S. Heby O. Ullman B. Mol. Biochem. Parasitol. 2001; 115: 217-226Crossref PubMed Scopus (75) Google Scholar, 20Roberts S.C. Scott J. Gasteier J.E. Jiang Y. Brooks B. Jardim A. Carter N.S. Heby O. Ullman B. J. Biol. Chem. 2002; 277: 5902-5909Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and purified antibodies to the L. donovani hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (35Shih 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) were diluted 1:500 with blocking solution and incubated with the chamber slides to which wild type L. donovani (for ODC, SPDSYN, and ADOMETDC localization) or L. mexicana transfected with pXG-GFP+2′-arginase (for HGPRT detection) cells were affixed. After incubation of fixed parasites with primary antibody for 1 h, all chamber slides were rinsed six times for 5 min with a PBS wash solution containing 0.1% Tween 20. Fixed cells were then incubated with secondary anti-rabbit antibody conjugated to Texas Red dye (Molecular Probes, Eugene, OR) that had been diluted 1:1000 in blocking buffer. The incubation and all subsequent steps were performed in the dark. Secondary antibody was removed by soaking chamber slides six times for 5 min in wash buffer. Chamber slides were washed in PBS and mounted with either Vectashield® mounting medium (Vector Laboratories, Inc., Burlingame, CA) or a solution of 50 mm Tris, pH 8.0, 8% n-propylgalate (Sigma-Aldrich Corp.), and 90% glycerol. Images shown in Fig. 2 (E and F) were obtained by Aurelie Snyder of the Oregon Health & Science University-Molecular Microbiology and Immunology Research Core Facility (www.ohsu.edu/core) with the Applied Precision Deltavision® image restoration system. Deconvolution was performed using the iterative constrained algorithm of Agard et al. (36Agard D.A. Hiraoka Y. Shaw P. Sedat J.W. Methods Cell Biol. 1989; 30: 353-377Crossref PubMed Scopus (565) Google Scholar), and additional image processing was performed on an Silicon Graphics Octane workstation. Images for Fig. 2 (A-D and G) were obtained by using a Zeiss Axiovert 200 inverted microscope and deconvolution accomplished using Axiovision 3.1 software (Carl Zeiss Optical, Chesterfield, VA). Molecular Constructs for the Replacement of the Arginase Alleles—The 5′- and 3′-flanking regions of the L. mexicana arginase ORF were identified from sequence analysis of the arginase locus and cloned into the appropriate sites within the pX63-HYG and pX63-PHLEO vectors. The 5′-flanking region was amplified by PCR with TaqDNA polymerase (Promega Corp., Madison, WI), pBluescript-arginase as a template, the sense primer, 5′-CCGCTGAAGCTTGGAGATACGCCCCCGAGG-3′ (HindIII site underlined), and the antisense primer, 5′-GTTCAACATGTCGACCTTGCCAT-3′ (SalI site underlined). The resulting ∼720-bp PCR product was first subcloned into the pCR® 2.1-TOPO® vector and then excised with HindIII and SalI and inserted into pX63-HYG and pX63-PHLEO that had been digested with HindIII and SalI. The resulting plasmids were designated pX63-HYG-5′F and pX63-PHLEO- 5′F. To generate the 3′ arginase flank for subcloning into pX63-HYG- 5′F, a sense primer, 5′-GTAAATCCCGGGAAGCTATAGACGCGTGTGTG-3′, and an antisense primer, 5′-GGCATTCCCGGGGCGTTTACACTCCCTGG-3′ (SmaI sites underlined), were synthesized, and pBlue-script-arginase was used as the template for PCR amplification. The ∼500-bp PCR product was subcloned into the pCR® 2.1-TOPO® vector, excised with SmaI, gel-purified, and ligated into SmaI-cut pX63-HYG-5′F vector to generate the allelic replacement vector pX63-HYG-Δarg. Because of the presence of a SmaI sites within the PHLEO coding region of pX63-PHLEO, a different cloning strategy was employed to insert the 3′-flanking region of arginase into pX63-PHLEO-5′F. A ∼1.0-kb sequence from the 3′-flanking region of arginase was amplified by PCR using the sense primer, 5′-TGCGCACACACAGATCTATATTTAT-3′, and the antisense primer, 5′-GCAACTCCGAAACCAGATCTCCTCC-3′ (BglII sites underlined). The 1.0-kb PCR fragment was subcloned into the pCR® 2.1-TOPO® vector, excised with BglII, and the fragment was cloned into pX63-PHLEO-5′F that had been digested with BamHI and BglII to generate pX63-PHLEO-Δarg. The correct orientations of the 5′- and 3′-flanking regions within the gene-targeting plasmids were confirmed by restriction mapping and limited nucleotide sequencing of the insert junctions. Generation of Δarg Null Mutants—The Δarg knockouts were generated by double-targeted gene replacement starting with wild type MNYC/BZ/62/M379 L. mexicana. pX63-HYG-Δarg and pX63-PHLEO-Δarg were digested with HindIII and BglII to liberate linear fragments, designated HYG-Δarg and PHLEO-Δarg, respectively, containing the drug resistance marker and the arginase flanking regions. HYG-Δarg and PHLEO-Δarg were isolated from agarose gels and then transfected into parasites using standard electroporation conditions for transfection of Leishmania promastigotes (17LeBowitz 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, 18Jiang Y. Roberts S.C. Jardim A. Carter N.S. Shih S. Ariyanayagam M. Fairlamb A.H. Ullman B. J. Biol. Chem. 1999; 274: 3781-3788Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). First, HYG-Δarg was transfected into wild type L. mexicana to create the arginase/arg heterozygote, and colonies were isolated by selection on plates of semi-solid DME-L-CS medium containing 50 μg/ml hygromycin. The genotype of the arginase/arg heterozygote was then confirmed by Southern blot analysis using arginase flanking regions as probes. A heterozygous clone was then subjected to a second round of transfection with PHLEO-Δarg and Δarg null lines selected in liquid DME-L-CS media containing 50 μg/ml hygromycin, 50 μg/ml phleomycin, and 200 μm ornithine to circumvent potential ornithine auxotrophy. The Δarg genotype was verified by Southern blotting, and clones were isolated on semi-solid DME-L-CS plates containing 50 μg/ml hygromycin, 50 μg/ml phleomycin, and 200 μm ornithine. A total of 12 clones were picked and screened by Southern blotting using the arginase ORF as a probe. The gene replacements of two of the Δarg clones were evaluated in more detail by probing Southern blots with the arginase flanks. ARG Assays—The ARG activities in wild type, knockout, and transfected parasites were determined using lysates prepared from 1 × 108 exponentially growing parasites that had been resuspended in 150 μl of 50 mm glycine/10 mm MnCl2/1 mm dithiothreitol, pH 9.5. A protease inhibitor mixture (Roche Applied Science) was added, and the cells were sonicated three times for 10 s each. Protein concentrations in the resultant cell lysates were determined according to the Bradford method (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217390) Google Scholar). ARG assays were initiated by the addition of 20 μl of parasite extract (2.5 mg of protein/ml) to an 80-μl reaction mixture containing 50 mm glycine/10 mm MnCl2/1 mm dithiothreitol, pH 9.5, and 10 mm l-[guanidino-14C]arginine (31.3 μCi/mmol). At various intervals, the enzyme reaction was terminated by the addition of 10-μl aliquots of the reaction mixture to 5 μl of glacial acetic acid. The [14C]urea product was separated from unreacted l-[guanidino-14C]arginine by paper chromatography using Whatman #1 chromatography paper as the matrix and n-butanol:acetic acid:water (8:1:2) as the mobile phase. RF values for urea and arginine were 0.31 and 0.00, respectively. The [14C]urea formed during the assay was detected by autoradiography and quantitated using a Beckman model LS 6500 liquid scintillation counter. Putrescine, Spermidine, S-Adenosylmethionine, Decarboxy-S-adenosylmethionine (dAdoMet), Ornithine, and Arginine Pool Analyses—Polyamine, AdoMet, and dAdoMet pools were measured as previously reported (19Roberts S.C. Jiang Y. Jardim A. Carter N.S. Heby O. Ullman B. Mol. Biochem. Parasitol. 2001; 115: 217-226Crossref PubMed Scopus (75) Google Scholar, 20Roberts S.C. Scott J. Gasteier J.E. Jiang Y. Brooks B. Jardim A. Carter N.S. Heby O. Ullman B. J. Biol. Chem. 2002; 277: 5902-5909Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 38Seiler N. J. Chromatogr. 1986; 379: 157-176Crossref PubMed Scopus (68) Google Scholar). Ornithine and arginine were separated using standard ion-exchange high-performance liquid chromatography with Li/citrate elution and quantification by post-column ninhydrin derivatization (39Slocum R.H. Cummings J.G. Hommes F.A. Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York1991: 87-126Google Scholar). Uptake Assays—The r