GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites

Abstract

Purine acquisition is an essential nutritional process for Leishmania. Although purine salvage into adenylate nucleotides has been investigated in detail, little attention has been focused on the guanylate branch of the purine pathway. To characterize guanylate nucleotide metabolism in Leishmania and create a cell culture model in which the pathways for adenylate and guanylate nucleotide synthesis can be genetically uncoupled for functional studies in intact cells, we created and characterized null mutants of L. donovani that were deficient in either GMP reductase alone (Δgmpr) or in both GMP reductase and its paralog IMP dehydrogenase (Δgmpr/Δimpdh). Whereas wild type parasites were capable of utilizing virtually any purine nucleobase/nucleoside, the Δgmpr and Δgmpr/Δimpdh null lines exhibited highly restricted growth phenotypes. The Δgmpr single mutant could not grow in xanthine, guanine, or their corresponding nucleosides, while no purine on its own could support the growth of Δgmpr/Δimpdh cells. Permissive growth conditions for the Δgmpr/Δimpdh necessitated both xanthine, guanine, or the corresponding nucleosides, and additionally, a second purine that could serve as a source for adenylate nucleotide synthesis. Interestingly, GMPR, like its paralog IMPDH, is compartmentalized to the leishmanial glycosome, a process mediated by its COOH-terminal peroxisomal targeting signal. The restricted growth phenotypes displayed by the L. donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.

Graphical abstract

The restricted growth phenotypes displayed by the Leishmania donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.

1. Introduction

Leishmania donovani, the causative agent of visceral leishmaniasis, is a digenetic protozoan parasite that exists as a flagellated extracellular promastigote within sandflies of the Phlebotomus and Lutzomyia genera and as immotile intracellular amastigotes within macrophages and other reticuloendothelial cells of infected mammalian hosts. Visceral leishmaniasis, if untreated, is a terminal disease. Unfortunately, the current assortment of anti-leishmanial drugs is far from ideal due to toxicity, invasive routes of administration, and emergent resistance [1–3]. Thus, the need for new drugs and for the identification of new chemotherapeutic targets is acute. One pathway that presents an attractive drug target is that for purine salvage because, unlike mammals, all protozoan parasites lack the enzymatic machinery for purine biosynthesis de novo and, therefore, the salvage of host purines is an indispensable nutritional requirement [4,5]. Accordingly, each genus of protozoan parasite has evolved a unique complement of transporters and enzymes that enables it to acquire purines from their hosts.

The purine salvage pathway of Leishmania is perhaps the most complex and convoluted among all the protozoan parasites [6–8]. As a consequence, virtually any purine nucleobase or nucleoside can serve as the sole source of purine to support growth of wild type parasites [4–7]. In addition to a multiplicity of purine interconversion enzymes [4–7,9], Leishmania expresses four enzymes capable of salvaging preformed purines to the nucleotide level: hypoxanthine-guanine phosphoribosyltransferase (HGPRT), xanthine phosphoribosyltransferase (XPRT), adenine phosphoribosyltransferase, and adenosine kinase [3,5,10,11]. A systematic genetic analysis of the functional roles of the four salvage enzymes in L. donovani has confirmed that none of these enzymes, by itself, is necessary for adenylate and guanylate nucleotide production [12,13]. However, a conditionally lethal Δhgprt/Δxprt double knockout in L. donovani exhibited a highly restrictive growth phenotype in which promastigotes could not propagate on any purine in the absence of pharmacologic intervention by 2′-deoxycoformycin (dCF), which served to prevent deamination of 6-aminopurine sources. This finding demonstrated that either HGPRT or XPRT alone is sufficient and necessary for parasite survival and growth [12–15]. The Δhgprt/Δxprt strain was also extremely compromised in its capacity to infect mice [14,16].

The strikingly aberrant growth and infectivity phenotype of the Δhgprt/Δxprt null mutant implied that the six nucleotide interconversion enzymes that function to balance adenylate and guanylate nucleotide pools could serve as bottlenecks for interconversion of salvaged purines into the intracellular nucleotide pools. These six enzymes include adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) that convert IMP to AMP, IMP dehydrogenase (IMPDH) and GMP synthetase that convert IMP to GMP, and GMP reductase (GMPR) and AMP deaminase that respectively back convert GMP and AMP to IMP (Fig. 1) [5,7,17–21]. The importance of the nucleotide interconversion pathways in growth and infectivity was validated by the isolation and characterization of Δadss and Δasl and Δimpdh null mutants [22,23]. The Δadss and Δasl parasites exhibited growth phenotypes essentially indistinguishable from that of the previously characterized Δhgprt/Δxprt double knockout, whereas the Δimpdh cells could utilize guanine, guanosine, xanthine or xanthosine, all guanylate precursors, to fulfill their purine requirements [14,22,23]. All three null mutants exhibited reduced infectivity phenotypes in macrophages; however, only Δasl parasites were severely incapacitated in their ability to establish a visceral infection in the mouse model, whereas parasite burdens in mice inoculated with Δadss or Δimpdh cells were robust [22,23].

Fig. 1. Purine metabolism in Leishmania.

The schematic depicts the enzymatic machinery in Leishmania parasites involved in the salvage and interconversion of metabolites that produce the adenylate and guanylate monophosphate nucleotides. 1, GMP reductase; 2, inosine monophosphate dehydrogenase; 3, GMP synthase; 4, xanthine phosphoribosyltransferase; 5, guanine deaminase; 6, hypoxanthine-guanine phosphoribosyltransferase; 7, adenylosuccinate synthetase; 8, adenylosuccinate lyase; 9, AMP deaminase; 10, adenine phosphoribosyltransferase; 11, adenine aminohydrolase; AMP, adenosine monophosphate; GMP, guanosine monophosphate; IMP, inosine monophosphate; XMP, xanthine monophosphate; Ade, adenine; Hyp, hypoxanthine; Xan, xanthine; Gua, guanine.

GMPR and IMPDH from L. donovani are paralogous proteins that possess a cystathionine-β-synthase (CBS) domain that is involved in nucleotide sensing and enzyme regulation [24], as well as a COOH-terminal tri-peptide peroxisomal targeting signal-1 (PTS1) that directs these enzymes to the glycosome [21,23,25,26], a peroxisomal-like microbody found in Leishmania and related trypanosomatids [27–30]. Recombinant L. donovani GMPR and IMPDH [21,24], as well as their recombinant counterparts from the related trypanosomatid Trypanosoma brucei [31,32], have been expressed, purified, characterized, and shown to exhibit unique kinetic, as well as structural, features distinct from their mammalian counterparts. Because the growth and infectivity profiles of the Δimpdh parasites indicated that these knockout lines utilized GMPR to back-convert guanylate nucleotides into IMP that could be used to produce adenylate nucleotides [23], we used targeted gene replacement strategies to introduce a Δgmpr lesion into wild type L. donovani in an effort to further validate the utility of the GMPR protein as a functional component of the nucleotide interconversion pathway. The Δgmpr knockout was subsequently used as a background strain to create an L. donovani Δgmpr/Δimpdh double null mutant in which the adenylate and guanylate branches of purine metabolism were genetically uncoupled. Unlike wild type L. donovani, the Δgmpr line failed to grow on xanthine, guanine, or guanosine as a purine source, while the Δgmpr/Δimpdh double mutant could not grow in any purine on its own. Permissive conditions for growth of the Δgmpr/Δimpdh double knockout required at least two purines, one providing a source of guanylate nucleotides and one a source of adenylate nucleotides. These findings confirm the general model for purine interconversion and metabolism in L. donovani and validate the functional utility of GMPR and IMPDH, which may serve as checkpoints to balance adenylate and guanylate nucleotide production in these parasites [24].

2. Materials and methods

2.1. Chemicals and Reagents

Purine bases and nucleosides were purchased from Sigma–Aldrich® (St. Louis, MO), or Fisher Scientific (Pittsburgh, PA), and restriction enzymes were bought from New England Biolabs (Beverley, MA). The TOPO® TA Cloning® kit and pCR®2.1-TOPO® vector were procured from Thermo Fisher Scientific (Waltham, MA). IMPDH and ODC antisera were produced in guinea pigs or rabbits, respectively, as previously reported [21,33], and the mouse monoclonal anti-α-tubulin (DM1A) antibody was obtained from EMD Millipore (Billerica, MA). Goat anti-rabbit HRP-conjugated and goat anti-mouse HRP-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA), and the goat anti-guinea pig HRP-conjugated antibody was acquired from Santa Cruz Biotechnology (Dallas, TX). The pX63-PHLEO, pX63-PAC, pX63-NEO, pX63-HYG, and pXG-BSD leishmanial expression vectors harboring the phleomycin/bleomycin binding protein (PHLEO), puromycin N-acetyltransferase (PAC), neomycin resistance (NEO), hygromycin phosphotransferase (HYG), and blasticidin deaminase (BSD) markers [34–36], respectively, were constructed and supplied by Dr. Stephen M. Beverley (Washington University, St. Louis, MO), and the pQE-80 vector was acquired from Qiagen (Valencia, CA). The goat anti-rabbit Oregon Green-, goat anti-guinea pig Rhodamine Red conjugated secondary fluorescent antibodies, and SYBR® Green were obtained from Thermo Fisher Scientific. All other chemicals and reagents were of the highest quality commercially accessible.

2.2. Axenic Parasite cell culture

The LdBob strain [37] adapted from the 1S2D clone of wild type L. donovani [38,39] was originally obtained from Dr. Beverley. LdBob promastigotes were cultured at 26 °C in modified Dulbecco’s Modified Eagle Medium-Leishmania (DME-L) [40] medium [12] that was supplemented with 5% FBS. Plating methods and single cell cloning protocols for L. donovani promastigotes have been described previously [40,41]. Wild type and Δgmpr parasites were routinely cultivated in 100 μM hypoxanthine as the purine nutrient, and the Δgmpr[pGMPR] and Δgmpr[pgmprΔSKL] cell lines were propagated in 100 μM guanine. The Δimpdh knockout parasites were maintained in 100 μM xanthine, while the Δgmpr/Δimpdh double knockout strain was grown in 100 μM hypoxanthine and 100 μM xanthine. Axenic amastigotes were propagated at 37 °C as detailed [12,37,38] in 100 μM of the same growth-permissive purines in which the corresponding promastigotes were grown. All cell lines were routinely cycled between promastigotes and axenic amastigotes [37] with their respective purine additions.

2.3. GMPR and IMPDH cloning and sequencing

The Leishmania infantum GMPR (LinJ.17.0870) was identified in the GeneDB database (http://www.genedb.org) by BLAST searches using the human GMPR1 protein sequence (NP_006868.3) [42,43]. The Leishmania donovani GMPR ORF was amplified by PCR from L. donovani 1S2D genomic DNA with the primers designed against the L. infantum coding sequence (Table S1), cloned into TOPO® TA and sequenced. IMPDH was cloned and sequenced as described [17,18]

2.4. DNA manipulations and Southern blotting

Genomic DNA from 5 × 10⁷ – 1 × 10⁸ parasites was isolated using the Quiagen DNeasy Blood and Tissue Kit (Waltham, MA), digested with appropriate restriction enzymes, fractionated on 0.8% agarose gels, transferred to nylon membranes, and Southern blotting analyses performed using conventional protocols [9]. Hybridization probes were the full length coding sequences for GMPR and IMPDH, both of which were generated by PCR, separated on 0.8% agarose gels, and gel-purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI). The template plasmids from which the hybridization probes were amplified harbored the full-length genes that had been previously cloned into the TOPO® TA Cloning® Kit with pCR™2.1-TOPO® vector (Thermo Fisher Scientific).

2.5. Gene targeting and complementation constructs

The 5′ and 3′ UTRs of GMPR were PCR-amplified from L. donovani genomic DNA using primers designed against the flanking regions of the L. infantum GMPR sequence that had been obtained from GeneDB (http://www.genedb.org). The PCR-generated flanking regions of L. donovani GMPR, containing either HindIII/SalI (5′ UTR) or SmaI/BglII (3′ UTR) restriction sites (Table S1) were ligated into the TOPO® TA vector and sequenced. The pX63-HYG-Δgmpr targeting plasmid used to create the Δgmpr cell line was generated by excising the 5′ or 3′ UTR from the TOPO® TA vector with HindIII/SalI or SmaI/BglII, respectively, followed by sequential insertion of the 5′ or 3′ UTR into the corresponding restriction sites within the pX63-HYG vector [35] that had also been digested with HindIII/SalI, followed by digestion with SmaI/BglII. Due to the presence of a SmaI restriction site in the pX63-PAC vector [34], a different strategy was used to generate the pX63-PAC-Δgmpr replacement plasmid. Briefly, the pX63-HYG-Δgmpr plasmid was digested with HindIII/BamHI, while the pX63-PAC vector was digested with HindIII/BglI, and the compatible DNA fragments containing either the 5′ and 3′ GMPR UTRs and the plasmid backbone or the PAC drug resistance cassette were ligated together to form the pX63-PAC-Δgmpr replacement plasmid. Complementation constructs that facilitated the expression of either GMPR or mis-localized gmprΔSKL in parasites containing a Δgmpr null mutation were created using the pXG-BSD leishmanial expression vector [36]. The GMPR ORF was amplified via PCR from genomic DNA with sense and antisense primers that added a 5′ SmaI or 3′ BamHI restriction site, respectively (Table S1), cloned into the pCR™2.1-TOPO® vector, and sequenced to ensure fidelity. To generate a mutant gmpr in which the last three amino acids (SKL) that comprise the PTS1 of GMPR were deleted, an antisense primer that encompassed a BamHI restriction site, an artificially introduced in-frame stop codon and amino acids 486–489 of the GMPR ORF was designed (Table S1). This antisense primer was used in conjunction with the sense primer described above to PCR-amplify the gmprΔSKL fragment which was then subcloned into the pCR™2.1-TOPO® vector, and sequenced to ensure fidelity. The GMPR and gmprΔSKL fragments were excised from the pCR™2.1-TOPO® vector via digestion with Sma/BamHI, gel purified and ligated into the pXG-BSD blasticidin resistance expression plasmid [36] that had also been digested with SmaI/BamHI and gel purified. Both the wild type pXG-BSD-GMPR and truncated pXG-BSD-gmprΔSKL constructs were verified by sequencing. The Δimpdh targeting constructs were generated as described using the pTRG vector and 4-way multi-fragment ligation system that was designed by our laboratory [23,44].

2.6. Creation of null mutants and complemented strains

5 × 10⁷ LdBob promastigotes were transfected using standardized electroporation conditions [7]. Electroporated parasites were maintained for 24 h in liquid medium prior to plating on semi-solid DME-L-based growth medium supplemented with a permissive purine at a concentration of 100 μM and the appropriate drugs. The drug concentrations employed for the genetic manipulations were 50 μg/ml hygromycin, 50 μg/ml phleomycin, 20 μg/ml puromycin, 20 μg/ml Geneticin (G418), and 20 μg/ml blasticidin, as applicable for the selective marker. The pX63-HYG-Δgmpr and pX63-PHLEO-Δgmpr knockout vectors were linearized by digestion with HindIII and BgIII while the pTRG-PAC-Δimpdh and pTRG-NEO-Δimpdh constructs were excised with PacI, and the fragments harboring the drug resistance cassette and the UTRs from the gene of interest were gel purified just prior to electroporation. The linearized targeting constructs used to introduce the genetic lesions were designated by the name of the plasmid minus the initial letter, e.g., X63-HYG-Δgmpr from pX63-HYG-Δgmpr. A GMPR/Δgmpr∷HYG (GMPR/gmpr) heterozygote was generated after transfecting wild type LdBob with X63-HYG-Δgmpr, and the Δgmpr∷HYG/Δgmpr∷PHLEO (Δgmpr) null mutant was isolated after transfection of the GMPR/gmpr heterozygote with X63-PHLEO-Δgmpr followed by selection in hygromycin, phleomycin and 100 μM hypoxanthine as a purine source. The IMPDH/Δimpdh∷PAC heterozygote was then created into the Δgmpr background using the linearized TRG-PAC-Δimpdh targeting construct, and the Δgmpr/IMPDH/impdh cell line was subsequently transfected with TRG-NEO-Δimpdh, selected in hygromycin, phleomycin, puromycin, and G418 with 100 μM concentrations of both hypoxanthine and xanthine to generate the Δgmpr/Δimpdh∷PAC/Δimpdh∷NEO (Δgmpr/Δimpdh) double knockout. The pXG-BSD-GMPR and pXG-BSD-gmprΔSKL plasmids were used to generate the Δgmpr[pGMPR], Δgmpr[pgmprΔSKL], Δgmpr/Δimpdh[pGMPR] and Δgmpr/Δimpdh[pgmprΔSKL] complemented cell lines from the Δgmpr and Δgmpr/Δimpdh knockouts, respectively. The complemented cell lines were selected in blasticidin, and 100 μM guanine was the added purine. Southern blot analysis was used to verify the genotypes of the heterozygous, knockout, and complemented strains as described above.

2.7. Antibody generation and western blotting

The L. major GMPR, which shows 98% amino acid sequence identity to LdGMPR was used for antibody production as previously described [24]. Open Biosystems (Huntsville, AL) was contracted to produce polyclonal LmGMPR antiserum in rabbits using purified recombinant LmGMPR as an immunogen and standard injection protocols. Antiserum to the L. donovani IMPDH antiserum was produced in guinea pigs as described [21], and mouse monoclonal anti-α-tubulin antibody (DM1A) was purchased from EMD Chemicals (Gibbstown, NJ). Western blotting was performed via standard protocols [45] using goat anti-rabbit HRP-conjugated, goat anti-guinea pig HRP-conjugated, and goat anti-mouse HRP conjugated secondary antibodies (Thermo Fisher Scientific).

2.8. Growth phenotypes

To evaluate the capabilities of the Δgmpr, Δgmpr[pGMPR], Δgmpr[pgmprΔSKL], Δimpdh, Δgmpr/Δimpdh, Δgmpr/Δimpdh[pGMPR], and Δgmpr/Δimpdh[pgmprΔSKL] cells to multiply in different purines, wild type and transgenic parasites were washed several times with PBS, resuspended at a density of 5 × 10⁴ parasites/ml in modified DME-L [12] containing 100 μM of one or more purines, supplemented with 5% dialyzed FBS, seeded in 1.0 ml aliquots into 24-well tissue culture plates (Sarstedt Inc., Newton, NC), and incubated at 26 °C. Parasites were allowed to grow for 7–10 days then enumerated visually via hemacytometer. Parasite growth in adenine or adenosine was evaluated in the presence of 20 μM dCF, an inhibitor of the L. donovani adenine aminohydrolase [46–49], in order to prevent deamination of the 6-aminopurines to hypoxanthine and inosine. Without added dCF, adenine and adenosine are converted to hypoxanthine and inosine, respectively, over the course of the 7–10 day experiment, so dCF is obligatory for preservation of the added 6-aminopurines in these growth experiments. Additionally, growth experiments were conducted with the Δgmpr/Δimpdh cell line in which the concentration of hypoxanthine or xanthine was fixed at 25 μM and the concentrations of xanthine or hypoxanthine were varied from 1.56 to 100 μM, respectively, and the final cell density was assessed using a SYBR® Green assay [50,51]. Briefly, SYBR® Green was diluted to 1X concentration in 1% Triton X-100 in PBS, and incubated with cells for 60 min while shaking. Fluorescence was quantified with a Molecular Devices SpectraMax M2 plate reader using wavelengths of 485_Ex and 538_Em.

2.9. Macrophage infections

J774 culture-form murine macrophages (ATCC, Manassas, VA) were seeded at a density of 2 × 10⁵ cells per well into 4-well Nunc™ Lab-Tek™ ‖ Chambered Coverglass slides (Thermo Fisher Scientific) placed in a 37 °C 5% CO₂ incubator and allowed to adhere for 8 h. The infection protocol with wild type, Δgmpr, Δgmpr[pGMPR], Δgmpr[pgmprΔSKL], Δimpdh, Δgmpr/Δimpdh, and Δgmpr/Δimpdh[pGMPR] parasites was performed as detailed [12,14]. 72 h post-infection, amastigotes were stained with the Diff-Quik kit (International Medical Equipment Inc., San Marcos, CA) and enumerated visually on a Zeiss Axiovert 200 inverted microscope (Carl Zeiss Microimaging, Thornwood, NY).

2.10. Subcellular localization

GMPR was localized in L. donovani promastigotes by indirect immunofluorescence as described [21,22,25,26] using a 1:500 dilution of anti-LmGMPR antibody and a 1:10,000 dilution of secondary goat anti-rabbit Oregon Green-conjugated antibody (Thermo Fisher Scientific). Parasites were costained with guinea pig antibodies to the L. donovani IMPDH protein (1:200) and goat anti-guinea pig Rhodamine Red-conjugated (1:10,000) secondary antibody (Thermo Fisher Scientific) to stain glycosomes. IMPDH has been previously validated as a glycosomal enzyme [23]. Cells were photographed on a Zeiss Axiovert 200 inverted microscope (Carl Zeiss Microimaging) using a 63× oil immersion lens and a Zeiss AxioCam MR camera coupled with Axiovision 4.2 software (Carl Zeiss Microimaging) and images were compiled using Adobe Photoshop Creative Suite 4.

3. Results

3.1. Confirmation of the Δgmpr genotype

Southern blot analysis of genomic DNA from wild type, GMPR/gmpr heterozygotes, Δgmpr knockouts, Δgmpr[pGMPR] or Δgmpr[pgmprΔSKL] complemented parasites probed with the full length GMPR ORF confirmed the expected genotypes of all five cell lines (Fig. 2A). The ≈ 3300 bp SmaI fragment excised from wild type or GMPR/gmpr genomic DNA encompasses the full length GMPR ORF as well as 123 bp and 1690 bp of the 5′ and 3′- UTR’s, respectively. The absence of a hybridization signal in genomic DNA from Δgmpr parasites confirmed the null genotype of this cell line, and episomal complementation of the Δgmpr cell line with either [pGMPR] or [pgmprΔSKL] was verified by the presence of a 1479 or 1470 bp SmaI/BamHI fragments, respectively. Western blot analysis with polyclonal anti-GMPR antisera confirmed the specificity of the reagent, the absence of GMPR protein in the Δgmpr mutant, and the presence of GMPR in wild type, Δgmpr[pGMPR] and Δgmpr[pgmprΔSKL] parasites. Equal loading of the blot was confirmed by probing the same samples with anti-tubulin antibody (Fig. 2B).

Fig. 2. Southern and Western analysis of Δgmpr knockouts.

(A) Total genomic DNA from wild type (lane 1), GMPR/gmpr heterozygotes (lanes 2 and 3), Δgmpr (lane 4), Δgmpr[pGMPR] (lane 5), or Δgmpr[pgmprΔSKL] (lane 6) parasites was digested with SmaI (lanes 1–4) or SmaI/BamHI (lanes 5 and 6), fractionated on a 0.8% agarose gel, transferred to nylon membranes, and hybridized under stringent conditions with a probe containing the full length GMPR ORF. (B) Lysates from exponentially growing wild type (lane 1), Δgmpr (lane 2), Δgmpr[pGMPR] (lane 3) or Δgmpr[pgmprΔSKL] (lane 4) parasites were loaded onto a 4–20% polyacrylamide gel and analyzed by immunoblotting using monospecific, polyclonal anti-GMPR antibodies that had been produced in rabbits. Equal loading of samples was verified by stripping and reprobing the same membrane with commercially available mouse anti-α-tubulin monoclonal antibody.

3.2. Growth phenotype of the Δgmpr null mutant

The impact of a Δgmpr lesion on the nutritional requirements of L. donovani promastigotes was evaluated by assessing the capacity of wild type, Δgmpr, and GMPR-complemented promastigotes to grow in medium supplemented with one of a selection of purines (Fig. 3). Growth in adenine or adenosine was evaluated in the presence of 20 μM dCF, which acts to prevent deamination of these 6-aminopurines into hypoxanthine or inosine, respectively, over the course of the growth experiments [46–49]. Whereas wild type, promastigotes could multiply in medium supplemented with any of the purine nucleobases or nucleosides tested, the Δgmpr null mutant could not propagate on guanine, guanosine, xanthine, or xanthosine as a sole external source of purine. Episomal complementation of Δgmpr parasites with Δgmpr[pGMPR] rescued the restrictive growth phenotypes in these 6-oxopurines. Furthermore, the Δgmpr[pgmprΔSKL] complemented cell line was also capable of robust growth in guanine, xanthine, and their corresponding ribonucleosides, indicating that deletion of the SKL COOH-terminal PTS1 tripeptide did not negatively impact the fitness of parasites expressing mislocalized gmprΔSKL protein (Fig. 3).

Fig. 3. Growth phenotype of Δgmpr knockouts.

The ability of wild type, Δgmpr, Δgmpr[pGMPR], or Δgmpr[pgmprΔSKL] to grow in defined medium containing different purine sources was evaluated. Parasites were seeded at a density of 5 × 10⁴/ml into medium containing 100μM purine, incubated for 7–10 days and enumerated via hemacytometer. Parasite proliferation in adenine (Ade), or adenosine (Ado), was evaluated in the presence of 20μM dCF.

3.3. Confirmation of the Δgmpr/Δimpdh genotype

To investigate the consequences of genetically uncoupling the adenylate and guanylate branches of the purine salvage pathway, we generated an L. donovani Δgmpr/Δimpdh double knockout. The Δimpdh mutation was created in the Δgmpr background after two rounds of targeted gene replacement and selection in permissive growth conditions that required the inclusion of two separate purines in the growth medium, one that could be metabolized to adenylate nucleotides and the other utilized to make guanylate nucleotides. The genotype of the resulting double knockout parasites was established by Southern blot analysis of genomic DNA from wild type and mutant parasites digested with SmaI or SmaI/BamHI and hybridized to the GMPR ORF as a probe (Fig. 4A). The ≈ 3300 bp wild type GMPR SmaI fragment encompassed the GMPR ORF, as well as a portion of the 5′ and 3′ flanking regions, and the 1479 bp SmaI/BamHI fragment included either the GMPR or gmprΔSKL ORFs that had been excised from the [pXG-BSD] episomes. The absence of a signal in the lanes with Δgmpr, Δgmpr/IMPDH/impdh, or Δgmpr/Δimpdh DNA confirmed the Δgmpr genotype in those strains. The same genomic DNA samples were digested with EcoRV/PstI and probed with the full length IMPDH sequence (Fig. 4B). The IMPDH ORF, as well as a portion of the IMPDH 5′ and 3′ UTRs, was excised from the genomic DNA and are within an approximately 2300 bp band in lanes containing genomic DNA from wild type, Δgmpr, and Δgmpr/IMPDH/impdh parasites. The lack of signal in the Δgmpr/Δimpdh, Δgmpr/Δimpdh[pGMPR], and Δgmpr/Δimpdh[pgmprΔSKL] cell lines confirmed the replacement of both IMPDH alleles in these strains (Fig. 4B). Western blot analysis the genotype of the Δgmpr/Δimpdh knockouts and verified that the Δgmpr/Δimpdh[pGMPR] and Δgmpr/Δimpdh[pgmprΔSKL] complemented cells produce the GMPR protein (Fig. 4C). The same samples were probed with anti-IMPDH antibody to confirm the presence of the IMPDH protein in wild type and Δgmpr parasites and the absence of IMPDH in Δimpdh, Δgmpr/Δimpdh, Δgmpr/Δimpdh[pGMPR], and Δgmpr/Δimpdh[pgmprΔSKL] cell lysates. Both blots were also hybridized with anti-ODC antibody to demonstrate equal loading.

Fig. 4. Southern and Western analysis of Δgmpr/Δimpdh knockouts.

Total genomic DNA from wild type (lane 1), Δimpdh (lane 2), Δgmpr (lane 3), Δgmpr/IMPDH/impdh (lane 4), Δgmpr/Δimpdh (lane 5), Δgmpr/Δimpdh[pGMPR] (lane 6), or Δgmpr/Δimpdh[pgmprΔSKL] (lane 7), parasites was digested with SmaI (Panel A, lanes 1–5), SmaI/BamHI (Panel A, lanes 6 and 7, Panel B, lanes 6 and 7), or EcoRV/PstI (Panel B, lanes 1–5) fractionated on a 0.8% agarose gel, and blotted onto nylon membrane. The blots were hybridized under stringent conditions with a probe containing either the full length GMPR (A) or IMPDH ORF (B). (C) Western blot analysis was performed on total cell lysates from wild type (lane 1), Δimpdh (lane 2), Δgmpr (lane 3), Δgmpr/Δimpdh (lane 4), Δgmpr/Δimpdh[pGMPR] (lane 5), or Δgmpr/Δimpdh[pgmprΔSKL] (lane 6), using monospecific, anti-GMPR or anti-IMPDH antibodies, and equal loading of the samples was verified by probing the same membranes with anti-ODC antibody.

3.4. Growth phenotype of Δgmpr/Δimpdh knockouts

The capacity of the Δgmpr/Δimpdh double knockout to utilize various purines was then compared to that of the wild type, Δgmpr, Δimpdh, Δgmpr/Δimpdh[pGMPR], and Δgmpr/Δimpdh [pgmprΔSKL] cell lines (Fig. 5A). Whereas wild type promastigotes could grow in medium with any of the purine nucleobases, nucleosides, or combinations tested, the Δgmpr/Δimpdh parasites could not propagate in medium supplemented with any individual purine, including hypoxanthine or xanthine (Fig. 5A). The Δgmpr/Δimpdh double knockout could, however, grow robustly when hypoxanthine and xanthine were combined in the growth medium (Fig. 5A). The Δgmpr control, as shown in Fig. 3, could not propagate in medium supplemented with either guanosine or xanthine but was capable of robust growth in either hypoxanthine or inosine and in adenine or adenosine in the presence of 20 μM dCF (Fig. 5A). In contrast, the Δimpdh null mutant was able to grow in guanine, guanosine or xanthine but not in adenine, adenosine hypoxanthine or inosine, although both Δgmpr and Δimpdh promastigotes grew robustly, as expected, in medium supplemented with a mixture of 100 μM hypoxanthine and 100 μM xanthine (Fig. 5A). The growth phenotypes of the complemented Δgmpr/Δimpdh[pGMPR] and Δgmpr/Δimpdh[pgmprΔSKL] cell lines mirrored those of Δimpdh parasites, indicating that the GMPR and gmprΔSKL proteins produced in these complemented cell lines provided the parasites with adequate levels of GMPR or gmprΔSKL to sustain their growth in guanine, guanosine, or xanthine, regardless of the subcellular location of the protein.

Fig. 5. Growth phenotype of Δgmpr/Δimpdh knockouts.

(A) Wild type, Δimpdh, Δgmpr, Δgmpr/Δimpdh, Δgmpr/Δimpdh[pGMPR] or Δgmpr/Δimpdh[pgmprΔSKL] parasites were inoculated at a density of 5 × 10⁴/ml into medium containing 100μM purine, incubated for 7–10 days and enumerated via hemacytometer. Parasite proliferation in adenine (Ade), or adenosine (Ado), was evaluated in the presence of 20 μM dCF. (B) Final cell density was assessed in a fixed concentration of 25 μM hypoxanthine (Hyp) (■) or xanthine (Xan) (▲), while the concentration of the other purine was varied from 1.56–100 μM, and fluorescence units were measured using a SYBR® Green assay.

The Δgmpr and Δimpdh lesions in the Δgmpr/Δimpdh double knockout effectively disconnect the adenylate and guanylate nucleotide biosynthetic branches in that strain affording the opportunity to dissect the relative contributions of both branches to nucleotide-driven cellular functions. In this study, we exploited this genetic disengagement of the purine branches in the Δgmpr/Δimpdh cell line to establish its nutritional requirements for hypoxanthine, which can only be incorporated into adenylate nucleotides, and xanthine, exclusively a source of guanylate nucleotides. Thus, growth of Δgmpr/Δimpdh promastigotes was determined by varying the concentrations of hypoxanthine and xanthine added to the culture medium in the presence of either 25 μM xanthine or 25 μM hypoxanthine, respectively (Fig. 5B). Noticeably, the requirements for xanthine to sustain Δgmpr/Δimpdh growth in the presence of 25 μM hypoxanthine were substantially lower than those for xanthine in the presence of 25 μM hypoxanthine (Fig. 5B).

3.5. Macrophage infections

To ascertain whether the Δgmpr lesion compromised the capacity of L. donovani to establish an infection in mammalian macrophages, the abilities of wild type, knockout cell lines, and complemented promastigotes to infect murine J774 culture form macrophages were appraised (Fig. 6). Whereas wild type, Δgmpr[pGMPR] and Δgmpr[pgmprΔSKL] parasites were capable of sustaining comparable and robust infections in J774 cells after 72 h, amastigote loads in macrophages infected with either the Δimpdh or Δgmpr lines were reduced by ~25%, while amastigote burdens of macrophages infected with Δgmpr/Δimpdh promastigotes were ~50% of levels achieved with wild type parasites (Fig. 6). The Δgmpr/Δimpdh[pGMPR] and Δgmpr/Δimpdh[pgmprΔSKL] parasites had amastigote loads in J774 cells that were equivalent to that obtained with the Δimpdh single knockout.

Fig. 6. Macrophage infections.

Culture form J774 murine macrophages were seeded at a density of 2 × 10⁵ cells/ml into 4-well chamber slides, and infected with stationary phase L. donovani parasites at a 10:1 ratio. The ability of wild type, Δimpdh, Δgmpr, Δgmpr[pGMPR], Δgmpr[pgmprΔSKL], Δgmpr/Δimpdh, Δgmpr/Δimpdh[pGMPR], or Δgmpr/Δimpdh[pgmprΔSKL], parasites to infect macrophages was assessed via hemacytometer.

3.6. Immunolocalization of GMPR

The L. donovani GMPR accommodates an archetypical PTS1 signal sequence (SKL). To confirm that this topogenic signal is targeting GMPR to the glycosome, immunofluorescence assays were performed on wild type, Δgmpr, Δgmpr[pGMPR] and Δgmpr[pgmprΔSKL] promastigotes (Fig. 7). Immunofluorescence analysis of wild type (Fig. 7, panels a–d) and Δgmpr[pGMPR] cells (Fig. 7, panels e–h) revealed a punctate staining pattern for GMPR protein in both of these cell lines. The glycosomal milieu of GMPR in wild type and Δgmpr[pGMPR] cells was corroborated by co-staining with anti-IMPDH antibodies (Fig. 7, panels i–j). Conversely, the staining pattern for the gmprΔSKL protein in the Δgmpr[pgmprΔSKL] parasites was diffuse (Fig. 7, panels m–p). The absence of both GMPR and IMPDH in the Δgmpr/Δimpdh cells was also confirmed in these immunofluorescence assays (Fig. 7, panels q–t).

Fig. 7. Localization of GMPR.

Wild type (a–d), Δgmpr (e–h), Δgmpr[pGMPR] (i–l), Δgmpr[pgmprΔSKL] (m–p), or Δgmpr/Δimpdh (q–t) L. donovani promastigotes were subjected to immunofluorescence analysis using rabbit anti-GMPR (a, e, i, m, and q) or guinea pig anti-IMPDH (b, f, j, n, and r) monospecific, polyclonal antibodies. Goat anti-rabbit Oregon Green-conjugated and goat anti-guinea pig Rhodamine Red-conjugated secondary antibodies were used to detect the GMPR and IMPDH primary antibodies, respectively. Overlays of the Oregon Green and Rhodamine Red staining are shown in panels c, g, k, o and s. Parasites were also stained with DAPI (d, h, l, p, and t).

4. Discussion

By virtue of serving as the key enzyme that initiates the back-conversion of guanylate nucleotides to adenylate nucleotides, GMPR plays a prominent role in ensuring a proper equilibrium between guanylate and adenylate nucleotide pools in cells. The role of GMPR in maintaining the nucleotide balance in protozoan parasites is particularly significant because these organisms do not synthesize purine nucleotides de novo and must rely on host purines to meet their nutritional needs and to maintain and proportion their intracellular nucleotide levels. In contrast to the mammalian GMPR, the leishmanial GMPR accommodates CBS motifs. These regulatory elements are found within a variety of channels and enzymes from bacteria to humans and have been speculated to sense changes in their nucleotide environments via the binding of adenosyl motifs, which in turn affects their activity [52]. In contrast to previous studies, the single CBS domain of the Leishmania GMPR, which is composed of two tandemly arranged~60 amino acid repeat CBS motifs, has been characterized biochemically and shown to bind GTP, GMP, and ATP. Binding of GTP and GMP resulted in GMPR activation, whereas ATP binding caused GMPR inhibition [24]. These nucleotide-induced kinetic alterations were also accompanied by changes in the quaternary structure of GMPR [24]. A similar analysis of the CBS domain of the paralogous L. donovani IMPDH also demonstrated nucleotide interaction, although IMPDH activity was inhibited by GTP and unaffected by ATP [24]. The biochemical analysis of the CBS domain of GMPR highlights the central role of this enzyme in nucleotide metabolism as a regulatory checkpoint between guanylate and adenylate metabolism in Leishmania

Despite the detailed in vitro characterization of the Leishmania GMPR enzyme [24], its role in intact parasites has not been evaluated. To test its metabolic significance, an L. donovani Δgmpr knockout was created by targeted gene replacement and characterized, establishing that GMPR is not essential for parasite viability under permissive conditions. As anticipated, Δgmpr promastigotes failed to grow when xanthine, guanine, or guanosine was provided as the sole purine source (Fig. 3) because a GMPR deficiency prevents conversion of these purines to adenylate nucleotides (see Fig. 1). Thus, GMPR must be essential for guanylate nucleotide incorporation into the amastigote adenylate nucleotide pool in infected hosts. All other purines were permissive for promastigote growth since adenylate nucleotide transformation to guanylate nucleotides remained intact in Δgmpr cells. The genetic lesion only slightly impacted the ability of L. donovani to infect macrophages (Fig. 6). Complementation of the Δgmpr lesion with an ectopic GMPR circumvented both the pronounced growth and slight infectivity deficits, and proper GMPR localization did not seem necessary for these phenotypic reversals, as the Δgmpr[pgmprΔSKL] expressing mislocalized gmpr in which the PTS1 triad had been deleted did not express the growth or infectivity shortfalls of the Δgmpr null line (Figs. 3, 6 and 7). It should be noted that the episomally encoded GMPR and gmpr in the Δgmpr[pGMPR] and Δgmpr[pgmprΔSKL] cells, respectively, were overexpressed compared to wild type GMPR, and this overexpression could have masked any fitness or growth defect conferred by cytosolic mislocalization of the gmpr in the Δgmpr[pgmprΔSKL] parasites in our experiments (Fig. 2B). However, the glycosomal milieu of other glycosomal enzymes such as HGPRT, XPRT, and IMPDH also does not seem to be essential for their function in either the promastigote or amastigote forms since PTS1 deletion mutants of ectopic hgprt, xprt, and impdh genes all resulted in mistargeting of their encoded proteins to the cytosol but complemented phenotypic deficiencies in the corresponding null mutant lines at expression levels comparable to wild type L. donovani [21,23,25,26]. Consequently, it remains unclear what the biological advantage is for kinetoplastids to compartmentalize some of the enzymes involved in purine salvage and metabolism in the glycosome.

The ability to generate independent Δgmpr and Δimpdh L. donovani clones offered the opportunity to create a conditionally lethal Δgmpr/Δimpdh double knockout line in which the adenylate and guanylate branches could be genetically uncoupled. Because the Δgmpr/Δimpdh null mutant, unlike wild type L. donovani, could not interconvert adenylate and guanylate nucleotides, the double knockout line could not be propagated in any single purine nucleobase or nucleoside provided to the culture medium although the cell line grew robustly in a combination of hypoxanthine and xanthine (Fig. 5A), which could serve as sources of adenylate and guanylate nucleotides, respectively (Fig. 1). The genetic uncoupling of the adenylate and guanylate nucleotide branches enabled us to determine that the amount of purine required to fulfill adenylate nucleotide requirements in L. donovani promastigotes was considerably greater than that required to fulfill guanylate nucleotide requirements, presumably due to the relatively higher concentrations of intracellular adenylate nucleotides in the parasite (Fig. 5B). This genetic disjunction of adenylate and guanylate nucleotide synthesis in Δgmpr/Δimpdh cells has also been instrumental in the evaluation of the consequences of selective nutritional depletion of parasite adenylate or guanylate nucleotides on fundamental biological processes such as the stress response of Leishmania to purine depletion [53]. Exploiting this strategy, Martin et al. proved that the purine stress response in which the L. donovani proteome is remodeled could be attributed to selective depletion of the adenylate nucleotide pathway [53].

• GMP reductase (GMPR) is essential for guanylate nucleotide salvage from the host

• Δgmpr promastigotes cannot grow on xanthine, guanine, or guanosine

• A Δgmpr/Δimpdh mutant cannot interconvert adenylate and guanylate nucleotides

• A Δgmpr/Δimpdh double knockout cannot grow on a single purine source

Abbreviations

HGPRT

hypoxanthine-xanthine phosphoribosyltransferase

XPRT

xanthine phosphoribosyltransferase

dCF

2′-deoxycoformycin

ADSS

adenylosuccinate synthetase

ASL

adenylosuccinate lyase

IMPDH

IMP dehydrogenase

GMPR

GMP reductase

CBS

cystathionine-β-synthase

PTS1

peroxisomal targeting signal-1

PHLEO

phleomycin/bleomycin binding protein

PAC

puromycin N-acetyltransferase

NEO

neomycin resistance

HYG

hygromycin phosphotransferase

BSD

blasticidin deaminase

DME-L

Dulbeccco’s modified eagle medium-Leishmania

G418

Geneticin