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Originally published In Press as doi:10.1074/jbc.M512911200 on January 19, 2006

J. Biol. Chem., Vol. 281, Issue 12, 7952-7959, March 24, 2006
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Leishmania major Expresses a Single Dihydroxyacetone Phosphate Acyltransferase Localized in the Glycosome, Important for Rapid Growth and Survival at High Cell Density and Essential for Virulence*

Rachel Zufferey1 and Choukri Ben Mamoun2

From the Department of Genetics and Developmental Biology and the Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3301

Received for publication, December 2, 2005 , and in revised form, January 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite major advances in the understanding of pathogenesis of the human protozoan parasite Leishmania major, little is known about the enzymes and the primary precursors involved in the initial steps of synthesis of its major glycerolipids including those involved in virulence. We have previously demonstrated that the initial step of acylation of the precursor glycerol 3-phosphate is not essential for the synthesis of ester and ether phospholipids in this parasite. Here we show that Leishmania expresses a single acyltransferase with high specificity for the precursor dihydroxyacetone phosphate and shows the best activity in the presence of palmitoyl-CoA. We have identified and characterized the LmDAT gene encoding this activity. LmDAT complements the lethality resulting from the loss of both dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferase activities in yeast. Recombinant LmDAT exhibits biochemical properties similar to those of the native enzyme of the promastigote stage parasites. We show that LmDAT is a glycosomal enzyme and its loss in a {Delta}lmdat/{Delta}lmdat null mutant results in complete abrogation of the parasite dihydroxyacetone phosphate acyltransferase activity. Furthermore, lack of LmDAT causes a major alteration in parasite division during the logarithmic phase of growth, an accelerated cell death during stationary phase, and loss of virulence. Together, our results demonstrate that LmDAT is the only dihydroxyacetone phosphate acyltransferase of the L. major localized in the peroxisome, important for growth and survival and essential for virulence.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Worldwide, protozoan parasites of the genus Leishmania cause a large spectrum of important human diseases collectively named leishmaniasis. These parasites develop within the digestive tract of the sand fly vector as flagellated, mobile promastigotes and differentiate into and multiply as non-motile amastigotes within the phagolysosomal compartment of vertebrate host macrophages.

Glycerolipids constitute 70% of total lipids in the protozoan parasite Leishmania (1–3). They are classified into ester and ether lipids depending on the substitution at position 1 of the glycerol backbone. Ester lipids harbor an acyl group, whereas ether lipids carry a fatty alcohol moiety. Lipids of Leishmania parasites have been a focus of extensive studies because some of their derivatives, such as lipophosphoglycan and glycosylphosphatidylinositol-anchored protease gp63, were shown to be important for parasite virulence and development (for review, see Refs. 4–8). Lipids are also essential cell constituents and, therefore, must be constantly synthesized to allow multiplication of the parasite. This suggests that the pathways leading to their synthesis are essential for parasite proliferation and pathogenesis and, thus, offer a reasonable target for rational design of new anti-leishmanial drugs. In fact, a lipid-based drug, miltefosine, is a potent antileishmanial compound that inhibits parasite growth in vitro and in vivo and is currently used for treatment of visceral and mucocutaneous forms of leishmaniasis (9–12). The acylation of dihydroxyacetone phosphate (DHAP)3 by a DHAP acyltransferase (DHAPAT) represents the initial and obligatory step in the biosynthesis of ether lipids in most organisms that synthesize alkylglycerolipids (13). The product of this first acylation reaction, 1-acyl-DHAP, is then converted to 1-alkyl-DHAP by a FAD-dependent alkyl DHAP synthase (14), which is further reduced to 1-alkyl-glycerol-3-phosphate (1-alkyl-G3P) by an NADPH-dependent 1-alkyl/acyl-DHAP reductase. 1-Alkyl-G3P serves as the obligate precursor for all ether phospholipids. Alternatively, 1-acyl-DHAP can be reduced to 1-acyl-G3P by an NADPH-dependent 1-alkyl/1-acyl-DHAP reductase, which is then used for the biosynthesis of ester phospholipids. The relative contribution of the DHAP acylation step in the biosynthesis of ester phospholipids has not yet been firmly established (15, 16).

DHAPAT activity has been characterized biochemically in different organisms (17–19). In most animal tissues, DHAPAT is found in a membrane-bound fraction (15, 20) and localized in the luminal side of peroxisomes (15, 21). This enzyme was also found to be part of a heterotrimeric complex that includes the 1-alkyl-DHAP synthase (22, 23). Alterations in DHAPAT function have been associated with various human diseases such as neonatal adrenoleukodystrophy, infantile Refsum, disease, hyperpipecolic academia, and rhizomelic chondrodyplasia punctata (24–26).

We have previously reported the characterization of the first acyltransferase LmGAT specific for the lipid precursor G3P. We showed that this activity was encoded by a single gene, LmGAT, whose deletion resulted in a major defect in the synthesis of triacylglycerols but had little or no effect on the biosynthesis of ester and ether phospholipids, suggesting that G3P is not the primary lipid precursor for membrane biogenesis in Leishmania promastigotes (27). In an attempt to evaluate the importance of dihydroxyacetone phosphate in parasite lipid metabolism and development, we have isolated and characterized the gene LmDAT encoding the DHAPAT enzyme involved in the acylation of this precursor. We provide evidence that LmDAT is the only dihydroxyacetone phosphate acyltransferase of the parasite, residing in peroxisome-like organelles termed glycosomes in Leishmania and related parasites, important for optimal growth during the exponential phase and for survival at high cell density and essential for virulence. These data suggest that acylation of DHAP may represent the major pathway for the synthesis of glycerolipids in Leishmania.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Conditions—Promastigotes of Leishmania major Friedlin V1 strain (MHOM/IL/80/Friedlin) were maintained in liquid and semi-solid M199-derived medium (28). Transfection was performed according to Ngo et al. (29), and selection was applied as appropriate in the presence of G418, nourseothricin, blasticidin, or puromycin (10, 150, 5, and 50 µg/ml, respectively). To examine parasite proliferation, parasites were collected at mid-log phase, diluted to 5 x 105 parasites/ml, and incubated at 26 °C. Growth was monitored by collecting samples over time and counting them using a hemacytometer. Limiting dilution assays were carried out by diluting parasite cultures with fresh media to 5 parasites/ml. 200-µl samples of this diluted culture were transferred into a 96-well plate. The plates were incubated for 2 weeks at 26 °C, and the presence of parasites was monitored by light microscopy.

Saccharomyces cerevisiae strains used in this study are listed in Table 1. Yeast was cultivated at 30 °C in YPD (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose), YPGal (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% galactose), or synthetic minimal media supplemented as required to maintain cell growth. Limiting dilutions on solid YPD and YPGal plates were performed as described previously (27).


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  		TABLE 1
Yeast strains used in this work

 
Plasmids—LmDAT was PCR-amplified from L. major genomic DNA using the primer pair BN-LmDHAPAT-5' ('5-CGGGATCCCATATGAGCTTCCCACCACCTCGG-3') and LmDHAPAT-B-3' ('5-CGGGATCCTCACATCTTGGATGGCTGTGTT-3'). The resulting PCR product was digested with BamHI and ligated to the BamHI site of pUC19 (New England Biolabs) in sense orientation to yield pUC19.LmDAT (Ec207). pBEVY-L.LmDAT (Ec210) was obtained by subcloning the 4.3-kilobase BamHI (partial)-XbaI fragment of pUC19.LmDAT into the BamHI and XbaI sites of pBEVY-L (30). The plasmid pL-BSD (Ec221) was constructed as follows; the BSD cassette was PCR-amplified with the primers 5-XbaI-BSD (5'-GCTCTAGATGCCTTTGTCTCAAGAAGAATC-3') and 3-XbaI-BSD (5'-GCTCTAGATTAGCCCTCCCACACATAAC-3') and pTEF1/BSD (Invitrogen) as template, digested with XbaI, and ligated into the SpeI sites of pXG.SAT (28) in sense orientation. To obtain the pL-BSD.HV-LmDAT (Ec247) plasmid, pL-BSD.LmDAT (Ec237) was first constructed by subcloning the 4.3 kilobases BamHI fragment of pUC19.LmDAT into the BamHI site of pL-BSD with the transcription orientation of the LmDAT gene similar to the BSD marker. Then a PCR reaction using the oligonucleotides B-His6-V5-LmDAT (5'-CGGGATCCATGCATCATCACCATCACCATGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGATGAGCTTC-CCACCACCTC-3') and LmDHAPAT-424r (5'-AGTTCGTCTCGCCAAGTG-3') and pUC19.LmDAT as a template was performed, and the resulting PCR product was digested with BamHI and EcoRI. The resulting 0.4-kilobase fragment was used to replace the corresponding non-tagged coding DNA region of pL-BSD.LmDAT digested with BamHI and EcoRI.

To delete the chromosomal LmDAT gene, the following plasmids were generated. First, the 5'-untranslated region of LmDAT was amplified with LmDAT-1778-B ('5-GGGGATCCAATGCATGTGTGC-3') and LmDAT-752-E ('5-CAGAATTCGAAGAGGGAGCTAAAGG-3'). The resulting PCR products were digested with BamHI and EcoRI and cloned into the corresponding sites of pBluescript II KS (+/-) (Stratagene), yielding pBS-5'U-LmDAT (Ec216). The 3'-untranslated region of LmDAT was amplified with LmDAT+1-S ('5-GAGGAGCTCGTGTAGACCTGTGTC-3') and LmDAT+801-K ('5-CGAGGTACCGCGAAGACATCAATATTG-3') followed by TA cloning into pCR2.1 (Invitrogen), resulting in pCR-3'U-LmDAT (Ec219). The 3'-untranslated region was excised from pCR-3'U-LmDAT after digestion with EcoRI and KpnI and ligated to the EcoRI and KpnI sites of pBS-5'U-LmDAT to give pBS.5'3'U-LmDAT (Ec220). The nourseothricin (SAT) and puromycin (PAC) cassettes were isolated from pXG.SAT (28) and pX63PAC (31), respectively, by digestion with EcoRI and SacI and ligated to the EcoRI and SacI sites of pBS.5'3'U-LmDAT yielding pBS.LmDAT:SAT (Ec231) and pBS.LmDAT:PAC (Ec225), respectively. All PCR-generated products were verified by sequencing.

Yeast Complementation—Cmy228 (gat1{Delta}gat2{Delta}+[pGAL1::GAT1 URA3] (32)) was transformed with pBEVY-L.LmDAT, yielding cmy228+[ADH1::LmDAT] (gat1{Delta}gat2{Delta}+[pGAL1::GAT1 URA3]+[p-BEVY-L.LmDAT]). Contra-selection to discard the (pGAL1::GAT1 URA3) episome was achieved in the presence of 5-fluoroorotate, yielding gat1{Delta}gat2{Delta}+[ADH1::LmDAT]. The presence of GAT1 was verified by PCR using the oligonucleotides ScSCT2–749 ('5-ATACGAAGGGCTGTGTAGG-3') and ScSCT2–1697 ('5-TCAACACCGATTTCACCG-3'). The presence of LmDAT was verified using the primer pair LmDAT-3531 ('5-GTTCGGCTTCGTCACAAC-3') and LmDHAPAT-B-3' ('5-CGGGATCCTCACATCTTGGATGGCTGTGTT-3'). The HNM1 gene was PCR-amplified using the primer pair OCHO47 ('5-CATTGCTTGTCACACTTGG-3') and OCHO78 ('5-CCGCTCGAGCTGCAGTCACTTCTTTCCCCACGGTAC-3').

Activity Assays—The yeast strain gat1{Delta}gat2{Delta}+[ADH1::LmDAT] was grown in YPD to an A600 of 0.8–1.0. Cells were washed twice in ice-cold water, and cell pellets were frozen at -80 °C until use. Yeast and Leishmania protein extracts were prepared as described previously (27, 33). Protein concentration was determined by the bicinchoninic acid assay using bovine serum albumin as a standard. Glycerol-3-phosphate acyltransferase (GPAT) activity was measured as previously described (27) using 100–200 µg of protein. DHAPAT activity was assessed by measuring the acylation rate of DHAP. This substrate was synthesized from fructose-1,6-bisphosphate as described by Bates and Saggerson (34). The assay was performed in a 0.5-ml final volume in a buffer containing 120 mM KCl, 50 mM Tris-HCl (pH 7.5), 4 mM MgCl2, 8 mM NaF, 4 mg/ml fatty acid poor albumin, 100 µM acyl-CoA, 0.25 unit of aldolase, 7 units of triose phosphate isomerase, 0.1% CHAPS, and 0.5–1 mg of protein. Assays were performed at 30 °C unless otherwise stated. For the pH-dependent DHAPAT assay, a 100 mM sodium phosphate buffer set up at different pH values was used. The specificity of L-[U-14C]glycerol 3-phosphate (Amersham Biosciences) and D-[fructose-U-14C] 1,6-bisphosphate (MP Biomedicals) were 149 and 295 mCi/mmol, respectively. DHAPAT and GPAT reaction products were verified by analytical thin layer chromatography using Silica Gel 60A plates (Whatman) and a solvent made of chloroform, methanol, acetic acid, 5% aqueous sodium metabisulfite (75:30:9:3, v/v/v/v). The plates were sprayed with EN3HANCE (PerkinElmer Life Sciences) before autoradiography. The Rf values for 1-acyl-G3P and 1-acyl-DHAP were 2.1 and 3.5, respectively.

Malate dehydrogenase assay was performed in 0.1 M potassium phosphate buffer, pH 7.4, containing 0.21 mM oxaloacetate and 0.26 mM NADH. The assay was initiated by the addition of 20 µg of Leishmania protein extracts, and the decrease in absorbance was monitored spectrophotometrically at a wavelength of 340 nm.

Microscopy—Differential interference contrast (DIC) analysis was performed using promastigotes fixed with 4% paraformaldehyde in phosphate-buffered saline and mounted on polylysine-coated slides. Immunofluorescence assay was performed with wild-type parasites alone or transformed with pL-BSD.HV-LmDAT (Ec247) as described previously (28). The recombinant His6-V5 tagged HV-LmDAT was revealed with V5 monoclonal antibodies (Invitrogen) and hypoxanthine-guanine phosphoribosyltransferase with specific rabbit polyclonal immunoglobulins (generous gifts of A. Jardim). Both antibodies were used at a 1:500 dilution. Images were taken with a Nikon fluorescence microscope.

Analysis of Cell Death—Parasites were washed once in phosphate-buffered saline and stained in the presence of 1 µg/ml propidium iodide (PrI) in phosphate-buffered saline for 10 min before being analyzed by flow cytometry.

Virulence Assays in Mice—Virulence assays were performed as described in Zufferey et al. (28). Briefly, parasites derived from a 3-day stationary culture were washed once in cold saline buffer. 1 x 106 or 1 x 107 cells were injected subcutaneously in the right rear footpad of 6–8-week-old female BALB/c mice (Charles River Laboratory). Infections were monitored by comparing the thickness of the footpad over time using a Vernier caliper.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the Dihydroxyacetone Phosphate Acyltransferase Activity in L. major Promastigotes—Our genetic and biochemical characterization of the L. major GPAT enzyme LmGAT suggested that the lipid precursor G3P does not play an essential function in parasite development or biosynthesis of its major acyl- and alkylglycerolipids. To examine the importance of the second lipid precursor DHAP in parasite membrane biogenesis and development, we characterized its first acylation step (DHAPAT activity) in whole cell extracts. DHAPAT activity was linear during ~8 min of incubation and reached a plateau thereafter when measured at 30 °C (Fig. 1A). When the assay was performed at 0 °C, only marginal activity could be detected, indicating that this reaction is enzymatic. Kinetic studies were conducted by varying the concentration of fructose 1,6-bisphosphate to obtain a substrate saturation curve. DHAPAT activity reached its maximum when the substrate concentration was ~2 mM (Fig. 1B). From these data, a Vmax of 208.8 ± 33.6 pmol/min x mg and a Km of 0.47 ± 0.1 mM could be calculated, indicating that DHAPAT activity in Leishmania involves a relatively low affinity DHAPAT enzyme(s). The specificity of DHAPAT was assessed by testing different fatty acyl-CoA donors that differ in length and/or saturation. The best activities were obtained when palmitoyl-CoA was present in the assay, whereas myristoyl-CoA, stearoyl-CoA, and linoleoyl-CoA all resulted in lower activities. The least effective fatty acyl-CoA donors were dodecyl-, palmitoleoyl-, oleoyl-, and arachidoyl-CoA (Fig. 1C).


Figure 1
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  		FIGURE 1.
DHAPAT activity in L. major promastigotes. The acylation rate of DHAP produced by catabolism of fructose 1,6-bisphosphate by an aldolase and triose phosphate isomerase was measured as described under "Experimental Procedures." A, time-dependent acylation of DHAP in Leishmania whole cell extracts carried out at 0 or 30 °C (open and closed circles, respectively) in the presence of 100 µM stearoyl-CoA and 1 mg of protein as described under "Experimental Procedures". B, kinetics of DHAPAT activity in Leishmania protein extracts. Substrate saturation curve was fitted to the Michaelis-Menten equation, V = Vmax x S/(Km + S). The Lineweaver-Burk double reciprocal plot is shown in the inset. Km and Vmax were calculated to be 0.47 ± 0.1 mM and 208.8 ± 33.6 pmol/min x mg. C, substrate specificity of DHAPAT activity. The reactions were incubated for 5 min at 30 °C in the presence of 100 µM fatty acyl-CoA. All assays were performed at least twice in duplicate, and only one representative experiment is shown here. S.D. are depicted.

 
Identification of LmDAT—To isolate the gene encoding the L. major DHAPAT activity, we searched the L. major genome for proteins harboring the four signature motifs important for acyltransferase activity (35). In addition to the LmGAT gene, we identified an open reading frame of ~4.3 kilobases encoding a protein of 1436 amino acids, which we termed LmDAT (for dihydroxyacetone phosphate acyltranferase) that exhibits significant similarity (~35% identity and ~55% similarity) to other eukaryotic DHAPATs (Fig. 2B). The most conserved region in the enzyme encompasses amino acids 680–975 and harbors the four canonical domains involved in substrate recognition and catalysis (Fig. 2B (35, 36)). Interestingly, unlike human and other higher eukaryotic DHAPATs, LmDAT contains an unusual N-terminal domain of ~650 amino acids. Homologs of LmDAT in the Trypanosomatidae family such as Trypanosoma cruzi and Trypanosoma brucei also harbor this N-terminal domain and share a high degree of identity and similarity with LmDAT over the whole protein length. A specific homology search of available protein databases with this N-terminal domain failed to identify any similarity to known proteins. The C-terminal domain of LmDAT contains a signature tripeptide SKM for localization to the glycosomes (37).


Figure 2
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  		FIGURE 2.
DHAPAT sequence alignment. A, schematic representation of DHAPAT orthologs from T. cruzi, T. brucei, human, rat (Rattus norvegicus), Caenorhabditis elegans, and fruit fly (Drosophila melanogaster). The domain containing the four conserved blocks diagnostic of G3P and DHAP acyltransferases are depicted as black rectangles. The C-terminal glycosomal/peroxisomal tripeptide is represented by an open rectangle. B, sequence alignment of the catalytic/substrate recognition domain of DHAPATs of L. major, T. cruzi, T. brucei, human, rat, C. elegans, and D. melanogaster. Numbers correspond to the amino acid position of L. major LmDAT protein sequence. Amino acids identical to LmDAT are in bold, and shaded motifs represent the four GPAT/DHAPAT diagnostic blocks.

 
Subcellular Localization of LmDAT—To determine the cellular localization of LmDAT, a hexahistidine-V5 tag was added to the N-terminal portion of the enzyme, and the resulting recombinant protein (HV-LmDAT) was expressed in wild-type Leishmania. Immunofluorescence analysis using a V5-specific monoclonal antibody revealed a signal diagnostic of glycosomal proteins. Indeed colocalization studies revealed a perfect colocalization between HV-LmDAT and the glycosomal marker, hypoxanthine-guanine phosphoribosyltransferase (38) (Fig. 3). No signal could be detected in non-transfected cells using anti-V5 antibody (Fig. 3). These data suggest that LmDAT is localized to the glycosomes (21).

Functional Analysis of LmDAT in Yeast—We have previously shown that the yeast S. cerevisiae can be used as a surrogate system to genetically and biochemically analyze the function of heterologous GPAT enzymes (27, 33). Yeast harbors two acyltransferases, GAT1 and GAT2, which catalyze the acylation of both G3P and DHAP (32, 39). GAT1+gat2{Delta} and gat1{Delta}GAT2+ single deletion mutants are viable, whereas a double mutant, gat1{Delta}gat2{Delta}, is lethal. To genetically assess the function of LmDAT, we examined whether LmDAT can rescue the lethal phenotype of the yeast double mutant gat1{Delta}gat2{Delta} lacking the GAT1 and GAT2 genes (32, 39). To this end, we generated a strain gat1{Delta}gat2{Delta}+[GAL1::GAT1]+[ADH1::LmDAT] that expresses LmDAT under the constitutive ADH1 promotor in the strain cmy228 (gat1{Delta}gat2{Delta}+[pGAL1::GAT1 URA3] (32)). This strain bears chromosomal deletions of GAT1 and GAT2 but expresses episomally the endogenous GAT1 under the control of the inducible GAL1 promotor. A control strain, gat1{Delta}gat2{Delta}+[GAL1::GAT1]+[ADH1], which carries the vector alone, was also generated. Both strains were serially diluted on complete media containing either glucose or galactose as a sole carbon source. As expected, both strains grew on galactose-containing media, which promotes expression of the GAT1 transgene (Fig. 4A). However, whereas the gat1{Delta}gat2{Delta}+[GAL1::GAT1]+ [ADH1::LmDAT] strain was capable of growing on glucose-containing media that represses GAT1 expression, the gat1{Delta}gat2{Delta}+ [GAL1::GAT1] +[ADH1] failed to give colonies on this medium, suggesting that LmDAT complements the loss of the endogenous GAT1 and GAT2 genes. We next selected 5-fluoroorotate-resistant derivatives of the gat1{Delta}gat2{Delta}+[GAL1::GAT1]+[ADH1::LmDAT] strain that lost pGAL1::GAT1 URA3 plasmid. Consequently, a strain gat1{Delta}gat2{Delta}+ [ADH1::LmDAT], which lacks yeast GAT1 and GAT2 genes and relies for survival solely on LmDAT, was successfully obtained, thus eliminating the possibility of complementation due to expression of the endogenous GAT1 gene. The presence of LmDAT or the absence of the episome expressing the GAT1 gene in this strain was verified by PCR analyses using primers specific to GAT1 and LmDAT (Fig. 4B). As a positive control, PCR analyses using primers specific for the choline transporter gene HNM1 showed the presence of this gene in all strains (Fig. 4B). Together, these data demonstrate that LmDAT can functionally complement the loss of the yeast dual DHAPAT/GPAT acyltransferase activity.


Figure 3
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  		FIGURE 3.
LmDAT is localized to the glycosomes. Wild-type cells expressing recombinant HV-LmDAT fusion were analyzed by DIC (panel 1) or immunofluorescence microscopy using anti-V5 monoclonal antibody (panel 2) or hypoxanthine-guanine phosphoribosyltransferase polyclonal antibodies (panel 3). Panel 4, merge of panels 2 and 3. Panels 5 and 6, DIC (panel 5) and immunofluorescence analysis using anti-V5 antibody (panel 6) in wild-type non-transfected control cells.

 


Figure 4
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  		FIGURE 4.
LmDAT complements the lethal phenotype of a yeast double mutant lacking GPAT and DHAPAT activity. A, growth assay of gat1{Delta}gat2{Delta}+[GAL1::GAT1]+[ADH1] (1) and gat1{Delta}gat2{Delta}+[GAL1::GAT1]+[ADH1::LmDAT] (2) serially 10-fold diluted on complete medium containing either glucose or galactose as sole carbon source. The highest dilution contains 3 x 104 cells. Plates were incubated at 30 °C for 4 days. B, agarose gel electrophoresis of PCR products obtained by amplifying genomic DNA from gat1{Delta}gat2{Delta}+[GAL1::GAT1] (1), gat1{Delta}gat2{Delta}+[GAL1::GAT1]+[ADH1::LmDAT], (2), and gat1{Delta}gat2{Delta}+[ADH1::LmDAT](3) with GAT1-, LmDAT-, and HNM1-specific (positive control) primers as described under "Experimental Procedures."

 


Figure 5
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  		FIGURE 5.
GPAT and DHAPAT activities of LmDAT expressed in S. cerevisiae. The acylation rate of DHAP produced by catabolism of fructose 1,6-bisphosphate by an aldolase and triose phosphate isomerase measured as described under "Experimental Procedures." A, time-dependent DHAPAT activity from cell extracts of gat1{Delta}gat2{Delta}+[ADH1::LmDAT] grown in YPD. The assay was performed as described under "Experimental Procedures." B, kinetics of DHAPAT activity of LmDAT. The substrate saturation curve was fitted to the Michaelis-Menten equation, and the double reciprocal plot is depicted as an inset. Km and Vmax were calculated to be 0.69 ± 0.06 mM and 2.39 ± 0.36 nmol/min x mg. C, fatty acyl-CoA donor specificity of LmDAT. Samples were incubated at 30 °C for 5 min in the presence of 100 mM fatty acyl-CoA. D, DHAPAT activity of LmDAT represents only 8.5±% of GPAT activity. GPAT activity of LmDAT performed in the presence of 100 mM palmitoyl-CoA during 5 min. E, LmDAT activity is pH-dependent. All assays were performed at least twice, and only one representative experiment is depicted. The S.D. is shown.

 


Figure 6
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  		FIGURE 6.
Generation of an LmDAT null mutant of L. major. A, genomic organization of the LmDAT locus in the wild-type and mutant versions of the allele. The probe (thick black line) and scale are depicted. B, Southern blot analysis of genomic DNA digested with BamHI and SalI. Lane 1, wild type; lane 2, the heterozygote mutant (LmDAT: PAC/LmDAT) with PAC-replaced allele; lane 3, homozygote mutant (LmDAT:PAC/LmDAT:SAT) with both LmDAT alleles replaced with PAC and SAT cassettes, respectively. The size and identity of the bands are shown. The probe used is depicted in A.

 
We next characterized the activity of recombinant LmDAT in yeast. LmDAT DHAPAT activity measured in the complemented cell extracts at 30 °C was linear for ~6 min before reaching a plateau. In contrast, only minimal activity could be detected when the assay was carried out at 0 °C (Fig. 5A). The DHAP concentration in the reaction mixture was varied to obtain a substrate saturation curve. DHAPAT activity was optimal at a substrate concentration of ~2 mM, and from the Michaelis-Menten representation, a Km of 0.69 ± 0.06 mM and a Vmax of 2.39 ± 0.36 nmol/min x mg were obtained (Fig. 5B). The specificity of the fatty acyl-CoA donor was then determined by testing different chain lengths and saturated/non-saturated fatty acyl-CoA donors. LmDAT showed the best activity with palmitoyl-CoA. Lower activity was obtained with lauroyl-, myristoyl-, palmitoleoyl-, stearoyl-, linoleoyl-, oleoyl-, and arachidoyl-CoA (Fig. 5C). These results are consistent with the fatty acid composition of Leishmania membranes where C16, C18 fatty acids prevail (3, 28) and with the activity of the endogenous enzyme. To assess whether LmDAT can also acylate G3P, GPAT assays were performed. Although GPAT activity could be detected in LmDAT-expressing cells, this activity represented only ~8.5% of its DHAPAT activity (Fig. 5D). LmDAT DHAPAT activity was optimal at a neutral pH, decreased by 30% at pH 9, and was almost absent at pH 5.5 (Fig. 5E).


Figure 7
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  		FIGURE 7.
{Delta}lmdat/{Delta}lmdat null mutant lacks DHAPAT activity. DHAPAT (A), GPAT (B), and malate dehydrogenase (C) activities of wild type (1), null mutant {Delta}lmdat/{Delta}lmdat (2), and complemented (3) strains were assayed as described under "Experimental Procedures." For DHAPAT and GPAT assays, samples were incubated at 30 °C for 5 min in the presence of 100 µM palmitoyl-CoA. The three assays were carried out twice in duplicate, and one representative experiment is depicted. S.D. are shown.

 
L. major Lacking LmDAT Gene Are Deficient in DHAPAT Activity—To assess the role of LmDAT in parasite physiology, a null mutant was generated. Two rounds of transformation and selection were performed to obtain the homozygous knock-out mutant. The first and second alleles were replaced by a puromycin (PAC) and a nourseothricin (SAT) cassette, respectively. A line, {Delta}lmdat/{Delta}lmdat, carrying both mutations was obtained, and the loss of the endogenous LmDAT alleles was verified by Southern blot analysis (Fig. 6). This result demonstrates that LmDAT is not essential for parasite viability. The DHAPAT activity of the {Delta}lmdat/{Delta}lmdat null mutant was compared with its isogenic parental strain (wild type) and to a {Delta}lmdat/{Delta}lmdat+[LmDAT BSD] complemented strain, carrying a wild-type LmDAT gene on an episome in the {Delta}lmdat/{Delta}lmdat knock-out background. Although the wild-type and complemented lines exhibited comparable DHAPAT activity levels, the mutant completely failed to acylate DHAP (Fig. 7A). This result suggests that LmDAT is the sole DHAPAT enzyme in L. major promastigotes. To assess whether the loss of DHAPAT activity in the {Delta}lmdat/{Delta}lmdat mutant could be compensated by an increase in the cellular GPAT activity catalyzed by the LmGAT enzyme, GPAT activity of the {Delta}lmdat/ {Delta}lmdat was compared with that of the wild-type and complemented strains. No differences in GPAT activity could be detected between the three strains (Fig. 7B). As a control, similar levels of the malate dehydrogenase activity that is not related to lipid metabolism were found in the three strains (Fig. 7C).

L. major Lacking LmDAT Gene Have Altered Growth during Exponential Phase and Increased Cell Death during Stationary Phase—Growth assays revealed that the null mutant {Delta}lmdat/{Delta}lmdat, although viable, grew at a ~45–50% reduced growth rate when compared with wild-type cells (Fig. 8A; Table 2). This growth defect could be compensated by the addition of the LmDAT gene. The doubling time of biomass accumulation was 14.2 ± 2.5 h for {Delta}lmdat/{Delta}lmdat mutant cells versus 7.6 ± 0.5 h for wild-type and 7.9 ± 0.7 h for complemented cells (Fig. 8A; Table 2). Furthermore, the {Delta}lmdat/{Delta}lmdat mutant reached only half the maximal cell density compared with that of the wild type and the complemented strains (~3 x 107 versus ~6 x 107cells/ml; Fig. 8A). These results demonstrate that LmDAT plays an important role in parasite development during the linear phase of cellular division. Interestingly, whereas the cell number of wild-type and complemented strains remained the same several days after they reached their maximum cellular density, the cell number of the {Delta}lmdat/{Delta}lmdat mutant dramatically decreased soon after reaching its maximum cellular density (Fig. 8A). Microscopic analysis revealed a major alteration in the morphology of the {Delta}lmdat/{Delta}lmdat cells with an increase in the number of unflagellated, rounded, and lysed cells in the culture (Fig. 8C). This defect was not observed during the linear phase of growth of the {Delta}lmdat/{Delta}lmdat mutant (data not shown). In contrast, the morphology of wild-type and complemented cells was unaltered during exponential and stationary phases of growth (Fig. 8C).


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  		TABLE 2
Leishmania doubling time (T) in hours

 


Figure 8
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  		FIGURE 8.
Growth defects of the {Delta}lmdat/{Delta}lmdat mutant. A, growth assay. Wild type (black circles), {Delta}lmdat/{Delta}lmdat (open circles), and {Delta}lmdat/{Delta}lmdat+[LmDAT BSD](gray circles) were seeded at 5 x 105/ml and enumerated as a function of time with a hemacytometer. B, limiting dilution assays. Wild type (1), {Delta}lmdat/{Delta}lmdat (2), and {Delta}lmdat/ {Delta}lmdat+[LmDAT BSD](3) strains were diluted to 5 parasites/ml 3 days after they reached maximum cell density and transferred into 96-well plates, and parasite growth was monitored 2 weeks later. The number of parasite-containing wells is represented in percentages. These assays were performed at least twice in duplicate, and a representative experiment with S.D. is represented. C, DIC images were taken from cells 3 days after they reached their maximum cell density.

 
We further analyzed cell survival during the stationary phase by plating wild type, knock-out, and complemented strains on fresh media and counting the number of resulting colonies. The null mutant {Delta}lmdat/ {Delta}lmdat gave ~70% decreased colony number when compared with wild-type and complemented cells (Fig. 8B). These results demonstrate that LmDAT is essential for cell viability during the stationary phase. Parasite death after maximum growth was further demonstrated using PrI to stain necrotic cells followed by quantification of PrI-positive cells by flow cytometry. Wild-type, knock-out, and complemented cells did not take up PrI during the logarithmic phase of development and replication (Fig. 9A). Upon entry into stationary phase, however, 14% of {Delta}lmdat/{Delta}lmdat cells were permeable to this dye, whereas only ~1–2% of wild-type and complemented cells were PrI-positive (Fig. 9B).

L. major Lacking LmDAT Gene Are Avirulent—To examine the role of LmDAT in L. major virulence, wild-type, null mutant, and complemented strains were examined for their ability to form a footpad lesion using a mouse model of cutaneous leishmaniasis. Whereas wild-type and complemented strains formed a lesion within 4 weeks after injection with 106 parasites, no lesions could be detected in mice injected with the null mutant 11 weeks post-injection with 106 or 107 parasites (Fig. 10).


Figure 9
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  		FIGURE 9.
Flow cytometry of PrI stained Leishmania parasites. End-log phase (A) or 3-day stationary phase (B) promastigotes were incubated in the presence of PrI as described under "Experimental Procedures." Then ~5,000 cells were analyzed by flow cytometry. 1, wild type; 2, {Delta}lmdat/{Delta}lmdat; 3, {Delta}lmdat/{Delta}lmdat+[LmDAT BSD].

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The characterization of the enzymatic machinery involved in the initiation of glycerolipid biosynthesis in Leishmania is essential for understanding how the parasite synthesizes its virulence factors and develops and multiplies to cause the pathological symptoms associated with human leishmaniasis. Two reactions initiate the glycerolipid biosynthetic pathway, acylation of G3P by a GPAT enzyme or of DHAP by a DHAPAT. We have previously shown that in Leishmania, the GPAT enzyme LmGAT is not essential for promastigote development and survival and that mutants lacking this gene were affected in the synthesis of triglycerides but had unaltered synthesis of the major ester and ether phospholipids and were as virulent as wild-type cells in a mouse model of cutaneous leishmaniasis (27). To understand the importance of the alternative pathway (acylation of DHAP) in parasite physiology, we have isolated the gene LmDAT encoding the DHAPAT protein, characterized its enzymatic properties, and generated a null mutant.

LmDAT and its orthologs from T. brucei and T. cruzi are unusually large enzymes harboring, in addition to their C-terminal DHAPAT domain, a large N-terminal domain of 650 amino acid residues. This domain does not share homology with any known proteins in the available databases. The presence of an unusual polypeptide extension has also been found in the subunit II of RNA polymerase I of T. brucei and L. major, which harbor an N-terminal 250 amino acid extension (40, 41), and the 3-mercaptopyruvate sulfur-transferase enzymes of L. major and Leishmania mexicana, which contain 70 additional amino acids at their C terminus (42). So far no function could be assigned to these domains.


Figure 10
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  		FIGURE 10.
The {Delta}lmdat/{Delta}lmdat mutant is avirulent. Groups of four mice were injected with 106 wild type (black circles), 106 complemented line {Delta}lmdat/{Delta}lmdat+[LmDAT BSD] (gray circles) or either 106 (open circles) or 107 (open triangles) {Delta}lmdat/{Delta}lmdat mutant parasites as described under "Experimental Procedures," and lesion formation was followed. The thickness of the footpad was measured with a vernier caliper.

 
Our results showed that LmDAT complements the lethal phenotype of the yeast double mutant gat1{Delta}gat2{Delta}. Although yeast cells lack ether phospholipids, their GAT1 and GAT2 enzymes are capable of acylating both G3P and DHAP (32, 39). The 1-acyl-DHAP formed from DHAP is reduced by an NADPH-dependent reductase to 1-acyl-G3P, which is subsequently used for the synthesis of ester phospholipids (43, 44). Because of the strong DHAPAT activity of LmDAT, its complementation of the gat1{Delta}gat2{Delta} mutant could most likely occur through the synthesis of 1-acyl-DHAP. However, we cannot exclude that the minor GPAT activity of LmDAT might also contribute to this complementation. Biochemical analysis revealed that similar to mammalian DHAPATs, LmDAT exhibits a low affinity for DHAP with a Km of ~0.7 mM and shows the best activity in the presence of palmitoyl-CoA (15, 18, 45, 46). The latter result contrasts with LmGAT, that was more promiscuous in term of fatty acyl-CoA donor, preferring longer unsaturated fatty-acyl-CoAs (27).

Our studies provide several lines of evidence supporting the idea that LmDAT is a single DHAPAT enzyme in L. major. First, the biochemical properties of recombinant LmDAT are identical to those measured from Leishmania promastigote extracts. Second, the null mutant {Delta}lmdat/{Delta}lmdat lacking LmDAT gene is deficient in DHAPAT activity. Third, low stringency hybridization analyses revealed that LmDAT is a single copy gene in the L. major genome (data not shown). Fourth, no other DHAPAT homologs could be identified in the available L. major sequence data base.

Mammalian cells harbor one GPAT in the endoplasmic reticulum, two in the mitochondria, and a DHAPAT in peroxisomes, whereas plants lack DHAPAT but have a battery of GPAT isoforms localized in different subcellular compartments such as chloroplast, cytoplasmic membranes, and mitochondria and expressed in a tissue-specific manner (Refs. 47 and 48; for review, see Refs. 49–51). On the other hand, yeast expresses two unique enzymes, GAT1 and GAT2, that acylate both G3P and DHAP with similar efficiency (32, 39). Interestingly, Leishmania represents an unusual situation, harboring a single GPAT enzyme encoded by LmGAT and a single DHAPAT, encoded by LmDAT (Ref. 27 and this work).

Previous studies showed that deletion of the LmADS gene of L. major, which encodes an alkyl dihydroxyacetone phosphate synthase (ADS), results in severe alteration in the synthesis of ether lipids but had no effect on the synthesis of ester phospholipids or promastigote growth (28). Furthermore, we demonstrated that a {Delta}lmgat/{Delta}lmgat mutant lacking GPAT activity, while affected in the synthesis of triglycerides, was unaltered in the synthesis of ester phospholipids and showed normal growth in logarithmic and stationary phases (27). Thus, the slow growth phenotype of the {Delta}lmdat/{Delta}lmdat mutant and its accelerated death in stationary phase are unlikely to be due to loss of synthesis of ether lipids or reduced synthesis of triglycerides. Therefore, we propose that the acylation of DHAP by LmDAT may represent the primary route for the synthesis of ester phospholipids in this parasite and that abrogation of this metabolic pathway is responsible for the slow growth phenotype. Alternatively, because glycolysis in Leishmania and related protozoa occurs within the glycosomes (52), it is possible that deletion of LmDAT may lead to increased levels of DHAP within these organelles. Accumulation of this precursor has previously been shown to be deleterious to Leishmania (53). Studies to evaluate these two hypotheses are warranted.

The slow growth phenotype and accelerated death after entry into the stationary cell stage of the {Delta}lmdat/{Delta}lmdat strain are reminiscent of a Leishmania mutant devoid of sphingolipids (54). Similar to fungi, Leishmania contains mainly inositol phosphoceramide instead of sphingo-myelin or glycosphingolipid in its membranes. Deletion of LmDAT may impair inositol phosphoceramide biosynthesis indirectly because of a lack of the precursor phosphatidylinositol.

A significant finding reported in this paper is the attenuated virulence phenotype of L. major resulting from the deficiency of LmDAT. Although the exact mechanism by which LmDAT promotes virulence remains to be elucidated, the {Delta}lmdat/{Delta}lmdat mutant might be considered as a vaccine candidate.

The phenotype of the {Delta}lmdat/{Delta}lmdat mutant is more severe than that of the {Delta}ads/{Delta}ads mutant, which lacks ether lipids, or that of the {Delta}lpg1/{Delta}lpg1 mutant, which lacks lipophosphoglycan (28, 55). This difference is not surprising because LmDAT-deficient parasites die rapidly upon entry into the stationary cell stage. Cessation of growth at high cell density has been shown to trigger differentiation to metacyclics (56). Thus, very likely the {Delta}lmdat/{Delta}lmdat mutant does not differentiate into the virulent metacyclic form of the parasite, which is pre-adapted for survival in the vertebrate host. Also, because L. major harbors ether-lipid-based virulence factors such as lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins, we surmise that their biosynthesis might be affected in this strain and, thus, contribute to the attenuation of virulence.


   FOOTNOTES
 
* This project was supported by grants from the National Institutes of Health and Department of Defense (to C. B. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Dept. of Biochemistry, Kansas State University, 104 Willard Hall, Manhattan, KS 66506. Back

2 To whom correspondence should be addressed: Dept. of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030-3301. Tel.: 860-679-3544; Fax: 860-679-8345; E-mail: Choukri{at}up.uchc.edu.

3 The abbreviations used are: DHAP, dihydroxyacetone phosphate; DHAPAT, DHAP acyltransferase; G3P, glycerol 3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; PrI, propidium iodide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PrI, propidium iodide. Back


   ACKNOWLEDGMENTS
 
We thank A. Jardim for the generous gift of hypoxanthine-guanine phosphoribosyltransferase antiserum. S. M. Beverley is greatly appreciated for the plasmids pXG.SAT and pX63.PAC. We thank Dr. Asis Das, University of Connecticut Health Center and the anonymous reviewers for constructive comments on the manuscript.



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