PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of plosonePLoS OneView this ArticleSubmit to PLoSGet E-mail AlertsContact UsPublic Library of Science (PLoS)
 
PLoS One. 2017; 12(7): e0181432.
Published online 2017 July 17. doi:  10.1371/journal.pone.0181432
PMCID: PMC5513551

The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids

Rachel Zufferey, Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing,1,2,* Karim Pirani, Investigation, Methodology, Writing – review & editing,1 Melanie Cheung-See-Kit, Investigation, Methodology, Writing – review & editing,2 Sungsu Lee, Investigation, Methodology, Writing – review & editing,2 Tyler A. Williams, Investigation, Methodology, Writing – review & editing,2 Daniel G. Chen, Investigation, Methodology, Writing – review & editing,2 and Md. Faruk Hossain, Writing – review & editing2
Pedro L. Oliveira, Editor

Abstract

Glycerophospholipids are the most abundant constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans and nagana in cattle. They are essential cellular components that fulfill various important functions beyond their structural role in biological membranes such as in signal transduction, regulation of membrane trafficking or control of cell cycle progression. Our previous studies have established that the glycerol-3-phosphate acyltransferase TbGAT is dispensable for growth, viability, and ester lipid biosynthesis suggesting the existence of another initial acyltransferase(s). This work presents the characterization of the alternative, dihydroxyacetonephosphate acyltransferase TbDAT, which acylates primarily dihydroxyacetonephosphate and prefers palmitoyl-CoA as an acyl-CoA donor. TbDAT restores the viability of a yeast double null mutant that lacks glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferase activities. A conditional null mutant of TbDAT in T. brucei procyclic form was created and characterized. TbDAT was important for survival during stationary phase and synthesis of ether lipids. In contrast, TbDAT was dispensable for normal growth. Our results show that in T. brucei procyclic forms i) TbDAT but not TbGAT is the physiologically relevant initial acyltransferase and ii) ether lipid precursors are primarily made by TbDAT.

Introduction

Trypanosoma brucei is a protozoan parasite of the Trypanosomatidae family, which is responsible for important diseases termed sleeping sickness in humans and nagana in domestic animals in Africa. This microorganism alternates between two hosts during its complex digenetic life cycle, the tsetse fly insect vector and the bloodstream of a vertebrate animal.

T. brucei membranes are made predominantly of glycerophospholipids, which represent 80% of total cellular lipids [1, 2]. The main function of glycerophospholipids is structural by forming the bilayer of the biological membrane. In addition, they fulfill various essential functions in cell cycle progression or in shaping the morphology of organelles such as mitochondria or endoplasmic reticulum; they also regulate signal transduction pathways as second messengers and control membrane trafficking [311]. T. brucei membranes contain the typical phospholipids found in most eukaryotic cells, phosphatidylcholine being the most abundant (PC, 45–60%) followed by phosphatidylethanolamine (PE, 10–20%), phosphatidylinositol (PI, 6–12%), phosphatidylserine (PS, <4%), and cardiolipin (<3%) [2, 10, 11].

Ether glycerophospholipids are a subset of glycerophospholipids that are characterized by the presence of a fatty alcohol instead of a fatty acid at the sn-1 position of the glycerol backbone. In T. brucei, they are primarily found in PE and PS classes [1, 2]. Ether lipids have been shown to be important for the integrity of the endoplasmic reticulum and Golgi apparatus, endocytosis, cholesterol trafficking in mammalian cell lines [12]. While cancer cells overproduce ether lipids for pro-survival purposes, Alzheimer’s disease has been associated with a decline in ether lipid production [13]. Ether glycerophospholipids are also essential for the myelination of nerve cells, development of the brain, eye lens, testes, and for spermatogenesis in mice [14, 15].

The biosynthesis of ether glycerophospholipids initiates with the acylation of dihydroxyacetonephosphate (DHAP) by a DHAP acyltransferase (DHAPAT). The acyl group is then exchanged with a fatty alcohol moiety by the subsequent action of an alkyl-DHAP synthase to yield alkyl-DHAP. The reduction of the latter to alkyl-glycerol-3-phosphate (alkyl-G3P) is executed by an acyl/alkyl-DHAP reductase. All these enzymes are associated with the peroxisomes. The DHAPAT can also participate in the production of ester glycerophospholipids in some organisms [1618]. In that case, 1-acyl-DHAP is directly converted into 1-acyl-G3P by an acyl/alkyl-DHAP reductase.

The DHAPAT enzyme was purified and characterized originally from mammals (human, rat, guinea pig) as a luminal, membrane associated, peroxisomal enzyme with specificity for DHAP [19]. It interacts directly with the alkyl-DHAP synthase with which it forms a multimeric complex [20]. Mutations in the encoding gene have been associated with a human disease called rhizomelic chondrodysplasia punctate, which is characterized by mental retardation, short stature, and dysmorphic facial appearance [2123].

In the related parasite Leishmania major, the two enzymes LmGAT and LmDAT are the only initial acyltransferases [18, 24, 25]. LmGAT specifically acylates G3P, contributing to ester glycerophospholipid biosynthesis only, and is dispensable for parasite growth and pathogenesis [18]. In contrast, LmDAT preferentially adds acyl groups to DHAP, participates in ester as well as ether glycerophospholipid biosynthesis, which is important for normal growth, survival during stationary phase and virulence [25].

In T. brucei, DHAPAT, alkyl-DHAP synthase and alkyl/acyl-DHAP reductase activities were found to be associated with peroxisome-like organelles, termed glycosomes in trypanosomatidae [2629]. Only the gene for the alkyl-DHAP synthase has been identified in this parasite [30]. Furthermore, the G3P acyltransferase (GPAT) TbGAT, which initiates the ester glycerophospholipid biosynthesis is dispensable for viability and glycerophospholipid production, suggesting that T. brucei possesses an additional initial acyltransferase [31]. This work presents the characterization of the alternative initial acyltransferase TbDAT that begins the ether glycerophospholipid biosynthetic pathway in T. brucei.

Results and discussion

Identification of TbDAT

Our previous studies established that the GPAT TbGAT, which initiates the ester glycerophospholipid biosynthesis, is dispensable for viability and glycerophospholipid production, suggesting that T. brucei possesses an additional initial acyltransferase(s) [31]. Furthermore, T. brucei bears a DHAPAT, which may be one of the alternative initial acyltransferase(s), but its molecular identity has not been established [28, 31]. Thus, a DHAPAT ortholog gene was searched in T. brucei genome using L. major LmDAT protein sequence (LmjF.34.1090) as a bait, which lead to the identification of Tb927.4.3160 that was named TbDAT. The latter encodes a protein of 1242 amino acids with a calculated molecular mass of 137 kDa. TbDAT is much larger than the orthologs of higher eukaryotes such as mouse or fly [25]. The N-terminal domain does not show any similarity to any known proteins as the leishmanial ortholog while the C-terminal domain contains the four typical conserved motifs that are likely involved in catalysis and substrate recognition [25].

T. brucei TbDAT suppresses the lethal phenotype of a S. cerevisiae double null mutant lacking endogenous GPAT and DHAPAT activities

S. cerevisiae possesses two initial acyltransferases ScGAT1 and ScGAT2 that have the capability to acylate both DHAP and G3P with similar efficiencies [32, 33]. Single deletion of either gene is not deleterious for life but inactivation of both genes is lethal. Thus, TbDAT was expressed under the control of the constitutive promoter ADH1 in a S. cerevisiae conditional double null mutant scgat1Δscgat2Δ [GAL1:ScGAT1] that synthesizes its endogenous ScGAT1 in the presence of galactose but not in the presence of glucose [32]. Double transformants scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1], scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:TbDAT] and scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:LmDAT] [25] gave colonies on galactose containing medium, while scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1]failed to grow on glucose containing medium as expected (Fig 1A). TbDAT conferred survival of the double null mutant scgat1Δscgat2Δ on glucose similar to the positive control scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:LmDAT] [25]. This assay demonstrates that TbDAT suppresses the lethal phenotype of a S. cerevisiae double null mutant lacking both endogenous initial acyltransferases.

Fig 1
TbDAT complements the lethal phenotype of a yeast double null mutant scgat1Δscgat2Δ.

TbDAT restores normal growth, survival in stationary phase and production of normal migrating lipophosphoglycan of the Leishmania null mutant Δlmdat/Δlmdat

The function of TbDAT was further investigated by expressing it in a L. major null mutant Δlmdat/Δlmdat. This strain exhibits a slow growth phenotype, poor survival during the stationary phase of growth and synthesizes an altered form of the ether lipid-anchored virulence factor lipophosphoglycan that migrates slower in a SDS-polyacrylamide gel [25]. Expression of TbDAT in the null mutant Δlmdat/Δlmdat restored normal growth and survival during the stationary phase of growth (Fig 2A). Furthermore, similar to the complemented strain Δlmdat/Δlmdat [LmDAT NEO], Δlmdat/Δlmdat [TbDAT NEO] produced normal forms of lipophosphoglycan, used here as a reporter for ether lipids while the null mutant produced slower migrating lipophosphoglycan (Fig 2B; [34]). These data suggest that TbDAT fulfills similar functions as LmDAT.

Fig 2
TbDAT complements the slow growth phenotype and restores survival during the stationary phase of growth of the L. major null mutant Δlmdat/Δlmdat.

TbDAT encodes a DHAPAT enzyme that exhibits preference for palmitoyl-CoA

To assess whether TbDAT acts as a DHAPAT enzyme, the gene was expressed in a constitutive fashion in the S. cerevisiae double null mutant scgat1Δscgat2Δ, which lacks GPAT and DHAPAT activities, in the absence of the [GAL1:ScGAT1] episome. GPAT and DHAPAT activity assays were then performed. TbDAT acylated DHAP with very high efficiency but not G3P (Fig 1C), demonstrating that DHAP rather than G3P is the preferred substrate of TbDAT.

The fatty acyl-CoA specificity of TbDAT was further investigated. The preferred acyl-CoA donor was palmitoyl-CoA followed by myristoyl-CoA, palmitoleoyl-CoA and oleoyl-CoA (Fig 1D). Lauryl-CoA, stearoyl-CoA, linoleoyl-CoA and eicosanoyl-CoA were the least effective fatty acyl-CoA donors.

The fatty acyl-CoA specificity of the DHAPAT activity present in T. brucei cell extracts gave a slightly different pattern than that of TbDAT. The best activity was achieved in the presence of oleoyl-CoA and palmitoyl-CoA, followed by myristoyl-CoA, palmitoleoyl-CoA, linoleoyl-CoA, stearoyl-CoA, eicosanoyl-CoA and lastly myristoyl-CoA (Fig 1E). This result suggests that TbDAT is not the only DHAPAT enzyme in this parasite and that another DHAPAT enzyme(s) with different fatty acyl-CoA preference may exist.

TbDAT localizes to the glycosomes

To determine the subcellular localization of TbDAT, the enzyme was tagged with a V5 epitope at its N-terminus. A C-terminal tag was not attempted due to the presence of a putative peroxisomal targeting tripeptide SRM, suggesting that TbDAT resides in the glycosomes, which are peroxisomal related organelles in trypanosomes [36]. Immunofluorescence assay was carried out in the presence of anti-V5 antibodies, which gave a punctated stain within the parasite cell, a signal that overlapped nicely with that of glycosomes revealed with anti-glycosomal specific antibodies (Fig 3; [37]). This assay demonstrates that TbDAT resides in the glycosomes and is consistent with cell fractionation data established previously by Opperdoes and colleagues [26, 28, 29]. In contrast to other eukaryotes, most of the glycolytic enzymes are compartmentalized in the glycosomes in trypanosomatidae. Thus, the substrate DHAP, resulting from the breakdown of fructose 1,6-bisphosphate by a glycosomal aldolase and triose isomerase, is readily available for the TbDAT enzyme and does not need to be imported into the glycosomes [36, 38, 39]. In contrast, acyl-CoAs need to be transported into the glycosomes by unknown transport proteins as they are produced in the mitochondria by a type II fatty acid synthase complex and in the endoplasmic reticulum by an unusual elongase system [11, 4042].

Fig 3
V5:TbDAT localizes to the glycosome.

A conditional null mutant of TbDAT is viable but survives poorly in the stationary phase of growth

To assess the role of TbDAT in parasite’s biology, a tetracycline-dependent conditional null mutant was created by successive transformation and selection in the presence of appropriate antibiotics as described in the Methods’ section. This strategy was chosen as no null mutant could be obtained by traditional successive deletion of the TbDAT alleles and RNA interference was unsuccessful. The conditional null mutant bears replacement of both TbDAT alleles with antibiotic cassettes BSD, that mediates blasticidin resistance, and PAC, that relieves puromycin sensitivity, and expresses an N-terminal V5-tagged TbDAT under the control of a tetracycline TetO regulated promoter (Fig 4B). The genotype of the conditional null mutant Δtbdat/Δtbdat/TetO:V5:TbDAT was verified by PCR using appropriate primers (Fig 4C). Three clones were obtained and showed similar phenotypes. Only one clone is described in details in this work. V5-TbDAT expression is maintained in the presence of doxycycline but it is abolished after 5–6 days in the absence of antibiotic as demonstrated by RT-PCR and Western blot analysis (Fig 4D and 4E). The fact that V5-TbDAT expression took 5–6 days to reach undetectable levels seems to suggest that this enzyme or its mRNA may have a very low turnover or a very long half-life.

Fig 4
Generation of a conditional null mutant of TbDAT in T. brucei.

The importance of TbDAT in cell viability and growth was then investigated. The conditional null mutant Δtbdat/Δtbdat/TetO:V5:TbDAT exhibited similar growth rate in the absence as in the presence of doxycycline (Fig 5A), demonstrating that TbDAT is dispensable for normal growth. This result contrasts with that of L. major null mutant Δlmdat/Δlmdat, which exhibited a twice as long generation time compared to that of the wild type [25]. However, the conditional null mutant reached only half of the wild-type maximal cell density and survived poorly during the stationary phase of growth (Fig 5B and 5C) during which cells become round and necrotic (Fig 5D). The latter phenotype is reminiscent of that of the L. major null mutant Δlmdat/Δlmdat [25].

Fig 5
TbDAT is dispensable for normal growth but is important for survival during the stationary phase.

A conditional null mutant of TbDAT exhibited decreased DHAPAT activity

The DHAPAT activity of T. brucei conditional null mutant of TbDAT was then quantified. This enzymatic activity was present in cells grown in the presence of doxycycline but was diminished in cells grown in its absence by approximately 70% (Fig 6A), indicating that TbDAT is the main DHAPAT and that another enzyme(s) accounts for the remaining activity in procyclic trypanosomes. This result differs from that of L. major, in which LmDAT was the only DHAPAT enzyme in the parasite [18]. In contrast, GPAT activity levels were comparable in cells grown in the presence or absence of doxycycline, consistent with the fact that TbDAT acylates G3P very inefficiently (Fig 6A).

Fig 6
TbDAT is the main DHAPAT enzyme in procyclic trypanosomes.

The fatty acyl-CoA specificity of the remaining DHAPAT activity was then assessed. Best activity was achieved with oleoyl-CoA followed by linoleoyl-CoA, palmitoyl-CoA, myristoyl-CoA, palmitoleoyl-CoA, and stearoyl-CoA. Lauryl-CoA and eicosanoyl-CoA mediated the lowest DHAPAT activity (Fig 6B). This profile is reminiscent of that of the GPAT enzyme TbGAT [31]. Thus, the G3P and DHAP substrate specificity of TbGAT was reinvestigated using Leishmania as a heterologous expression system [31]. Expression of TbGAT in the null mutant Δlmgat/Δlmgat lead to a five-fold increase in GPAT activity but also to a modest but significant increase in DHAPAT activity, suggesting that TbGAT can contribute to the remaining DHAPAT activity in the Δtbdat/Δtbdat/TetO:V5:TbDAT cells grown in the absence of doxycycline (Fig 6C). In addition, TbGAT expression in a Δlmdat/Δlmdat mutant, which lacks DHAPAT activity, conferred slight DHAPAT activity as well (Fig 6D). In terms of substrate specificity, TbGAT resembles S. cerevisiae initial acyltransferases ScGAT1 and ScGAT2 that acylate both G3P and DHAP with similar efficiency [32, 33] and differs from L. major LmGAT, which acylates primarily G3P and not DHAP [18]. However, it is not excluded that an additional DHAPAT enzyme may exist in this parasite beside TbDAT and TbGAT. The creation of a double null mutant of TbDAT and TbGAT will give a definitive answer.

T. brucei was shown to also scavenge and remodel lipid precursors from the medium as an alternative way to synthesize its cellular glycerophospholipids [2, 8, 9, 44]. Thus, the importance of lipid scavenging was addressed by growing the conditional null mutant in delipidated medium (Fig 5E). Removal of doxycycline did not affect the growth rate, demonstrating that scavenging and remodeling of extracellular lipids are dispensable for the production of parasite’s glycerophospholipids in the absence of TbDAT.

TbDAT is essential for ether lipid biosynthesis

The consequence of a lack of TbDAT expression on ester and ether glycerophospholipid cellular composition was then evaluated. Total cellular lipids were isolated and analyzed by comprehensive mass spectrometry. Levels of ether glycerophospholipids PC, PI, PS, and PE were considerably decreased after doxycycline withdrawal (Table 1). In contrast, amounts of ester lipids were similar when TbDAT is expressed or not expressed, except that PI quantities were slightly but significantly increased in cells grown in the absence of doxycycline, maybe to compensate for the lower levels of the corresponding ether glycerophospholipids. Such a mechanism has been observed in mammalian cells defective in ether lipid metabolism and in T. brucei depleted for the ethanolamine-specific phosphotransferase gene TbEPT, which produced increased levels of ester PE to counteract the lower quantities of ether PE [44, 45].

Table 1
Glycerophospholipid quantification.

Our relative percentages of the individual glycerophospholipid species differ slightly from those reported in [46]. This may be explained by the different lipid extraction protocols applied to isolate total lipids (Folch based versus Bligh and Dyer) or the use of a different type of instrumentation. Lastly, the medium composition (especially FBS content) may account for these differences as well.

Lipid species are represented as percentages of total glycerophospholipids. The assay was carried out in quadruplicate and standard deviations are shown. WT, wild type; + doxy, Δtbdat/Δtbdat/TetO:V5:TbDAT grown in the presence of doxycycline;—doxy, Δtbdat/Δtbdat/TetO:V5:TbDAT grown in the absence of doxycycline for 10 days. *, the p value resulting from the comparison of Δtbdat/Δtbdat/TetO:V5:TbDAT grown in the presence or absence of doxycycline was < 0.05.

Organellar proteomics have established that TbDAT is also expressed in the bloodstream form of the parasite [29]. In addition, ether lipids were detected in lipidomics analyses of bloodstream form Trypanosomes, suggesting that TbDAT is active in this parasite form as well [1, 46]. Glycolysis is much more active in the bloodstream form of the parasite, thus producing copious amounts of DHAP in the glycosomes to be readily available for TbDAT. It would make sense that the parasite would use TbDAT rather than TbGAT to produce most of its glycerophospholipids, as G3P is a downstream product of DHAP. Thus, TbDAT is likely to be important as well in the bloodstream form of the parasite. The creation of a (conditional) null mutant in the bloodstream form will assess this hypothesis.

In conclusion, this work shows that DHAPAT activity in the procyclic form of T. brucei is primarily mediated by TbDAT, whose role is chiefly in ether lipid biosynthesis (Fig 7). In the absence of TbGAT however, TbDAT can also contribute to ester glycerophospholipid production via reduction of acyl-DHAP to acyl-G3P [31]. Our studies also suggest that TbGAT is a minor, alternative DHAPAT enzyme, even though it does not contribute to ether but only to ester glycerophospholipid biosynthesis [31].

Fig 7
Contribution of TbGAT and TbDAT to glycerophospholipid biosynthesis.

Materials and methods

Strains and growth conditions

The wild-type strain of T. brucei used in this work is the 29–13 cell line [47]. Parasites were grown in a SD79.3 medium. Delipidated medium was prepared using delipidated fetal bovine serum, which was extracted twice with the same volume of a chloroform/methanol mixture (2:1). The organic solvent’s contaminants were removed by blowing N2 (gas) for 3 hr. Leishmania mutants Δlmdat/Δlmdat and Δlmgat/Δlmgat, and complemented lines Δlmdat/Δlmdat [LmDAT NEO] and Δlmgat/Δlmgat [TbGAT NEO] were described elsewhere and propagated in a liquid M199-based medium in the presence of 20 μg/ml G418, as appropriate [18, 31, 48]. Growth, transformation, and limiting dilution of Trypanosome and Leishmania parasites were accomplished according to [49]. T. brucei transformants were selected and/or maintained in the presence of 15 g/ml G418, 25 μg/ml hygromycin, 5 μg/ml blasticidin, 5 μg/ml phleomycin, 1 μg/ml puromycin, 1 μg/ml doxycycline, as appropriate. For growth curves, cells from a mid-log phase culture were diluted to 1x106/ml and cells were counted with a hemocytometer as a function of time.

Saccharomyces cerevisiae strains used in this work are scgat1Δscgat2Δ [GAL1:ScGAT1], scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:LmDAT] and scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1] were described previously [25, 32].

Plasmids

The plasmid pBEVY-L-TbDAT (Ec312) for expression in S. cerevisiae was constructed by PCR amplifying the TbDAT gene followed by cloning in sense orientation into the BamHI site of pBEVY-L [50]. The same DNA fragment was also inserted in sense orientation into the BamHI site of pXG1a [51] and pLEW100-V5 (Ec877) to give pXG.TbDAT (Ec516) and pLew100-V5:TbDAT (Ec879) for expression in Leishmania and T. brucei, respectively. The episome pLEW100-V5 was created by amplifying the V5 encoding epitope gene by PCR and cloning into the HindIII site of pLEW100 [47].

For inactivation of the TbDAT alleles, antibiotic resistance cassettes BSD and PAC were flanked by approximately 500 bp of TbDAT UTR regions to give the plasmids pUC.TbDAT:BSD (Ec383) and pUC.TbGAT:PAC (Ec874), respectively. The antibiotic cassettes were then integrated into the TbDAT locus by double crossing over. All PCR-amplified DNAs were verified by sequencing.

Creation of S. cerevisiae scgat1Δscgat2Δ [ADH1-TbDAT]

The S. saccharomyces mutant scgat1Δscgat2Δ [GAL1:ScGAT1 (Sc28 = cmy228) was transformed with pBEVY-L-TbDAT and selected on leucine prototrophy to yield scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:TbDAT] (Sc31; [32]). Then, 5-fluorouracil was applied to remove the [GAL1:ScGAT1] episome to give scgat1Δscgat2Δ [ADH1:TbDAT] (Sc157). The genotype of the resulting cell line was verified by PCR (Fig 1B).

Creation of the conditional null mutant Δtbdat/Δtbdat/TetO:V5:TbDAT

The conditional null mutant of TbDAT was created by the subsequent transformation of the TbDAT:PAC and the TbDAT:BSD cassettes that were amplified by PCR using pUC.TbDAT:PAC and pUC.TbDAT:BSD as template, respectively, and oligonucleotides 190 (5’-CCGAAAATGCAAACCACACAC-3’) and 192 (5’-TCCTGTAGGTGACAGATAATGG-3’). The PCR product was directly used for transformation of wild-type procyclic form parasites followed by selection in the presence of puromycin. The resistant heterozygous TbDAT:PAC/TbDAT clone was verified by PCR and then subjected to a second round of transformation with pLew100-V5:TbDAT, which was previously linearized with NotI, to give phleomycin resistant TbDAT:PAC/TbDAT/TetO:V5:TbDAT cells. Lastly, the latter strain was transformed with the TbDAT:BSD cassette and parasites resistant to blasticidin, phleomycin and hygromycin were selected. Also, a concentration of 1 μg/mg of doxycycline was applied to maintain V5:TbDAT expression as needed. The proper chromosomal integrations were all verified by PCR (Fig 4C).

Reverse transcription (RT) PCR

RNA was purified from mid-log phase parasites with Trizol (Thermo Fisher Scientific, Waltham, MA) as described by the manufacturer’s protocol. DNA contaminant was degraded using a DNA removal kit and RT reaction was carried out by random priming using the SuperScript III reverse transcriptase, both from Thermo Fisher Scientific (Waltham, MA). PCR was carried out with primers 138 (‘5-ACCCGGGCTCACAGAAGAG-3’) and 328 (‘5 CTCTAGACCCATCACCAGGAGCAAATAG-3’) specific to TbDAT, or oligonucleotides 539 (5’-ACGCCAAGCTAATACATGAACC-3’) and 540 (5’-TATTTCTCAGGCTCCCTCTCC-3’) specific to 18S rRNA gene using the Titanium Taq polymerase (Clontech Laboratories Inc., Mountain View, CA).

Enzymatic assays

Leishmania, S. cerevisiae and Trypanosoma whole cell extracts were prepared as described previously [25]. Protein concentration was determined by the bicinchoninic acid assay using bovine serum albumin as a standard.

DHAPAT and GPAT activity assays were performed as described in [18, 25, 31]. Briefly, GPAT assay was performed in a buffer system containing 20 mM TrisHCl pH7.5, 4 mM NaF, 1 mM DTT, 2 mM MgCl2, 1 mg/ml fatty acid free BSA, 100 μM fatty acyl-CoA, 0.4 mM G3P (including 5.7 μM of [U14C]L-G3P; specific activity of 25 mCi/mmol; MP Biomedicals, LLC, Santa Ana, CA), and 200 μg of protein extract in a total volume of 200 μl. The reaction was incubated at 30°C and stopped after10 min with 700 μl of 1% HClO4. The products were extracted with 2 ml of a methanol:chloroform (1:1) mixture and the lower, organic phase was washed with 700 μl of 1% HClO4. The organic phase was air dried and the radioactivity was quantified with a scintillation counter.

DHAPAT activity was assessed by measuring the acylation rate of DHAP based on a protocol established by Bates and Saggerson [25, 31, 52]. First, DHAP was produced by catabolism of fructose 1,6-bisphosphate by the enzymes triose isomerase (28 U; Millipore-Sigma, St Louis, MO) and aldolase (0.44 U; Millipore-Sigma, St Louis, MO) in a buffer system containing 50 mM TrisHCl pH7.5, 0.12 M KCl, 1 mM NaF, 4 mM MgCl2, 2 mg/ml fatty acid free BSA, 150 μM fatty acyl-CoA, and 0.5 mM fructose 1,6-bisphosphate (including 0.13 µM of [U-14C]D-fructose 1,6-bisphosphate; specific activity of 295 mCi/mmol; MP Biomedicals, LLC, Santa Ana, CA). The assay was incubated at 30°C for 15 min after which 0.1% CHAPS and 1.5 mg of cell extracts were added. The reaction was stopped after 10 min by adding 400 μl of 1% HClO4. The samples were then extracted as described above for the GPAT assay. Both enzymatic assays were performed at least twice in duplicate. The incorporated radioactivity was quantified by liquid scintillation counting.

Electrophoresis and Western blot analysis

Proteins were separated by SDS-PAGE (7.5–12.5%/4%) and subjected to Western blot analyses in the presence of antisera specific to the arginase [43], GPEET procyclin (5H3, [53]) or of monoclonal antibodies against the V5 epitope (Thermo Fisher Scientific, Waltham, MA) or lipophosphoglycan (WIC79.3, [35]) according to [25].

Lipid analysis

Parasites were grown in quadruplicate cultures to late log phase and washed three times with cold PBS. Total cellular lipids were purified according to Schneiter et al. [54]. Lipids were profiled by electrospray ionization tandem mass spectrometry (ESI-MS/MS) using the method described by [34], except that internal standards were (with some small variation in amounts in different batches of internal standards): 0.6 nmol di12:0-PC, 0.6 nmol di24:1-PC, 0.6 nmol 13:0-lysoPC, 0.6 nmol 19:0-lysoPC, 0.3 nmol di12:0-PE, 0.3 nmol di23:0-PE, 0.3 nmol 14:0-lysoPE, 0.3 nmol 18:0-lysoPE, 0.3 nmol 14:0-lysophosphatidylglycerol (lysoPG), 0.3 nmol 18:0-lysoPG, 0.3 nmol di14:0-PA, 0.3 nmol di20:0 (phytanoyl)-PA, 0.2 nmol di14:0-PS, 0.2 nmol di20:0(phytanoyl)-PS, 0.23 nmol 16:0–18:0-PI, 0.3 nmol di14:0-PG, and 0.3 nmol di20:0(phytanoyl)-PG. In addition to the scans previously described [34], a scan for PG as [M+NH4]+ in the positive mode with NL 189.0 was performed with collision energy of 20 V, declustering potential of 100 V, and exit potential of 14 V. Ether-linked (alk(en)yl, acyl) lipids were quantified in comparison to the diacyl compounds with the same head groups without correction for response factors for these compounds as compared to their diacyl analogs.

Immunofluorescence assay

Immunofluorescence assay was carried out as described previously [31]. The monoclonal antibody specific to V5 (Thermo Fisher Scientific, Waltham, MA, #R96025) and glycosomes were used at a 1:500 and 1:100 dilution, respectively [37, 53].

For viability assay, T. brucei cells were labeled with the Zombie Green Fixable Viability Kit (BioLegend, San Diego, CA) and washed as stated by the manufacturer’s instructions. Cells were then fixed for 10 min in the presence of 4% paraformaldehyde in PBS and mounted on poly-lysine coated cover slips. Green and red cells were visualized with a fluorescence microscope and quantified with a hemocytometer. At least 200 cells were counted. This assay was performed twice in duplicate.

Statistical analyses

Data are represented as mean ± SE. The significance of means’ differences was calculated by two-tailed paired t-test using Microsoft Excel data analysis. Differences were considered significant when the p-value was < 0.05.

Acknowledgments

We are grateful to A. Gunzel for providing the T. brucei strain 29–13 and the plasmid pLEW100. The arginase specific antiserum, anti-glycosomal antibodies, and monoclonal antibodies WIC79.3 were a gift of B. Ullman, M. Parsons, and S. Turco, respectively. We thank C. McMaster for sharing the S. cerevisiae strain cmy228. Adelisa Franchitti, Jennifer Page, Kelly Dahlstrom, and Kara Dunlap are acknowledged for their excellent technical contribution.

Funding Statement

This work was supported by the National Institute of Health, SC3GM113743 to RZ, (https://www.nih.gov); National Institute of Health, T36GM101995 to RZ (https://www.nih.gov/); The lipid analyses described in this work were performed at the Kansas Lipidomics Research Center Analytical Laboratory. Instrument acquisition and method development at the Kansas Lipidomics Research Center was supported by National Science Foundation (EPS 0236913, MCB 0455318, DBI 0521587; https://www.nsf.gov), Kansas Technology Enterprise Corporation (http://cdm16884.contentdm.oclc.org/ui/custom/default/collection/default/resources/custompages/ag), K-IDeA Networks of Biomedical Research Excellence (INBRE) of National Institute of Health (P20RR16475; https://www.nih.gov), and Kansas State University (http://www.k-state.edu/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

Data Availability

All relevant data are within the paper. There are no supporting information.

References

1. Patnaik PK, Field MC, Menon AK, Cross GA, Yee MC, Butikofer P. Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms. Mol Biochem Parasitol. 1993;58(1):97–105. . [PubMed]
2. Smith TK, Butikofer P. Lipid metabolism in Trypanosoma brucei. Mol Biochem Parasitol. 2010;172(2):66–79. doi: 10.1016/j.molbiopara.2010.04.001 ; PubMed Central PMCID: PMCPMC3744938. [PMC free article] [PubMed]
3. Zhang K, Beverley SM. Phospholipid and sphingolipid metabolism in Leishmania. Mol Biochem Parasitol. 2010;170(2):55–64. doi: 10.1016/j.molbiopara.2009.12.004 ; PubMed Central PMCID: PMCPMC2815228. [PMC free article] [PubMed]
4. Coppolino MG, Dierckman R, Loijens J, Collins RF, Pouladi M, Jongstra-Bilen J, et al. Inhibition of phosphatidylinositol-4-phosphate 5-kinase Ialpha impairs localized actin remodeling and suppresses phagocytosis. J Biol Chem. 2002;277(46):43849–57. doi: 10.1074/jbc.M209046200 . [PubMed]
5. Krauss M, Kukhtina V, Pechstein A, Haucke V. Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes. Proc Natl Acad Sci U S A. 2006;103(32):11934–9. doi: 10.1073/pnas.0510306103 ; PubMed Central PMCID: PMCPMC1567676. [PubMed]
6. Santarius M, Lee CH, Anderson RA. Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools. Biochem J. 2006;398(1):1–13. doi: 10.1042/BJ20060565 ; PubMed Central PMCID: PMCPMC1525017. [PubMed]
7. Gibellini F, Hunter WN, Smith TK. The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei. Mol Microbiol. 2009;73(5):826–43. doi: 10.1111/j.1365-2958.2009.06764.x ; PubMed Central PMCID: PMCPMC2784872. [PMC free article] [PubMed]
8. Signorell A, Gluenz E, Rettig J, Schneider A, Shaw MK, Gull K, et al. Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei. Mol Microbiol. 2009;72(4):1068–79. doi: 10.1111/j.1365-2958.2009.06713.x . [PubMed]
9. Farine L, Butikofer P. The ins and outs of phosphatidylethanolamine synthesis in Trypanosoma brucei. Biochim Biophys Acta. 2013;1831(3):533–42. doi: 10.1016/j.bbalip.2012.09.008 . [PubMed]
10. Serricchio M, Butikofer P. Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis. FEBS J. 2011;278(7):1035–46. doi: 10.1111/j.1742-4658.2011.08012.x . [PubMed]
11. Ramakrishnan S, Serricchio M, Striepen B, Butikofer P. Lipid synthesis in protozoan parasites: a comparison between kinetoplastids and apicomplexans. Prog Lipid Res. 2013;52(4):488–512. doi: 10.1016/j.plipres.2013.06.003 ; PubMed Central PMCID: PMCPMC3830643. [PMC free article] [PubMed]
12. Thai TP, Rodemer C, Jauch A, Hunziker A, Moser A, Gorgas K, et al. Impaired membrane traffic in defective ether lipid biosynthesis. Hum Mol Genet. 2001;10(2):127–36. . [PubMed]
13. Braverman NE, Moser AB. Functions of plasmalogen lipids in health and disease. Biochim Biophys Acta. 2012;1822(9):1442–52. doi: 10.1016/j.bbadis.2012.05.008 . [PubMed]
14. Watschinger K, Werner ER. Orphan enzymes in ether lipid metabolism. Biochimie. 2013;95(1):59–65. doi: 10.1016/j.biochi.2012.06.027 ; PubMed Central PMCID: PMCPMC3520006. [PMC free article] [PubMed]
15. Gorgas K, Teigler A, Komljenovic D, Just WW. The ether lipid-deficient mouse: tracking down plasmalogen functions. Biochim Biophys Acta. 2006;1763(12):1511–26. doi: 10.1016/j.bbamcr.2006.08.038 . [PubMed]
16. Jones CL, Hajra AK. Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver. Arch Biochem Biophys. 1983;226(1):155–65. . [PubMed]
17. Liu D, Nagan N, Just WW, Rodemer C, Thai TP, Zoeller RA. Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids. J Lipid Res. 2005;46(4):727–35. doi: 10.1194/jlr.M400364-JLR200 . [PubMed]
18. Zufferey R, Mamoun CB. The initial step of glycerolipid metabolism in Leishmania major promastigotes involves a single glycerol-3-phosphate acyltransferase enzyme important for the synthesis of triacylglycerol but not essential for virulence. Mol Microbiol. 2005;56(3):800–10. doi: 10.1111/j.1365-2958.2005.04579.x . [PubMed]
19. Hajra AK. Dihydroxyacetone phosphate acyltransferase. Biochim Biophys Acta. 1997;1348(1–2):27–34. . [PubMed]
20. Biermann J, Just WW, Wanders RJ, Van Den Bosch H. Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes. Eur J Biochem. 1999;261(2):492–9. . [PubMed]
21. Ofman R, Hettema EH, Hogenhout EM, Caruso U, Muijsers AO, Wanders RJ. Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2. Hum Mol Genet. 1998;7(5):847–53. . [PubMed]
22. Sztriha L, Al-Gazali LI, Wanders RJ, Ofman R, Nork M, Lestringant GG. Abnormal myelin formation in rhizomelic chondrodysplasia punctata type 2 (DHAPAT-deficiency). Dev Med Child Neurol. 2000;42(7):492–5. . [PubMed]
23. Elias ER, Mobassaleh M, Hajra AK, Moser AB. Developmental delay and growth failure caused by a peroxisomal disorder, dihydroxyacetonephosphate acyltransferase (DHAP-AT) deficiency. Am J Med Genet. 1998;80(3):223–6. . [PubMed]
24. Zufferey R, Al-Ani GK, Dunlap K. Leishmania dihydroxyacetonephosphate acyltransferase LmDAT is important for ether lipid biosynthesis but not for the integrity of detergent resistant membranes. Mol Biochem Parasitol. 2009;168(2):177–85. doi: 10.1016/j.molbiopara.2009.08.006 ; PubMed Central PMCID: PMCPMC2764253. [PMC free article] [PubMed]
25. Zufferey R, Ben Mamoun C. 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. J Biol Chem. 2006;281(12):7952–9. doi: 10.1074/jbc.M512911200 . [PubMed]
26. Opperdoes FR. Localization of the initial steps in alkoxyphospholipid biosynthesis in glycosomes (microbodies) of Trypanosoma brucei. FEBS Lett. 1984;169(1):35–9. . [PubMed]
27. Zomer AW, Opperdoes FR, van den Bosch H. Alkyl dihydroxyacetone phosphate synthase in glycosomes of Trypanosoma brucei. Biochim Biophys Acta. 1995;1257(2):167–73. . [PubMed]
28. Heise N, Opperdoes FR. The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in Leishmania mexicana promastigotes. Mol Biochem Parasitol. 1997;89(1):61–72. . [PubMed]
29. Vertommen D, Van Roy J, Szikora JP, Rider MH, Michels PA, Opperdoes FR. Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei. Mol Biochem Parasitol. 2008;158(2):189–201. doi: 10.1016/j.molbiopara.2007.12.008 . [PubMed]
30. Zomer AW, Michels PA, Opperdoes FR. Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase. Mol Biochem Parasitol. 1999;104(1):55–66. . [PubMed]
31. Patel N, Pirani KA, Zhu T, Cheung-See-Kit M, Lee S, Chen DG, et al. The glycerol-3-phosphate acyltransferase TbGAT is dispensable for viability and the synthesis of glycerolipids in Trypanosoma brucei. J Eukaryot Microbiol. 2016. doi: 10.1111/jeu.12309 . [PMC free article] [PubMed]
32. Zaremberg V, McMaster CR. Differential partitioning of lipids metabolized by separate yeast glycerol-3-phosphate acyltransferases reveals that phospholipase D generation of phosphatidic acid mediates sensitivity to choline-containing lysolipids and drugs. J Biol Chem. 2002;277(41):39035–44. doi: 10.1074/jbc.M207753200 . [PubMed]
33. Zheng Z, Zou J. The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae. J Biol Chem. 2001;276(45):41710–6. doi: 10.1074/jbc.M104749200 . [PubMed]
34. Al-Ani GK, Patel N, Pirani KA, Zhu T, Dhalladoo S, Zufferey R. The N-terminal domain and glycosomal localization of Leishmania initial acyltransferase LmDAT are important for lipophosphoglycan synthesis. PLoS One. 2011;6(11):e27802 doi: 10.1371/journal.pone.0027802 ; PubMed Central PMCID: PMCPMC3219689. [PMC free article] [PubMed]
35. Kelleher M, Bacic A, Handman E. Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes. Proc Natl Acad Sci U S A. 1992;89(1):6–10. ; PubMed Central PMCID: PMCPMC48163. [PubMed]
36. Opperdoes FR, Borst P. Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett. 1977;80(2):360–4. . [PubMed]
37. Parker HL, Hill T, Alexander K, Murphy NB, Fish WR, Parsons M. Three genes and two isozymes: gene conversion and the compartmentalization and expression of the phosphoglycerate kinases of Trypanosoma (Nannomonas) congolense. Mol Biochem Parasitol. 1995;69(2):269–79. . [PubMed]
38. Visser N, Opperdoes FR. Glycolysis in Trypanosoma brucei. Eur J Biochem. 1980;103(3):623–32. . [PubMed]
39. Swinkels BW, Gibson WC, Osinga KA, Kramer R, Veeneman GH, van Boom JH, et al. Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei. EMBO J. 1986;5(6):1291–8. ; PubMed Central PMCID: PMCPMC1166939. [PubMed]
40. Cronan JE. Avant garde fatty acid synthesis by trypanosomes. Cell. 2006;126(4):641–3. doi: 10.1016/j.cell.2006.08.001 . [PubMed]
41. Lee SH, Stephens JL, Englund PT. A fatty-acid synthesis mechanism specialized for parasitism. Nat Rev Microbiol. 2007;5(4):287–97. doi: 10.1038/nrmicro1617 . [PubMed]
42. Lee SH, Stephens JL, Paul KS, Englund PT. Fatty acid synthesis by elongases in trypanosomes. Cell. 2006;126(4):691–9. doi: 10.1016/j.cell.2006.06.045 . [PubMed]
43. D'Antonio EL, Ullman B, Roberts SC, Dixit UG, Wilson ME, Hai Y, et al. Crystal structure of arginase from Leishmania mexicana and implications for the inhibition of polyamine biosynthesis in parasitic infections. Arch Biochem Biophys. 2013;535(2):163–76. doi: 10.1016/j.abb.2013.03.015 ; PubMed Central PMCID: PMCPMC3683356. [PMC free article] [PubMed]
44. Signorell A, Rauch M, Jelk J, Ferguson MA, Butikofer P. Phosphatidylethanolamine in Trypanosoma brucei is organized in two separate pools and is synthesized exclusively by the Kennedy pathway. J Biol Chem. 2008;283(35):23636–44. doi: 10.1074/jbc.M803600200 ; PubMed Central PMCID: PMCPMC3259767. [PMC free article] [PubMed]
45. Dorninger F, Brodde A, Braverman NE, Moser AB, Just WW, Forss-Petter S, et al. Homeostasis of phospholipids—The level of phosphatidylethanolamine tightly adapts to changes in ethanolamine plasmalogens. Biochim Biophys Acta. 2015;1851(2):117–28. doi: 10.1016/j.bbalip.2014.11.005 ; PubMed Central PMCID: PMCPMC4331674. [PMC free article] [PubMed]
46. Richmond GS, Gibellini F, Young SA, Major L, Denton H, Lilley A, et al. Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei. Parasitology. 2010;137(9):1357–92. doi: 10.1017/S0031182010000715 ; PubMed Central PMCID: PMCPMC3744936. [PMC free article] [PubMed]
47. Wirtz E, Leal S, Ochatt C, Cross GA. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol. 1999;99(1):89–101. . [PubMed]
48. Zufferey R, Allen S, Barron T, Sullivan DR, Denny PW, Almeida IC, et al. Ether phospholipids and glycosylinositolphospholipids are not required for amastigote virulence or for inhibition of macrophage activation by Leishmania major. J Biol Chem. 2003;278(45):44708–18. doi: 10.1074/jbc.M308063200 . [PubMed]
49. Lee JH, Nguyen TN, Schimanski B, Gunzl A. Spliced leader RNA gene transcription in Trypanosoma brucei requires transcription factor TFIIH. Eukaryot Cell. 2007;6(4):641–9. doi: 10.1128/EC.00411-06 ; PubMed Central PMCID: PMCPMC1865645. [PMC free article] [PubMed]
50. Miller CA 3rd, Martinat MA, Hyman LE. Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae. Nucleic Acids Res. 1998;26(15):3577–83. ; PubMed Central PMCID: PMCPMC147745. [PMC free article] [PubMed]
51. Ha DS, Schwarz JK, Turco SJ, Beverley SM. Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol. 1996;77(1):57–64. . [PubMed]
52. Bates EJ, Saggerson ED. A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment. Biochem J. 1979;182(3):751–62. ; PubMed Central PMCID: PMCPMC1161409. [PubMed]
53. Butikofer P, Vassella E, Ruepp S, Boschung M, Civenni G, Seebeck T, et al. Phosphorylation of a major GPI-anchored surface protein of Trypanosoma brucei during transport to the plasma membrane. J Cell Sci. 1999;112 (Pt 11):1785–95. . [PubMed]
54. Schneiter R, Daum G. Extraction of yeast lipids. Methods Mol Biol. 2006;313:41–5. doi: 10.1385/1-59259-958-3:041 . [PubMed]

Articles from PLoS ONE are provided here courtesy of Public Library of Science