Chemicals were purchased from Sigma-Aldrich or Fluka. Restriction endonucleases and DNA modifying enzymes were from New England Biolabs or Promega. [2-3H] ethanolamine (50 Ci mmol−1) and d-[2-3H] mannose (15 Ci mmol−1) were purchased from Amersham. [9,10–3H(N)]- tetradecanoic acid (myristic acid) (47 Ci mmol−1) was from Perkin Elmer, while l-[3–3H]-serine (20 Ci mmol−1) and GDP-[3H]mannose (20 Ci mmol−1) were from ARC. l-[35S] methionine (1175 Ci mmol−1) was from MP Biochemicals. l-[2,2,3–2H]-serine (d3-Ser) was from CDN Isotopes.
The non-natural phospholipids standards 1,2-diheptadecanoyl-sn-glycero-3-phosphate, GPA(17:0/17:0); 1,2-dipentadecanoyl-sn-glycerol-3-phosphoethanolamine GPEtn(15:0/15:0); 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], GPGro(14:0/14:0); 1,2-diheneicosanoyl-sn-glycero-3-phosphocholine, GPCho(21:0/21:0); 1,2-dimyristoyl-sn- glycerol-3-[phospho-l-serine], GPSer(14:0/14:0); and 1-dodecanoyl-2-tridecanoyl- sn-glycero-3-phospho-(1′-myo-inositol), GPIno(12:0/13:0) were purchased from Avanti Polar Lipids.
Cloning and sequencing of T. brucei ECT
The putative TbECT gene was amplified from T. brucei strain 427 genomic DNA together with the 5′-and 3′ untranslated regions (UTRs) of 278 and 402 bp, respectively, using Pfu DNA polymerase and the forward and reverse primers 5′-ATAAGTAAgcggccgcGCTAAAGGTGTTGGTGAAACTAGCGC-3′ (F1) and 5′-ATAAGTAAgcggccgcTGGTGAAACAAAACGTTAGTACA-3′ (R2) each containing a NotI restriction site (lower case). The resulting 1.8 kb (ECT and UTRs) fragment was cloned into pCR-Blunt-II TOPO vector (Invitrogen) yielding the pCR-Blunt-II-flECT construct. Clones were sequenced and compared with the annotated Gene Data Bank sequences.
T. brucei cell culture
Bloodstream form T. brucei brucei
strain Lister 427, previously genetically modified to express T7 polymerase and the tetracycline repressor protein (Wirtz et al., 1999
), is referred to here as wild-type. This cell line allows inducible expression of ectopic genes under the control of the T7 promoter and tetracycline operator. Cells were cultured at 37°C and 5% CO2
in HMI-9 medium supplemented with 2.5 μg ml−1
of G418 to maintain the neomycin drug pressure. TbECT
conditional null mutant culturing media was supplemented with puromycin, phleomycin, hygromycyn and tetracycline at the concentrations in the section below. Cells were counted daily and the average cell volume recorded with a CASY Cell Counter and Analyser System Model TT using lower and upper cell dimension limits of 2.40 and 5.70 μm.
Stable isotope labelling of bloodstream form T. brucei
2.5 × 107T. brucei
bloodstream form cells at a density of 0.5 × 106
were incubated overnight at 37°C in HMI-9 media supplemented with 1 mM d3
-Ser. Total lipids were extracted by the method of Bligh and Dyer (1959
), and samples were analysed with a Micromass Quattro Ultima triple quadrupole mass spectrometer equipped with a nano-electrospray source. [M-H] adducts of unlabelled GPSer (d3
)-labelled GPSer, unlabelled GPEtn and (d3
)-labelled GPEtn were monitored by neutral loss scanning for m/z 96 and 99 and by parent ion scanning for m/z 196 and 199 respectively. Each spectrum encompasses at least 50 repetitive scans.
Construction of T. brucei ECT conditional gene knockout
To construct the T. brucei gene replacement cassettes, the 5′ and 3′ UTRs adjacent to the TbECT ORF were amplified from the pCR-Blunt-II-flECT construct using the primers F1 and 5′-GGATCCGTTTAAACTTACGGACCGTCAAGCTTCAAAGGGGTAAAACCCACAGATAC-3′ (R1) for the 5′-UTR; and the primers 5′-AAGCTTGACGGTCCGTAAGTTTAAACGGATCC ACTTTTATCCTTCGTGAGGAAT-3′ (F2) and R2 for the 3′ UTR. The amplified products were then used in a knitting PCR, that utilized a short linker region (underlined) containing the restriction sites BamHI, HindIII and PmeI in order to anneal together the 5′-to the 3′ UTR.
A HindIII restriction site internal to the sequence of the 3′-UTR was silenced by site directed mutagenesis using the forward primer 5′-CCATCAAAAAAGAAGGAGAAGCCTTGCATGGATAAGTAGAG-3′, the reverse primer 5′-CTCTACTTATCCATGCAAGGCTTCTCCTTCTTTTTTGATGG-3′ and the QuickChange Site Directed Mutagenesis Kit (Stratagene).
The stitched UTRs were then ligated into pGEM-5Zf(+) (Promega) via the NotI sites and the antibiotic resistance markers hygromycin phosphotransferase (HYG) or puromycin acetyltransferase (PAC) were ligated between the BamHI and HindIII restriction sites.
To generate the tetracycline inducible ectopic copy of the TbECT
gene, the ORF was amplified by PCR from the pCR-Blunt-II-flECT
construct using Pfu
polymerase and the forward and reverse primers 5′-CCCAAGCTT
GGGATGAAACGGTCGGTGTCGAAGGT-3′ and 5′-CCTTAATTAA
GGCACCTCTCTGACATTTCTGTACA-3′. The PCR product was then cloned into the vector pLew100, which contained a phleomycin resistance cassette (Wirtz et al., 1999
), using the HindIII and Pac
I sites (underlined).
These constructs were purified using a QIAprep Miniprep Plasmid Kit (Qiagen), linearized with NotI, precipitated with sodium acetate/ethanol and dissolved in sterile water to a final concentration of 2 mg ml−1. The DNA was then electroporated into T. brucei bloodstream form cells: 3 × 107 cells were re-suspended in T-cell Nucleofector solution and transfected using the program X-001 on an AMAXA Biosystems nucleofector. Transfected cells were left to recover in HMI-9 medium overnight or for 6 h; a 10-fold dilution of the cells was also applied at this stage. After recovery, drug selection was achieved in the presence of a final concentration of 0.1 μg ml−1 of puromycin for cells containing the PAC gene; 2.5 μg ml−1 of phleomycin for cells transfected with the pLew100 construct; and 4 μg ml−1 of hygromycyn for cells containing the HYG gene. Before deletion of the second TbECT allele expression of ectopic TbECT was ensured by addition of 1 μg ml−1 of tetracycline. This concentration was maintained for culturing of the TbECT conditional null mutant, whereas arrest of ECT gene expression was achieved by washing cells three times in tetracycline-free HMI-9 prior to culturing in tetracycline-free HMI-9.
RNA isolation and cDNA synthesis
Total RNA was isolated from bloodstream form T. brucei using the RNeasy mini kit (Qiagen). TbECT specific cDNA was generated and amplified using the specific forward 5′-GAGATATACATATGAAACGGTCGGTGTCGAAGG-3′ and reverse 5′-CCGGATCCTCACACCTCTCTGACATTTCTGTA C-3′ primers using the SuperScript III One step RT-PCR kit with Platinum Taq (Invitrogen). As a negative control to exclude DNA contamination of the RNA sample, reverse transcriptase was omitted from the reaction and replaced with GoTaq polymerase (Promega). Tubulin tyrosine-ligase (TbTTL) cDNA was also amplified using the forward and reverse primers 5′-GGAATTCCATATGATGAGTGAGTTCCCGTTGGTGTC-3′ and 5′-CGCGGATCCGCGTCACGTGCTCCCGATAGGCAATTC-3′ to show equal RNA input. The PCR products were then run on a 1% agarose gel.
Southern and Northern blotting
Southern and Northern blotting were performed essentially as described before (Martin and Smith, 2006a
), using TbECT
specific probes generated by PCR using the primers previously described for the amplification of the ECT
ORF for ligation into pLew100.
Subcellular localization studies
Immunofluorescence, subcellular fractionation and differential centrifugation were carried out on the TbECT
cKO cells grown in the presence of tetracycline as described before (Martin and Smith, 2006b
Mid-log bloodstream form T. brucei parental SM cells and TbECT cKO cells grown in the presence or absence of tetracycline for 36 and 42 h were incubated for 10 min at 37°C in HMI-9 medium containing 50 nM Mitotracker Red CMXRos (Molecular Probes). Cells were then harvested, washed in HMI-9 medium and incubated for further 30 min in the absence of Mitotracker. Cells were collected by centrifugation (800 g, 10 min), washed in PBS and fixed with 4%(w/v) paraformaldehyde in PBS. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 2 μg ml−1), washed in PBS, let adhere to polylysine slides and the slides mounted.
In vivo T. brucei metabolic labelling
For metabolic labelling 2 × 107 mid-log cells were centrifuged (800 g, 10 min) and washed in: serine free Minimal Essential Media (MEM), for [3H]serine and [3H]ethanolamine labelling; methionine free MEM, for [35S]methionine labelling; glucose free MEM, for [3H]mannose labelling and MEM supplemented with defatted BSA precoupled with [3H]myristate for [3H]myristate labelling. The cells were then re-suspended in the appropriate media at the final concentration of 1 × 107 cells ml−1. Cells were labelled for 1 h at 37°C with 50 μCi ml−1 of the relevant radiolabelled species. Two aliquots containing each 1 × 107 cells were then collected from each labelling experiment by centrifugation (800 g, 10 min) and used for lipid and protein analysis.
Lipids were extracted in chloroform:methanol:water (10:10:3) for 1 h, the supernatant removed and the pellet extracted with fresh 10:10:3 for 1 h. The supernatants were pooled and dried under a stream of nitrogen prior to desalting using butanol/water partitioning. Lipids were separated by HPTLC using silica 60 HPTLC plates with chloroform : methanol : water (10:10:3 v/v) as the solvent. The aqueous phases containing the ethanolamine metabolites were dried under a nitrogen stream and separated by HPTLC using silica 60 HPTLC plates in a solvent system composed by 100% ethanol : 0.5% sodium chloride : 25% ammonium hydroxide (10:10:1). Cold ethanolamine, ethanolamine-phosphate and CDP-ethanolamine standards were run in parallel and detected with ninhydrin. Radiolabelled species were detected by fluorography at −80°C, after spraying with En3hance™ and using Kodak Biomax MS film with an intensifying screen. Proteins were separated on a 4–12% SDS-PAGE gel and visualized by Coomassie blue staining. Destained gel was soaked in En3hance™ (NEN) for 30 min, washed with water twice, soaked in 10% glycerol and dried. The dried gel was then exposed to Kodak Biomax MS film for 7 days at −80°C.
For scanning electron microscopy the samples were fixed directly in HMI-9 media by adding glutaraldehyde to a final concentration of 2.5% (v/v) and processed as described before (Urbaniak et al., 2006
). Samples were then examined using a Philips XL 30 environmental scanning electron microscope operating at an accelerating voltage of 15 Kv.
For transmission electron microscopy cell pellets were fixed for up to 24 h with Peter's fixative (1.25% glutataldehyde, 1% paraformaldehyde in 0.08 M sodium cacodylate pH 7.2 with 0.02% calcium chloride). Samples were rinsed twice in 0.08 M sodium cacodylate pH 7.2 then postfixed/stained with 1% osmium tetraoxide (aq) for 1 h. Specimens were then rinsed twice in distilled water before fixation/staining in 3% uranyl acetate (aq) for 16–18 h. Samples were rinsed twice in distilled water, dehydrated through a graded ethanol series then washed twice in propylene oxide. Samples were then infiltrated with a 50% (v/v) mixture of Durcopan resin and propylene oxide for 24 h on a rotary mixer at room temperature. After infiltration for a further 24 h in 100% Durcopan resin, samples were polymerized at 60°C for 24 h. Sections were cut using a Leica Ultracut UCT microtome, mounted on pioloform coated copper grids, post stained with 3% uranyl acetate (aq) and Reynold's lead citrate and examined using a JEOL-1200 EX transmission electron microscope operating at a accelerating voltage of 80 Kv.
Electrospray tandem mass spectrometry
Total lipids from 1 × 108
trypanosomes were extracted by the method of Bligh and Dyer (1959
) with un-natural lipid standards added prior to lipid extraction [1 nmol of standard per sample, with the exception of gpi
no(12:0 13:0) of which only 0.2 nmol were added per sample]. Samples were analysed with a Micromass Quattro Ultima triple quadrupole mass spectrometer equipped with a nano-electrospray source, as described previously (Guler et al., 2008
). Each spectrum encompasses at least 50 repetitive scans.
TbECT recombinant protein expression and purification
The ECT ORF was PCR amplified from the pCR-Blunt-II-flECT construct with Pfu polymerase using the forward primer 5′-GGAATTCCATATGATGAAACGGTCGGTGTCGAAG-3′ containing a NdeI restriction site (underlined) and the reverse primer 5′-GGATCCCCTTGAAAATACAGGTTTTCGCCGCCGGTACCCACCTCTCTGACATTTCTGTACACATCTGGC-3′, which contains a BamHI restriction site (underlined) and encodes a product carrying a TEV protease cleavage site (bold). The amplicon was purified (QIAquick PCR purification kit, Qiagen), subcloned into pCR-Blunt II TOPO (Invitrogen) and sequenced. Using the NdeI and BamHI restriction sites the putative TbECT was ligated into the expression vector pET-20b (Novagen), generating the construct pET20b-TbECT-TevP-His6, which incorporates a C-terminal TEV cleavable hexa-histidine tag when expressed.
construct was transformed in BL21(DE3) Codon plus RIL cells and clones selected on LB-agar plates containing carbenicillin (100 μg ml−1
) and chloramphenicol (25 μg ml−1
). Starter cultures were grown at 37°C from single colonies in 10 ml of LB medium containing carbenicillin (50 μg ml−1
) and chloramphenicol (12.5 μg ml−1
), for at least 5 h. The starter culture was then used to inoculate 1 l of autoinduction media (Studier, 2005
) and the cells were grown at room temperature for 24 h. Cells were harvested by centrifugation (3500 g
, 20 min, 4°C) and re-suspended in buffer A (50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol and 5% glycerol). Cells were lysed in the presence of Dnase I using a French press and the lysate cleared by centrifugation (35 000 g
, 30 min, 4°C).
The cleared lysate was applied to a 5 ml HisTrap™ column (GE-Healthcare) preloaded with Ni2+. Unbound proteins were removed by washing the column with 15 column volumes of buffer A, while TbECT was eluted with an imidazole gradient in the same buffer.
Fractions containing TbECT were pooled and the His-tag was cleaved with TEV protease (1 mg of protease per 10–20 mg of protein substrate) containing an uncleavable His-tag, while dialysing against buffer A. The cleaved TbECT was purified from the remaining His-tagged version of TbECT, the tag and the His-tagged TEV protease with a second round of nickel ion affinity chromatography, as described above. Fractions containing cleaved TbECT were pooled, the purity of the sample was checked by SDS-PAGE, and aliquots were snap frozen in liquid nitrogen and stored at −80°C.
In order to assess the molecular weight and the oligomeric state in solution of Tb
ECT, the protein was purified as above but glycerol, which can interfere with such analyses, was omitted from the purification buffers. Exact molecular weight was assessed by MALDI time-of-flight mass spectrometry. Sedimentation velocity experiments were performed (wavelength of 280 nm, rotor AN50-TI at 45 000 r.p.m. and 20°C), using a Beckman Coulter XL-1 analytical ultracentrifuge. The samples were run in 25 mM HEPES pH 7.5, 50 mM NaCl, 2 mM DTT, 2 mM MgCl2
at concentrations of 0.25, 0.5 and 0.75 mg ml−1
. Samples were centrifuged simultaneously and A280
measurements taken at five-minute intervals for 16 h. The resultant data were analysed using the programs SEDFIT and SEDNTERP (Schuck, 2000
; Lebowitz et al., 2002
Assay of TbECT activity
Ethanolamine-phosphate cytidylyltransferase activity was measured by a malachite green colorimetric assay in 96-well plate format using a SpectraMAX 340PC plate reader (Molecular Devices). The assay contained 25 mM Bis-tris propane pH 7.5, 5 mM MgCl2, 1.425 mg ml−1 of recombinant TbECT, 0.1 unit of inorganic pyrophosphatase (Sigma) and varying concentrations of CTP and Etn-P, in a total volume of 50 μl. The reaction was carried out for 5 min at room temperature and stopped by the addition of 100 μl of malachite green reagent (Biomol Green), and the absorbance recorded at 620 nm. Controls were conducted to make sure enzyme activity and product formation displayed a linear relationship in the time span of the assay. All enzyme assays were conducted at least in triplicate. Enzyme Kinetics, SigmaPlot (SPSS), was used to analyse and fit the kinetic data. Substrate recognition was investigated by substituting either Etn-P or CTP with substrate analogues or alternative nucleotide phosphates.
The pH optimum of the reaction was assessed by measuring TbECT activity at various pHs: Bis-Tris methane (pH 6.5, 7.0), Bis-Tris propane (pH 7.5, 8.0, 8.5) or Glycine (pH 9.0).
The production of CDP-ethanolamine by the ethanolamine-phosphate cytidylyltransferase assay was assessed using a modified method of Tijburg et al. (1992
). Briefly, 2 μg of purified protein are incubated with a reaction mixture (total volume 50 μl) of 100 mM Tris pH 8, 10 mM MgCl2
, 5 mM CTP, and 4 mM Etn-P at 37°C for 20 min and quenched by boiling at 100°C for 5 min. Substrates and products were separated by HPTLC using silica 60 plates with 100% ethanol : 0.5% sodium chloride : 25% ammonium hydroxide (10:10:1, v/v) as solvent. Substrates and products were visualized by spraying with ninhydrin (0.2% in water-saturated butan-1-ol).
Cell-free system assay of GPI biosynthesis
Membranes of T. brucei
wild-type and TbECT
conditional null mutants grown in the presence or absence of tetracycline for 30, 36 and 42 h were isolated and prepared as described previously (without the addition of tunicamycin prior to lysis) (Smith et al., 1997
), snap frozen in liquid nitrogen and stored at −80°C until required. The cell-free system assay was carried out and visualized as described before (Smith et al., 1997
) using 1 × 107
cell equivalents per assay, GDP-[3
H]Mannose (0.3 μCi per 107
cell equivalents) with or without 1 mM PMSF.