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Vol. 17, Issue 12, 5265-5274, December 2006
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*Division of Biological Chemistry and Molecular Microbiology, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom; Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow G11 6NU, Scotland, United Kingdom; and Institute of Biomedical and Life Science, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
Submitted August 11, 2006;
Revised September 18, 2006;
Accepted September 29, 2006
Monitoring Editor: John York
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Vanhamme et al., 2001). The bloodstream-form parasites exist as dividing "slender" forms and nondividing "stumpy" forms that are preadapted for survival in the tsetse fly. After ingestion in a blood meal, the stumpy trypomastigote-form parasites differentiate into dividing procyclic-form parasites that colonizes the tsetse midgut. The procyclic trypanosomes express a radically different cell surface coat, thought to be made up 
3 x 106 procyclin glycoproteins (Roditi et al., 1987; Mowatt et al., 1987; Richardson et al., 1988) and a smaller number (1 x 106) of poly-N-acetyllactosamine–containing free GPIs (Lillico et al., 2003; Vassella et al., 2003; Nagamune et al., 2004; Roper et al., 2005). The procyclins are polyanionic, rod-like (Roditi et al., 1989; Treumann et al., 1997), proteins encoded by procyclin genes (Roditi and Clayton, 1999). In T. brucei strain 427, used in this study, the parasites contain (per diploid genome) two copies of the GPEET1 gene encoding 6 Gly-Pro-Glu-Glu-Thr repeats, one copy each of the EP1-1 and EP1-2 genes, encoding EP1 procyclins with 30 and 25 Glu-Pro repeats, respectively, two copies of the EP2-1 gene, encoding EP2 procyclin with 25 Glu-Pro repeats and two copies of the EP3-1 gene, encoding EP3 procyclin with 22 Glu-Pro repeats (Acosta-Serrano et al., 1999). The EP1 and EP3 procyclins contain a single N-glycosylation site, occupied exclusively by a conventional Man5GlcNAc2 oligosaccharide, at the N-terminal side of the Glu-Pro repeat domain (Treumann et al., 1997; Acosta-Serrano et al., 1999). Whereas neither EP2 nor GPEET procyclin is N-glycosylated, GPEET1 procyclin is phosphorylated on six of seven Thr residues (Butikofer et al., 1999; Mehlert et al., 1999; Schlaeppi et al., 2003). In culture, the procyclin expression profile depends on the carbon source (Vassella et al., 2000) and metabolic state of the cells (Morris et al., 2002; Vassella et al., 2004) and in the tsetse fly there appears to be a program of procyclin expression such that GPEET procyclin is expressed early, giving way to EP1 and EP3 procyclin expression (Acosta-Serrano et al., 2001; Vassella et al., 2001). GPEET and EP procyclins contain similar GPI membrane anchors. These are based on the ubiquitous ethanolamine-P-6Man
1–2Man1-6Man1-4GlcN1-6PI core (where, in this case, the phosphatidylinositol [PI] lipid is a 2-O-acyl-lyso-PI structure; Treumann et al., 1997), but they also contain the largest and most complex known GPI side chains. These side chains are large poly-disperse branched poly-N-acetyllactosamine structures (with an average of 8–12 repeats, depending on the preparation) that can terminate with 2-3–linked sialic acid residues (Ferguson et al., 1993; Treumann et al., 1997). Sialic acid is transferred from serum sialoglycoconjugates to terminal 
Gal residues by the action of a cell surface GPI-anchored trans-sialidase enzyme (Engstler et al., 1993; Pontes de Carvalho et al., 1993; Montagna et al., 2002). Trans-sialylation of surface components plays a role in the successful colonization of the tsetse fly (Nagamune et al., 2004). In vivo, the N-termini of the procyclins are removed by tsetse fly gut proteases (Acosta-Serrano et al., 2001), though the role of this event is unclear (Liniger et al., 2004), and it is thought that the underlying (protease resistant) anionic repeat units and associated GPI anchor side chains might protect the parasite from the approach tsetse fly gut hydrolases (Acosta-Serrano et al., 2001).
In previous studies, the cell surface architecture of procyclic trypanosomes has been manipulated by gene knockout of the procyclin genes themselves (Vassella et al., 2003) or genes encoding enzymes that act in the later parts of the GPI biosynthetic pathway, i.e., TbGPI10 and TbGPI8 (Nagamune et al., 2000, 2004; Lillico et al., 2003), or by galactose-starvation (Roper et al., 2005). The procyclin, TbGPI10 and TbGPI8 knockouts all resulted in parasites devoid of GPI-anchored procyclins, but this was apparently compensated for by an up-regulation in free GPI expression.
In this article, we describe the phenotype of procyclic trypanosome TbGPI12 null mutants that cannot synthesize GPI structures beyond GlcNAc-PI. To our surprise, these mutants are viable in culture, though unable to colonize the tsetse midgut. The mutant also revealed that the procyclic trypanosome surface coat contains molecules other than GPI-anchored proteins and free GPIs. Adsorbed serum components were excluded as coat components and [3H]glucosamine labeling revealed a hereto unidentified high-molecular-weight glycoconjugates that may constitute some or all of the residual surface coat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Plasmid maxi-preps were prepared using a Qiagen kit (Qiagen, Crawley, West Sussex, United Kingdom), digested with NotI, heated at 65°C for 30 min, precipitated with ethanol, redissolved in sterile water, and used for electroporation of procyclic forms of T. brucei strain 427 clone 29.13, referred in this article as wild-type (WT) cells. These cells were grown in SDM-79 (Brun and Schonenberger, 1979) in the presence of 15% fetal bovine serum (FBS), haemin 7.5 mg/L at 28°C, containing the appropriate antibiotics for selection (G418 at 15 µg/ml to maintain the T7 RNA polymerase; hygromycin at 50 µg/ml to maintain the tetracycline repressor protein; phleomycin at 2.5 µg/ml for pLew100; puromycin at 1 µg/ml; and blasticydin at 10 µg/ml). Cell densities were measured using a hemocytometer and plotted as the product of cell density and dilution.
Southern Blotting
Genomic DNA was prepared using DNAzol (Helena Biosciences, Gateshead, United Kingdom). Probes were amplified by PCR, gel-purified, and dUTP-fluorescein–labeled by random priming (Gene Images Kit, GE Healthcare, Little Chalfont, United Kingdom).
Cell-free System Experiment
Procyclic-form T. brucei membranes were prepared (Masterson et al., 1989) from wild-type cells, TbGPI12 null mutant, and TbGPI12 conditional null mutant grown with daily addition of 1 µg/ml tetracycline, or without tetracycline for 16 d, in media containing 15% FBS certified tetracycline-free (Clontech, Palo Alto, CA). Trypanosome membranes were washed twice and resuspended at 1 x 10–9/ml in 2x incorporation buffer (Güther and Ferguson, 1995). Aliquots (2 x 107 cells) were added to equal volume of water containing 2 µCi of UDP[3H]GlcNAc (41.6 Ci/mmol, Perkin Elmer-Cetus) and 2 mM GDP-Man and labeled for 20 min at 30°C. Subsequent chloroform/methanol/water and butan-1-ol extractions were performed as described before (Güther et al., 1994). Samples and glycolipid standards were run on aluminum-backed silica gel-60 HPTLC (Merck, Rahway, NJ) and developed using chloroform/methanol/1 M ammonium acetate/13 M ammonia/water (180:140:9:9:23, vol/vol). Radiolabeled compounds were detected by fluorography at –80°C after spraying with En3Hance (Perkin-Elmer Cetus, Norwalk, CT) using Kodak XAR-5 film (Eastman Kodak, Rochester, NY) and intensifying screens.
Anti-myc Western Blotting
Washed parasites were lysed in 20 mM Tris-HCl, pH 7.2, 2% SDS, boiled and adjusted to 0.3% SDS, and 1% Triton X-100 in 20 mM Tris-HCl, pH 7.2, and aliquots equivalent to 2 x 108 cells were immunoprecipitated with 1 µg anti-myc mAb (Upstate Biotechnology, Lake Placid, NY) and protein G agarose (Sigma, Poole, Dorset, United Kingdom). The washed beads were boiled in SDS sample buffer and applied to an Invitrogen gel (Paisley, United Kingdom; 4–12%, NuPage with MOPS buffer). Proteins were transferred to nitrocellulose, and the blocked filter was probed with anti-myc mAb (10 ng/ml) and developed with an anti-mouse secondary antibody conjugated to horseradish peroxidase (Sigma; diluted 1:10,000) and ECL reagent (Amersham).
Mass Spectrometry of Lipid Extracts
Wild-type procyclic-form T. brucei, TbGPI12 null mutant cells (5 x 108 total) were washed and resuspended in 0.1 ml phosphate-buffered saline and extracted with chloroform/methanol/water (10:10:3 vol/vol/vol) overnight at 4°C and then sonicated for 15 min in a sonicating water bath. The extract was centrifuged for 5 min at full speed in an Eppendorf microfuge, and the supernatant was transferred to a fresh Eppendorf tube. After drying under a stream of nitrogen, the products were partitioned between butan-1-ol and water (0.2 ml each). The aqueous phase was extracted twice more with 0.2 ml water-saturated butan-1-ol. The combined butan-1-ol phases were back-washed three times with 0.4 ml butanol-saturated water. The washed butan-1-ol phases were dried under nitrogen and dissolved in 200 µl chloroform/methanol/water (10:10:3 vol/vol/vol). Small aliquots were transferred to Waters nanotips (Millipore, Milford, MA; type F) and analyzed by electrospray ionization–mass spectrometry (ES-MS) in negative ion mode on an ABI Q-StarXL mass spectrometer (Surrey, United Kingdom). Individual ions were subjected to collision induced dissociation (CID) and tandem mass spectrometry (ES-MS/MS). The product ion spectra were used to identify phospholipid type and molecular species.
FACS Analysis
For FACS analysis, an aliquot of wild-type and TbGPI12 null mutant cells were centrifuged, resuspended in 1 ml of fresh SDM-79 media, and incubated with anti-EP mAb 247 (Richardson et al., 1988) diluted 1:100 (30 min, room temperature). Parasites were washed with cold PBS and incubated with FITC anti-mouse (Sigma; diluted 1:10000) for 1 h at room temperature. After incubation, parasites (at the concentration of 5 x 106 cells/ml) were analyzed in a Becton Dickinson FACSCalibur (Cockeysville, MD) using detector FL1-A.
Metabolic Labeling of Live T. brucei Procyclic Forms
T. brucei procyclic forms (5 ml at 107/ml) were washed twice with SDM-79 media depleted of L-proline, D-mannose, D-glucose, D-glucosamine, L-serine, L-ethanolamine and containing 2% FBS and resuspended in 10 ml of the same media and split into 2-ml aliquots. One aliquot was labeled with 5 µCi/ml [14C]proline for 2 h at 28°C. Another aliquot, after addition of L-proline and L-hydroxy-proline, was labeled either with [3H]ethanolamine (50 µCi/ml) or [3H]glucosamine (200 µCi/ml) for 20 h at 28°C, in the latter case with the further addition of L-serine. The final concentrations of all supplements were as described for SDM-79 (Brun and Schönenberger, 1979). After labeling, cells were washed and resuspended in PBS. An equal volume of 2x SDS-sample buffer containing 0.2 M DTT was added, and the samples were boiled and applied into 4–12% Nupage BisTris polyacrylamide gels and run using MOPS running buffer (Invitrogen). Gels were stained with Coomassie blue, soaked in En3Hance (Perkin Elmer-Cetus), dried, and placed in contact with Kodak XAR-5 film and intensifying screen at –80°C.
Ruthenium Red Stain and Ultramicroscopy
Parasites in culture medium were washed twice in cold PBS and fixed at 4°C for 1 h in 2.5% glutaraldehyde, 0.1 M cacodylate buffer containing 0.15% ruthenium red, 5 mM CaCl2, and 5% sucrose and processed according to Zufferey et al., (2003) through 1% osmium tetroxide/ruthenium red. Further processing included 30-min en bloc staining of the cells with 1% aqueous uranyl acetate before a graded ethanol dehydration, rinsing with propylene oxide (2 x 5 min), and embedding in Epon-Araldite resin. Ultrathin sections were prepared on 200-mesh grids, stained in Reynold's lead citrate for 5 min, and viewed at 120 kV on a Zeiss 912 Omega transmission electron microscope (Thornwood, NY), recording zero-loss images on a 2K Proscan digital camera system (Proscan, Lagerlechfeld, Germany).
Surface Biotinylation of Procyclic Form T. brucei
Wild-type and TbGPI12 null mutant procyclic cells (10 ml cultures at 3 x 107/ml for each) were washed three times with 20 ml PBS and resuspended in 20 ml of EZ-link sulfo-NHS-biotin (Pinpoint Cell Surface Protein Isolation Kit, Pierce, Rockford, IL) in PBS and rotated at 4°C for 30 min. After quenching, lysate preparation, and centrifugation, the supernatant was purified using Neutravidin beads. Proteins were eluted by boiling the beads with SDS-sample buffer containing DTT and were applied into 4–12% Nupage BisTris polyacrylamide gels run with MOPS buffer (Invitrogen). The gel was stained with Coomassie blue, and the more abundant bands in the null mutant lane were cut out of the gel, alkylated, digested with trypsin, and processed for mass-fingerprinting. (Fingerprints Proteomic Facility, University of Dundee, Scotland). Identical samples were applied into a duplicate gel to develop with silver to visualize procyclins.
Tsetse Fly Infections
Pupae of Glossina morsitans morsitants were obtained from the Institute of Zoology, Slovak Academy of Science (Bratislava, Slovakia). Newly hatched (teneral) flies were fed with an infected bloodmeal, which consisted of 107 parasites mixed with washed defribinated horse blood (containing 10% FBS). In the case of flies infected with TbGPI12 conditional null mutant cells, the bloodmeal also contained 25 µg/ml tetracycline. The latter was always included, at the same concentration, in successive bloodmeals until flies were dissected. Tetracycline is not harmful for tsetse flies, and it has been successfully used in the past to induce trypanosome gene expression within tsetse compartments (Peacock et al., 2005). Infected flies were fed with bloodmeals every 2–3 d. After 2 wk, midguts were isolated from infected flies and disrupted by mechanical force in cold SDM-79 containing 10% FBS. Isolated parasites from individual midguts were kept on ice until counted on a hemocytometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We replaced one allele with puromycin acetyltransferase (PAC) and, after antibiotic selection, we replaced the second allele with blasticydin deaminase (BSD). After dual antibiotic selection, a Southern blot with a TbGPI12 ORF probe showed that both alleles were replaced and that we had created a 
TbGPI12::PAC/TbGPI12::BSD null mutant (Figure 1B). A tetracycline-inducible TbGPI12 conditional null mutant was also created by introducing an ectopic C-terminally myc-tagged version of TbGPI12, targeted to the ribosomal DNA locus of the null mutant using the pLew100 vector (Wirtz et al., 1999). A Southern blot with a TbGPI12 ORF probe showed the presence of the ectopic gene and the absence of endogenous copies, i.e., a genotype of TiTbGPI12myc/TbGPI12::PAC/TbGPI12::BSD (Figure 1C).
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), TbGPI12 is a nonessential gene in cultured procyclic T. brucei. The mutant cells also had normal morphology, as judged by light and scanning electron microscopy (data not shown).
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First, cell-free systems of washed parasite membranes made from wild-type and TbGPI12 null and conditional null mutants (grown with and without tetracycline) were labeled with UDP-[3H]GlcNAc in the presence of excess unlabeled GDP-Man. Under these conditions, the wild-type cell-free system incorporates [3H]GlcNAc into [3H]GlcNAc-PI and downstream de-N-acetylated products such as GlcN-PI, with label accumulating in the GPI intermediate PP3, i.e., EtNP-Man3GlcN-(acyl)PI (Field et al., 1992; Güther and Ferguson, 1995 and Figure 3A, lane 1). In the TbGPI12 null mutant, the label accumulated only in GlcNAc-PI (Figure 3A, lane 2), suggesting that these membranes do not contain any residual GlcNAc-PI de-N-acetylase activity. The de-N-acetylation of GlcNAc-PI to GlcN-PI is an essential step in GPI biosynthesis, and no further reactions of GlcNAc-PI are expected without de-N-acetylation (Sharma et al., 1997). The same result was observed with the TbGPI12 conditional null mutant in the absence of tetracycline (Figure 3A, lane 4), indicating the complete absence of detectable TbGPI12 expression under nonpermissive conditions. On the other hand, the same mutant grown under permissive (plus tetracycline) conditions yielded a cell-free system that did not accumulate any GlcNAc-PI (Figure 3A, lane 3), suggesting some overexpression of GlcNAc-PI de-N-acetylase activity in these cells compared with wild type. The expression of the myc-tagged GlcNAc-PI de-N-acetylase protein under permissive conditions, but not under nonpermissive conditions, is shown by anti-myc Western blot (Figure 3B). The band with an apparent molecular weight of 28 kDa (Figure 3B, lane 2) is consistent with the predicted molecular weight of the TbGPI12-myc translation product (29.5 kDa). It is worth noting that the level of protein expression under permissive conditions was insufficient for direct Western blotting of whole cell lysates with anti-myc antibody and that immunoprecipitation of whole cell lysates from 2 x 108 cells with anti-myc was necessary to preconcentrate the target protein before Western blotting.
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The negative ion ES-MS and ES-MS/MS analyses also identified the major phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and PI components of procyclic-form T. brucei (Figure 4, A and B), which appear to present in comparable relative amounts in the two samples. Of particular note is a cluster of ions at m/z 750, 752, 778, and 780. These produced intense inositol-1,2-cyclic phosphate and inositol-monophosphate product ions at (m/z 241 and 259) in ES-MS/MS (data not shown), characteristic of inositolphosphoceramide (IPC) ions. These are discussed later. Negative product ion spectra of IPCs do not provide fine detail on the long-chain base and fatty acid compositions of the ceramide portions of IPCs, other than total carbon numbers and degrees of unsaturation (which are C32:1, C32:0, C34:1, and C34:0 for the T. brucei IPCs). These are, on average, smaller than those found in T. cruzi epimastigotes IPCs, which are mainly C34:1, C34:0, C36:1, and C36:0 (Bertello et al., 1995), and Leishmania promastigote IPCs, which are mainly C34:1 and C36:1 (Zufferey et al., 2003, Denny et al., 2004). The ES-MS/MS product ion spectra of the two major PE ions at m/z 726 and 728 (data not shown) showed these to be alkenylacyl-PE species (also known as plasmenylethanolamines) that appear to be common in kinetoplastid organisms (Villas Boas et al., 1999; Zufferey et al., 2003).
TbGPI12 Null Mutants Lack Cell Surface Procyclin and Free GPIs
In the absence of a functional GPI anchor biosynthetic pathway, we would not expect the expression of cell surface procyclins, as has been described for TbGPI10 and TbGPI8 knockout procyclic trypanosomes (Nagamune et al., 2000, 2004; Lillico et al., 2003). Analysis of cells by flow cytometry using anti-procyclin antibodies and FITC-labeled secondary antibody revealed a strong signal for wild-type cells (Figure 5A) and only background labeling for the TbGPI12 conditional null mutant grown under nonpermissive conditions (Figure 5B). Strong labeling was restored in the TbGPI12 conditional null mutant cells under permissive (plus tetracycline) conditions (Figure 5C).
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; Nagamune et al., 2004), TbGPI12 null mutants should not be able to synthesize any kind of GPI structure beyond GlcNAc-PI. Labeling of cells with [3H]ethanolamine confirmed this supposition. Thus, in wild-type cells, [3H]ethanolamine labeled procyclin and free GPIs (Figure 6B, lane 1), whereas these were absent in the TbGPI12 null mutants (Figure 6B, lane 2). The labeled band running above procyclin in both wild-type and mutant cells is most likely protein synthesis elongation factor 1a (Dever et al., 1989).
Attempts To Find Molecules That Might Replace Procyclin and Free GPIs in TbGPI12 Null Mutants
Transmission electron microscopy (TEM) of ultrathin sections of wild-type and TbGPI12 null procyclic trypanosomes stained with ruthenium red, a stain used to preserve prokaryote and eukaryote glycocalyx structure (Luft, 1966, 1971; Szubinska and Luft, 1971; Zufferey et al., 2003), revealed that the cell surface coats of these preparations were indistinguishable (Figure 7). This was a surprising finding because procyclins have been assumed to be the major component of the procyclic trypanosome surface coat (Roditi et al., 1989; Mehlert et al., 1998).
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; Guha-Niyogi and Turco, 2001; McConville et al., 2002; Atrih et al., 2005; Mendonca-Previato et al., 2005). Strong labeling of procyclin was apparent in the wild-type cells (Figure 9, lane 1), as expected, whereas this band was completely absent in the TbGPI12 null mutant, consistent with the cytometric and [14C]proline-labeling experiments described above (Figure 9, lane 2). Although [3H]glucosamine labeling did not reveal any obviously up-regulated glycoconjugates in the TbGPI12 null mutant, it did reveal a smear of labeled glycoconjugates, ranging in apparent molecular weight from 90 to >200 kDa, that were labeled in both the wild-type and TbGPI12 null mutant parasites.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the TbGPI12 gene clearly encodes all the GlcNAc-PI de-N-acetylase activity of procyclic-form trypanosome and its deletion prevents GPI synthesis beyond the early intermediate GlcNAc-PI (Figure 3). The accumulation of substantial quantities of GlcNAc-PI in the TbGPI12 null mutants, detected by ES-MS (Figure 4), indicates that the first step of GPI biosynthesis (i.e., the transfer of GlcNAc from UDP-GlcNAc to PI) is not sensitive to product inhibition. In the cell-free system and living cells, GlcNAc-PI de-N-acetylase activity may be a rate-limiting step in GPI biosynthesis and procyclin surface expression, respectively, because both are slightly higher in the TbGPI12 conditional null mutant when the expression of the ectopic copy of TbGPI12 is fully induced by tetracycline (Figures 3 and 5).
The molecular species of GlcNAc-PI that accumulate in the TbGPI12 null mutant mostly contain C18:0/C18:1 and C18:0/C18:2 diacyl-PI lipids, together with a small amount of C18:0/C18:2 alkylacyl-PI (Figure 4B). Interestingly, the relationship of C18:0/C18:2 (m/z 861) > C18:0/C18:1 (m/z 863) for the major diacyl-PI species is reversed for the GlcNAc-PIs that accumulate, where C18:0/C18:2 (m/z 1064) < C18:0/C18:1 (m/z 1066). In addition, the C16:0/C18:2 and C16:0/C18:1 diacyl-PI species (m/z 833 and 835) do not appear to be converted to GlcNAc-PI species, whereas the less-abundant C18:0/C18:2 alkylacyl-PI is converted to the corresponding GlcNAc-PI at m/z 1050. This apparent selection of particular PI species for GlcNAc addition may be consistent with a recent report (Martin and Smith, 2006b) that suggests that, at least in bloodstream-form T. brucei, two distinct pools of PI are synthesized, one for bulk membrane lipid and one for GPI anchor biosynthesis.
An unexpected finding from the lipid mass spectrometry data are the identification of IPC in T. brucei, although these are clearly not converted to GlcNAc-IPC in the TbGPI12 null mutant. To our knowledge, this is the first evidence for the presence of IPCs in T. brucei, but it is consistent with the very recent discovery of novel IPC synthase genes in this and other kinetoplastid organisms (Denny et al., 2006). In T. brucei, IPCs appear to be specific to the procyclic form because mass spectrometric analyses of bloodstream-form T. brucei lipids show them to be mostly C18:0/C18:2 and C18:0/C22:4 diacyl-PIs (Martin and Smith, 2006a, 2006b).
Thus far, the molecular architecture of procyclic-form T. brucei has been manipulated in four ways: 1) Knockout of the multiple procyclin genes themselves (Vassella et al., 2003); 2) Knockout of TbGPI10, encoding the third -mannosyltransferase of GPI biosynthesis (Nagamune et al., 2000, 2004); 3) Knockout of TbGPI8, encoding the catalytic subunit of the GPI transamidase complex (Lillico et al., 2003); and 4) Conditional knockout of TbgalE, encoding UDP-Glc 4'-epimerase, producing galactose starvation under nonpermissive conditions (Roper et al., 2005). In this article, we describe altering the cell surface in a distinct way by knocking out the TbGPI12 gene that, uniquely, simultaneously prevents the cell surface expression of procyclins and poly-N-acetyllactosamine–containing free GPI glycolipids (Figures 5 and 6).
The procyclin and TbGPI10 and TbGPI8 knockouts all result in the complete loss of cell surface procyclins, but this is compensated for by an up-regulation in the expression of wild-type (procyclin and TbGPI8 knockout) or slightly modified (TbGPI10 knockout) poly-N-acetyllactosamine–containing free GPI glycolipids. In the TbGPI12 knockout, reported here, poly-N-acetyllactosamine–containing free GPI glycolipids cannot be made and, therefore, cannot compensate for the loss of surface procyclins. We were surprised that the TbGPI12 gene was not essential for the growth of procyclic-form T. brucei, because deletion of this gene simultaneously wipes out both surface procyclin and poly-N-acetyllactosamine–containing free GPI expression. Currently, these molecules are assumed to represent the majority of the cell surface coat glycocalyx of this lifecycle stage of the organism (Mowatt and Clayton, 1987; Roditi et al., 1987, 1989; Richardson et al., 1988; Mehlert et al., 1998; Lillico et al., 2003; Vassella et al., 2003, Roper et al., 2005). It seems unlikely that the minimal glycolipid GlcNAc-PI, which accumulates in the mutant, could compensate for loss of these much larger surface molecules. Curiously, whereas the TbGPI10 and TbGPI8 knockouts have significantly and slightly, respectively, impaired cell culture growth kinetics compared with wild-type cells (Nagamune et al., 2004), the TbGPI12 knockout cells have perfectly normal growth characteristics (Figure 2). We have no obvious explanation for these differences between the mutants.
Analysis of the cell surface by ruthenium red staining and TEM showed that the density and depth of the cell surface glycocalyx did not appear to be significantly altered in the TbGPI12 null mutants compared with wild type (Figure 7). This, and the excellent growth characteristics of the TbGPI12 null mutants, prompted us to look for the possible up-regulation of other parasite surface molecules and/or the adsorption of serum components from the medium to compensate for the loss of procyclins and poly-N-acetyllactosamine–containing free GPIs. However, neither of these were apparent from surface biotinylation, followed by isolation on neutravidin and detection with Coomassie blue or silver stain (Figure 8). Because carbohydrate-rich proteoglycan- or mucin-like molecules might contain few primary amine groups for biotinylation, we took a different approach, namely biosynthetic radiolabeling with the ubiquitous trypanosomatid glycoconjugate precursor glucosamine. This revealed polydisperse glycoconjugates with high apparent molecular weights (90 to >200 kDa) on SDS-PAGE that were present in similar levels in TbGPI12 null and wild-type cells (Figure 9). It remains to be seen whether the majority of the surface coat observed by TEM is constituted by the novel glycoconjugates revealed by [3H]glucosamine labeling. This material is certainly a candidate, but the answer will have to await its isolation and characterization and the generation of mono-specific reagents that recognize its principal component(s).
TbGPI10 and TbGPI8 knockouts are partially and severely compromised, respectively, with respect to their ability to colonize the tsetse midgut (Nagamune et al., 2004). The difference in tsetse infectivity is thought to correlate with surface sialic acid content, governed by the status of the normally GPI-anchored transialidase (TS) enzyme (Nagamune et al., 2004). Thus, although TbGPI10 knockout parasites secrete anchorless TS and, therefore, still acquire surface sialic acid, the GPI-transamidase null TbGPI8 knockout parasites fail to remove the GPI-addition signal peptide from the TS precursor, to degrade it intracellularly, and to sialylate their surface. In the case of the TbGPI12 knockout cells, we observe normal growth kinetics but severely reduced tsetse infectivity (Figure 10). These cells will be able to secrete TS (like the TbGPI10 knockouts) but cannot express cell surface procyclin or free GPI sialic acid acceptors. The TbGPI12 null phenotype with respect to tsetse colonization is, therefore, like that of TbGPI8 null parasites, supporting the hypothesis that sialylation of procyclin GPI anchor side chains and/or free GPI side chains (that, uniquely, are both are absent in TbGPI12 mutant) are essential for tsetse midgut infection.
Although deletion of surface free GPIs and/or procyclins does not prevent procyclic trypanosome cell division in culture, galactose starvation is cytostatic (Roper et al., 2005). We previously suggested that galactose starvation might exert these cytostatic effects via inhibition of poly-N-acetyllactosamine side chain addition to procyclin GPI anchors and/or free GPIs, an idea that was consistent with the apparently compensatory 10-fold up-regulation in side chain–free procyclins before the cessation of cell division. However, this notion may need to be revised in the light of the healthy growth of the TbGPI12 knockout because this mutant has no GPI structures that can be modified with poly-N-acetyllactosamine side chains. Thus, it is possible that it is the effects of galactose starvation on one or more non-GPI–anchored glycoprotein(s), possibly the novel high apparent molecular weight molecules identified in this article, that cause cell division to cease. Alternatively, one could argue that the accumulation of procyclins without GPI side chains under galactose starvation might be toxic to the cells and that because this cannot occur in the TbGPI12 knockout cells, the latter grow normally.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Michael A.J. Ferguson (m.a.j.ferguson{at}dundee.ac.uk)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acosta-Serrano, A., Vassella, E., Liniger, M., Kunz Renggli, C., Brun, R., Roditi, I., Englund, P. T. (2001). The surface coat of procyclic Trypanosoma brucei: programmed expression and proteolytic cleavage of procyclin in the tsetse fly. Proc. Natl. Acad. Sci. USA 98, 1513–1518.
Atrih, A., Richardson, J. M., Prescott, A. R., Ferguson, M. A. (2005). Trypanosoma brucei glycoproteins contain novel giant poly-N-acetyllactosamine carbohydrate chains. J. Biol. Chem 280, 865–871.
Bertello, L. E., Goncalvez, M. F., Colli, W., de Lederkremer, R. M. (1995). Structural analysis of inositol phospholipids from Trypanosoma cruzi epimastigote forms. Biochem. J 310, Pt 1255–261.[Medline]
Brun, R. and Schönenberger, M. (1979). Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop 36, 289–292.[Medline]
Butikofer, P., Vassella, E., Ruepp, S., Boschung, M., Civenni, G., Seebeck, T., Hemphill, A., Mookherjee, N., Pearson, T. W., Roditi, I. (1999). Phosphorylation of a major GPI-anchored surface protein of Trypanosoma brucei during transport to the plasma membrane. J. Cell Sci 112, Pt 111785–1795.[Abstract]
Chang, T., Milne, K. G., Guther, M. L., Smith, T. K., Ferguson, M. A. (2002). Cloning of Trypanosoma brucei and Leishmania major genes encoding the GlcNAc-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol biosynthesis that is essential to the African sleeping sickness parasite. J. Biol. Chem 277, 50176–50182.
Cross, G. A. (1996). Antigenic variation in trypanosomes: secrets surface slowly. Bioessays 18, 283–291.[CrossRef][Medline]
Denny, P. W., Goulding, D., Ferguson, M. A., Smith, D. F. (2004). Sphingolipid-free Leishmania are defective in membrane trafficking, differentiation and infectivity. Mol. Microbiol 52, 313–327.[CrossRef][Medline]
Denny, P. W., Shams-Eldin, H., Price, H. P., Smith, D. F., Schwarz, R. T. (2006). The protozoan inositol phosphorylceramide synthase: a novel drug target which defines a new class of sphingolipid synthase. J. Biol. Chem 281, 28200–28209.
Dever, T. E., Costello, C. E., Owens, C. L., Rosenberry, T. L., Merrick, W. C. (1989). Location of seven post-translational modifications in rabbit elongation factor 1 alpha including dimethyllysine, trimethyllysine, and glycerylphosphorylethanolamine. J. Biol. Chem 264, 20518–20525.
Engstler, M., Reuter, G., Schauer, R. (1993). The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein. Mol. Biochem. Parasitol 61, 1–13.[CrossRef][Medline]
Ferguson, M. A., Murray, P., Rutherford, H., McConville, M. J. (1993). A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor. Biochem. J 291, 51–55.[Medline]
Field, M. C., Menon, A. K., Cross, G. A. (1992). Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein. J. Biol. Chem 267, 5324–5329.
Guha-Niyogi, A., Sullivan, D. R., Turco, S. J. (2001). Glycoconjugate structures of parasitic protozoa. Glycobiology 11, 45R–59R.
Güther, M. L. and Ferguson, M. A. (1995). The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei. EMBO J 14, 3080–3093.[Medline]
Güther, M. L., Masterson, W. J., Ferguson, M. A. (1994). The effects of phenylmethylsulfonyl fluoride on inositol-acylation and fatty acid remodeling in African trypanosomes. J. Biol. Chem 269, 18694–18701.
Lillico, S., Field, M. C., Blundell, P., Coombs, G. H., Mottram, J. C. (2003). Essential roles for GPI-anchored proteins in African trypanosomes revealed using mutants deficient in GPI8. Mol. Biol. Cell 14, 1182–1194.
Liniger, M., Urwyler, S., Studer, E., Oberle, M., Renggli, C. K., Roditi, I. (2004). Role of the N-terminal domains of EP and GPEET procyclins in membrane targeting and the establishment of midgut infections by Trypanosoma brucei. Mol Biochem. Parasitol 137, 247–251.[CrossRef][Medline]
Luft, J. H. (1966). Fine structures of capillary and endocapillary layer as revealed by ruthenium red. Fed. Proc 25, 1773–1783.
Luft, J. H. (1971). Ruthenium red and violet. II. Fine structural localization in animal tissues. Anat. Rec 171, 369–415.[CrossRef][Medline]
Martin, K. L. and Smith, T. K. (2006a). Phosphatidylinositol synthesis is essential in bloodstream form Trypanosoma brucei. Biochem. J 396, 287–295.[CrossRef][Medline]
Martin, K. L. and Smith, T. K. (2006b). The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream form Trypanosoma brucei is dependent on de novo synthesis of inositol. Mol. Microbiol 61, 89–105.[CrossRef][Medline]
Masterson, W. J., Doering, T. L., Hart, G. W., Englund, P. T. (1989). A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell 56, 793–800.[CrossRef][Medline]
McConville, M. J. and Ferguson, M. A. (1993). The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem. J 294, Pt 2305–324.[Medline]
McConville, M. J., Mullin, K. A., Ilgoutz, S. C., Teasdale, R. D. (2002). Secretory pathway of trypanosomatid parasites. Microbiol. Mol. Biol. Rev 66, 122–154 table of contents.
Mehlert, A., Treumann, A., Ferguson, M. A. (1999). Trypanosoma brucei GPEET-PARP is phosphorylated on six out of seven threonine residues. Mol. Biochem. Parasitol 98, 291–296.[CrossRef][Medline]
Mehlert, A., Zitzmann, N., Richardson, J. M., Treumann, A., Ferguson, M. A. (1998). The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei. Mol. Biochem. Parasitol 91, 145–152.[CrossRef][Medline]
Mendonca-Previato, L., Todeschini, A. R., Heise, N., Previato, J. O. (2005). Protozoan parasite-specific carbohydrate structures. Curr. Opin. Struct. Biol 15, 499–505.[CrossRef][Medline]
Montagna, G., Cremona, M. L., Paris, G., Amaya, M. F., Buschiazzo, A., Alzari, P. M., Frasch, A. C. (2002). The trans-sialidase from the African trypanosome Trypanosoma brucei. Eur. J. Biochem 269, 2941–2950.[Medline]
Morris, J. C., Wang, Z., Drew, M. E., Englund, P. T. (2002). Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library. EMBO J 21, 4429–4438.[CrossRef][Medline]
Mowatt, M. R. and Clayton, C. E. (1987). Developmental regulation of a novel repetitive protein of Trypanosoma brucei. Mol. Cell. Biol 7, 2838–2844.
Nagamune, K., Acosta-Serrano, A., Uemura, H., Brun, R., Kunz-Renggli, C., Maeda, Y., Ferguson, M. A., Kinoshita, T. (2004). Surface sialic acids taken from the host allow trypanosome survival in tsetse fly vectors. J. Exp. Med 199, 1445–1450.
Nagamune, K., Nozaki, T., Maeda, Y., Ohishi, K., Fukuma, T., Hara, T., Schwarz, R. T., Sutterlin, C., Brun, R., Riezman, H., Kinoshita, T. (2000). Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei. Proc. Natl. Acad. Sci. USA 97, 10336–10341.
Peacock, L., Bailey, M., Gibson, W. (2005). Tetracycline induction of gene expression in Trypanosoma brucei within the tsetse fly vector. Mol. Biochem. Parasitol 140, 247–249.[CrossRef][Medline]
Pontes de Carvalho, L. C., Tomlinson, S., Vandekerckhove, F., Bienen, E. J., Clarkson, A. B., Jiang, M. S., Hart, G. W., Nussenzweig, V. (1993). Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor. J. Exp. Med 177, 465–474.
Richardson, J. P., Beecroft, R. P., Tolson, D. L., Liu, M. K., Pearson, T. W. (1988). Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes. Mol. Biochem. Parasitol 31, 203–216.[CrossRef][Medline]
Roditi, I., Carrington, M., Turner, M. (1987). Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei. Nature 325, 272–274.[CrossRef][Medline]
Roditi, I. and Clayton, C. (1999). An unambiguous nomenclature for the major surface glycoproteins of the procyclic form of Trypanosoma brucei. Mol. Biochem. Parasitol 103, 99–100.[CrossRef][Medline]
Roditi, I. (1989). Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei. J. Cell Biol 108, 737–746.
Roper, J. R., Guther, M. L., Macrae, J. I., Prescott, A. R., Hallyburton, I., Acosta-Serrano, A., Ferguson, M. A. (2005). The suppression of galactose metabolism in procyclic form Trypanosoma brucei causes cessation of cell growth and alters procyclin glycoprotein structure and copy number. J. Biol. Chem 280, 19728–19736.
Schlaeppi, A. C., Malherbe, T., Butikofer, P. (2003). Coordinate expression of GPEET procyclin and its membrane-associated kinase in Trypanosoma brucei procyclic forms. J. Biol. Chem 278, 49980–49987.
Sharma, D. K., Smith, T. K., Crossman, A., Brimacombe, J. S., Ferguson, M. A. (1997). Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells. Biochem. J 328, Pt 1171–177.[Medline]
Szubinska, B. and Luft, J. H. (1971). Ruthenium red and violet. 3. Fine structure of the plasma membrane and extraneous coats in amoebae (A. proteus and Chaos chaos). Anat. Rec 171, 417–441.[CrossRef][Medline]
Treumann, A., Zitzmann, N., Hulsmeier, A., Prescott, A. R., Almond, A., Sheehan, J., Ferguson, M. A. (1997). Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei. J. Mol. Biol 269, 529–547.[CrossRef][Medline]
Vanhamme, L., Pays, E., McCulloch, R., Barry, J. D. (2001). An update on antigenic variation in African trypanosomes. Trends Parasitol 17, 338–343.[CrossRef][Medline]
Vassella, E., Acosta-Serrano, A., Studer, E., Lee, S. H., Englund, P. T., Roditi, I. (2001). Multiple procyclin isoforms are expressed differentially during the development of insect forms of Trypanosoma brucei. J. Mol. Biol 312, 597–607.[CrossRef][Medline]
Vassella, E., Butikofer, P., Engstler, M., Jelk, J., Roditi, I. (2003). Procyclin null mutants of Trypanosoma brucei express free glycosylphosphatidylinositols on their surface. Mol. Biol. Cell 14, 1308–1318.
Vassella, E., Den Abbeele, J. V., Butikofer, P., Renggli, C. K., Furger, A., Brun, R., Roditi, I. (2000). A major surface glycoprotein of Trypanosoma brucei is expressed transiently during development and can be regulated post-transcriptionally by glycerol or hypoxia. Genes Dev 14, 615–626.
Vassella, E., Probst, M., Schneider, A., Studer, E., Renggli, C. K., Roditi, I. (2004). Expression of a major surface protein of Trypanosoma brucei insect forms is controlled by the activity of mitochondrial enzymes. Mol. Biol. Cell 15, 3986–3993.
Villas Boas, M. H., Lara, L. S., Wait, R., Bergter, E. B. (1999). Identification of plasmenylethanolamine as a major component of the phospholipids of strain DM 28c of Trypanosoma cruzi. Mol. Biochem. Parasitol 98, 175–186.[CrossRef][Medline]
Wirtz, E., Leal, S., Ochatt, C., Cross, G. A. (1999). A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol 99, 89–101.[CrossRef][Medline]
Zufferey, R., Allen, S., Barron, T., Sullivan, D. R., Denny, P. W., Almeida, I. C., Smith, D. F., Turco, S. J., Ferguson, M. A., Beverley, S. M. (2003). Ether phospholipids and glycosylinositolphospholipids are not required for amastigote virulence or for inhibition of macrophage activation by Leishmania major. J. Biol. Chem 278, 44708–44718.
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), TbGPI12 null (
) and TbGPI12 conditional null cells under permissive (plus tetracycline, closed squares) and nonpermissive (minus tetracycline, 
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