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Eukaryotic Cell, March 2005, p. 556-566, Vol. 4, No. 3
1535-9778/05/$08.00+0     doi:10.1128/EC.4.3.556-566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Intracellular Glycosylphosphatidylinositols Accumulate on Endosomes: Toxicity of Alpha-Toxin to Leishmania major

Zhifeng Zheng,1 Rodney K. Tweten,2 and Kojo Mensa-Wilmot1*

Department of Cellular Biology, The University of Georgia, Athens, Georgia,1 Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma2

Received 22 September 2004/ Accepted 12 November 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur "free" or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using alpha-toxin (from Clostridium septicum). Alpha-toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for alpha-toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, alpha-toxin killed L. major with a 50% lethal concentration of 0.77 nM.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glycosylphosphatidylinositols (GPIs) exist in three forms in eukaryotes: (i) "free" (i.e., unattached to macromolecules), (ii) tethered to proteins (e.g., gp63 of Leishmania spp.), or (iii) linked to polysaccharides (e.g., lipophosphoglycan of Leishmania) (reviewed in references 21 and 26).

Free GPIs in Leishmania major consist of protein-linked GPIs and polysaccharide-linked GPIs. Protein-linked GPIs include biosynthetic intermediates, e.g., glucosamine (GlcNH2)-inositol(Ins)-1-phospho-diacylglycerol, mannose (Man){alpha}1-6Man{alpha}1-4GlcNH2-Ins-1-phospho-diacylglycerol, and Man{alpha}1-4GlcNH2-Ins-1-phospho-diacylglycerol, and the completed protein anchor ethanolamine-phospho-Man{alpha}1-2Man{alpha}1-6Man{alpha}1-4GlcNH2-Ins-1-phospho-diacylglycerol (21, 36). Polysaccharide-linked GPIs (e.g., lipophosphoglycan) contain the Man{alpha}1-3Man{alpha}1-4GlcNH2-Ins-1-phospho-diacylglycerol (reviewed inreference 21).

Free GPIs are detected on the plasma membrane of vertebrate and trypanosomatid cells (4, 22, 43, 46, 54). Other functions aside from the addition to protein and polysaccharide may be envisioned for free intracellular GPIs. In theory, enzymes that digest GPIs (e.g., phospholipases) could produce second messengers (e.g., arachidonic acid or diacylglycerol) that influence cell physiology. Knowing the subregion(s) of cells that GPIs accumulate in and localizing enzymes that catabolize the glycolipids may provide clues about novel functions of intracellular GPIs.

We present a general technique for determining the intracellular location of protein-linked GPIs (protein-GPIs) and apply it to L. major. Central to the protocol is the high specificity of alpha-toxin (from Clostridium septicum) for GPIs (25). When added to fixed or permeabilized cells, alpha-toxin recognizes intracellular protein-GPIs. In a GPI-deficient strain of L. major (40), alpha-toxin binding sites diminished dramatically, or were nonexistent, compared to binding sites in control cells. Concanavalin A (ConA), a control lectin, binds GPIs (20, 41, 50, 56, 57), GDP-mannose, and N-glycans (31, 48). As expected, ConA binding sites persisted in the GPI-deficient L. major, in contrast to the data obtained with alpha-toxin. By combining the alpha-toxin methodology with established protocols for identification of intracellular organelles, we demonstrate that GPIs concentrate on endosomes of L. major. In contrast, intracellular gp63 accumulated in the endoplasmic reticulum (ER).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids. Plasmid pUTK-GPIPLC was constructed by the insertion of a GPI-PLC coding region (8, 10) and a translation-enhancing 5' untranslated region (30) into a Leishmania expression plasmid, pXUTE-KanaR (pUTK) (41). The GPI-PLC insert was produced by PCR using forward primer KCR4 (5'-TAAGGATCCTTAACACAGGAGGCAGCTAatgtttggtggtgta-3') and reverse primer KCR5 (5'-TATGTGGATCCTTAtgaccttgcggtttggt-3'). KCR4 has a BamHI site (underlined) followed by a stop codon (italicized), an AUG-proximal region (e.g., lz-CTA [italicized]), and finally a sequence encoding the first four amino acids of GPI-phospholipase C polypeptide (PLCp) (lowercase) (24, 30). The reverse primer KCR5 contains a BamHI site (underlined), a stop codon (boldface italicized type), and the last 7 amino acids of GPI-PLCp (lowercase) (8, 10). The amplification product was digested with BamHI and ligated into a BglII site of pUTK (41).

Transfection of L. major. L. major promastigote strain LT252-CC1 (17) was cultivated at 27°C in M199 medium supplemented with 10% fetal bovine serum. Cells (107/ml) were harvested by centrifugation at 2,000 x g for 5 min. Cell pellets were washed once with ice-cold electroporation buffer (21 mM HEPES [pH 7.4], 137 mM NaCl, 5 mM KCl, 0.7 mM Na2PO4, 6 mM glucose) and electroporated under the following conditions: 475 V, 800 µF, and 13 {Omega} for one pulse (with a BTX ECM-600 apparatus) (40). The cells were incubated for 8 to 12 h at 27°C before the addition of G418 (30 µg/ml) to select stable transfectants. When specified, cells were grown in medium containing 200 µg of G418/ml for 21 to 28 days before use (see the appropriate figure legends).

Biotinylation of ConA and alpha-toxin. ConA or alpha-toxin (220 µg in 1 ml of phosphate-buffered saline [PBS]) was dialyzed against 40 mM of bicarbonate buffer (pH 8.6) with Slide-A-Lyzer cassette (Pierce) for 8 h at room temperature and then overnight at 4°C. After dialysis, 40 µl of ECL biotinylation reagent (Amersham) was added for each milligram of ConA or alpha-toxin, and the mixture was incubated at room temperature for 4 h with continuous mixing. Biotinylated protein was purified by size exclusion chromatography on a Sephadex G25 or Mini Bio-Spin column (Bio-Rad) and dialyzed against PBS.

Extraction of glycolipids from L. major. L. major promastigotes (108) were lysed with 1 ml of chloroform-methanol (2:1, vol/vol), pelleted, and reextracted with the same solvent (1:1, vol/vol). The pellet was then extracted with 1 ml of chloroform-methanol-water (1:2:0.8, vol/vol) for 2 h at 25°C, and insoluble material was pelleted (16,000 x g, 5 min) (45). The supernatant was dried under a stream of nitrogen and resuspended in a biphasic system of 1-butanol (200 µl) and water (100 µl). After mixing and centrifugation (16,000 x g, 30 s), the organic phase was saved, and the lower aqueous phase was extracted once with water-saturated 1-butanol (200 µl). The butanol phase from the last wash was combined with the other organic phases, back-extracted with water (200 µl), and dried under nitrogen gas. Dried glycolipids were resuspended in 40 µl of 40% 1-propanol and stored at –20°C until use.

Thin-layer chromatography and lectin blotting of glycolipids. Glycolipids (from 108 cell equivalents) were resolved on silica gel 60 aluminum-backed high-performance thin-layer chromatography (HPTLC) plates (Merck) using chloroform-methanol-1 M ammonium acetate-13 M NH4OH-water (40:30:2:2:5, vol/vol) as mobile phase (45). To detect glycolipids, dried HPTLC plates were immersed in polyisobutylmethacrylate (0.1% in hexane) for 10 s and dried in a fume hood. The HPTLC plates were blocked with 1% bovine serum albumin (BSA) in TTBS (20 mM Tris-HCl [pH 7.6], 300 mM NaCl, 0.05% Tween 20) for 1 h and incubated with ConA-biotin (10 µg/ml) in binding buffer (1% BSA in TTBS with 1 mM CaCl2, 1 mM MgCl2, and 0.4 mM MnCl2), or alpha-toxin-biotin (10 µg/ml in binding buffer) for 1 h at room temperature. After washing five times (each for 5 min) with TTBS, HPTLC plates were covered with a minimal solution of TTBS containing streptavidin-conjugated horseradish peroxidase (1:2,000) and developed by ECL chemiluminescence (Amersham). Plates were exposed to Kodak BioMax film for various amounts of time. Total phospholipids or glycolipids were revealed by spraying a dried HPTLC plate with 0.5% orcinol-3.5 N H2SO4 and heating (120°C, 5 min).

PI-PLC cleavage of glycolipids. Glycolipid extracts from L. major (108 cell equivalents) were dried and resuspended thoroughly in 100 µl of phosphatidylinositol (PI)-specific PLC (PI-PLC) buffer (25 mM HEPES-KOH [pH 7.5], 0.1% sodium deoxycholate) (34). Bacillus cereus PI-PLC (a gift from Mary Roberts, Boston University [32]) was added (at a final concentration of 50 U/ml), and the mixture was incubated at 37°C for 3 h. Reactions were terminated by extraction twice with 250 µl of water-saturated butanol and centrifuged (16,000 x g, 30 s), and butanol phases were pooled in a new tube. The butanol phase was back-extracted twice with 500 µl of water and dried under a stream of N2 (40). HPTLC of GPIs and lectin blotting with alpha-toxin-biotin or ConA were performed as described above.

Triton X-114 phase partition. Parasites (108 cells) were lysed in 200 µl of Triton X-114 buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10 mM EDTA, 0.5% precondensed Triton X-114, antipain [10 µg/ml], leupeptin [2 µg/ml], TLCK [N{alpha}-p-tosyl-L-lysine chloromethyl ketone] [37 µg/ml]) on ice for 15 min (13). The lysates were treated with PI-PLC (50 U/ml) or buffer for 2 h and precleared by centrifugation at 14,000 x g for 10 min at 4°C. The upper aqueous phase was overlaid onto a sucrose cushion (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10 mM EDTA, 6% [wt/vol] sucrose, 0.06% precondensed Triton X-114) in a fresh microcentrifuge tube. The samples were incubated at 30°C for 3 min and centrifuged at 10,000 x g for 3 min at room temperature. The upper aqueous layer was transferred to a fresh tube, and precondensed TX-114 was added (1.25% final concentration) to this sample which was incubated at 4°C for 15 min. The upper layer was overlaid back onto the original sucrose cushion, and the sample was incubated at 30°C for 3 min, followed by centrifugation for 3 min at 10,000 x g at room temperature. The total aqueous phase was transferred to a fresh tube, leaving a lower Triton X-114 phase (13).

Immunoprecipitation of gp63. Immunoprecipitation was carried out with anti-gp63 antibody as described previously (40). Briefly, cell pellets (108 cells) were resuspended in 1 ml of ice-cold immunoprecipitation dilution buffer (1.25% Triton X-100, 190 mM NaC1, 60 mM Tris-HCl [pH 7.5], 6 mM EDTA, antipain [10 µg/ml], leupeptin [2 µg/ml], TLCK [37 µg/ml]) on ice for 30 min. A total of 750 µl of 1.33x immunoprecipitation dilution buffer and 2 µl of anti-gp63 polyclonal antibody (a gift from K. P. Chang, University of Chicago Health Sciences Center [10]) were added to a 250-µl aliquot of cell lysate (2.5 x 107 cell equivalents). The solution was incubated at 4°C for 12 h with continuous inversion. A 50-µl portion of a 1:1 suspension of protein A-Sepharose (Sigma Chemical Co.) was added, and the incubation continued for 2 h at room temperature. Antibody complexes adsorbed to protein A-Sepharose were washed three times, each for 10 min, with 1 ml of immunoprecipitation wash buffer (0.1% Triton X-100, 0.02% sodium dodecyl sulfate [SDS], 150 mM Tris-HCl [pH 7.5], 5 mM EDTA, antipain [10 µg/ml], leupeptin [2 µg/ml], TLCK [37 µg/ml]). The beads received a final wash in Tris-buffered saline, after which 25 µl of 2.5x SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer was added. Beads were vortexed briefly and heated at 90°C for 3 min. Protein in 25 µl of eluate was analyzed by SDS-PAGE (14%).

Western blotting. Proteins (107 cell equivalents per lane) were separated by SDS-PAGE and electroblotted onto Immobilon-P membranes (Millipore) which were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 1 h at room temperature. Anti-gp63 polyclonal antibody (1:1,000 dilution in blocking buffer) was added, and the membranes were incubated for 1 h at room temperature. After washing three times (each for 10 min) with TTBS, alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:1,000 dilution) (Sigma) was added, followed by a 1-h incubation at room temperature. Color development was achieved with 5-bromo-4-chloro-3-indolylphosphate p-toluidine (BCIP) and p-nitroblue tetrazolium chloride (NBT) (Bio-Rad) (40).

Detection of the GPI of gp63 was performed with biotinylated alpha-toxin (2) and streptavidin-conjugated horseradish peroxidase coupled with ECL chemiluminescence (Amersham).

Organelle detection in live cells. Endolysosomal compartments were identified after uptake of fluorescent markers ConA-Alexa Fluor 594 and FM4-64 (42). For endocytosis of ConA-Alexa Fluor 594, cells at density of 107 were pelleted gently (2,000 x g, 2 min), resuspended in 500 µl of M199 medium containing ConA-Alexa Fluor 594 (100 µg/ml) (Molecular Probes Inc.), and incubated at 27°C for 30 min. For FM4-64 uptake, cells were incubated in serum-free M199 medium supplemented with dye (10 µM) (Molecular Probes Inc.) at 27°C for 30 min (42).

Golgi complex was detected with BODIPY-TR ceramide (Molecular Probes Inc.) (27). L. major was incubated with a BSA complex of BODIPY-TR ceramide (5 µM) for 90 min (1 h at 4°C and 30 min at 27°C). Cells were back-extracted with M199 medium containing 1.8% defatted BSA.

Immunofluorescence assays. Logarithmic-phase cells (107) were fixed in 2% paraformaldehyde for 8 min on ice, washed with PBS, and adhered to poly-L-lysine-coated coverslips. After permeabilization with 0.25% Triton X-100 (5 min, 4°C), nonspecific sites were blocked with 1% BSA in PBS for 1 h at room temperature.

The ER was identified with anti-Sec61p antibody (biotinylated). For this objective, fixed, permeabilized cells were incubated with anti-Sec61p-biotin (1:1,500 dilution in blocking buffer) for 1 h at room temperature. Cells were washed once with PBS, twice with high-salt buffer (PBS containing 500 mM NaCl), and twice with PBS. The ER was visualized with streptavidin-conjugated Alexa Fluor 594 (1:2,000 dilution) for 1 h.

gp63 was detected in permeabilized and nonpermeabilized cells as follows. To detect intracellular gp63, the fixed, permeabilized cells were incubated with anti-gp63 antiserum (1:1,000 dilution) (40). After washing with high-salt buffer (500 mM NaCl in PBS), the cells were covered with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:1,000 dilution) for 1 h.

To detect proteins on the plasma membrane, cells were fixed in glutaraldehyde (0.5% in PBS) for 10 min at 4°C before the addition of primary and secondary antibodies.

For detection of protein-GPIs, alpha-toxin (2.5 µg/ml) was added to permeabilized or nonpermeabilized cells and incubated for 30 min at 27°C. Alpha-toxin was detected with anti-alpha-toxin antibody (1:1,500 dilution) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody.

Cell nucleus and kinetoplast (mitochondrial DNA) were stained with 10 µm of 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) in antifade agent (Citifluor Ltd.). Labeled cells were viewed with a fluorescence microscope (DMIRBE; LEICA). Images were captured with an interline chip cooled charge-coupled-device camera (Orca 9545; Hamamatsu) and processed with OpenLab 3.1.2 software (Improvision Inc.).

For both organelle detection and immunofluorescence assays, data presented in each figure are representative of those found in over 90% of the population, unless otherwise noted.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A method for detecting intracellular GPIs. GPI-anchored proteins of L. major contain the glycolipid ethanolamine-phospho-Man{alpha}1-2Man{alpha}1-6Man{alpha}1-4GlcNH2-PI. GPIs of L. major are glycolipids that contain the core structure Man{alpha}1-4GlcNH2-Ins-1-phospho-diacylglycerol (36, 37); they include intermediates of the biosynthesis of the GPI anchor for L. major proteins as well as the prefabricated anchor that is transferred to protein. Synthesis of GPIs in eukaryotes is associated with the secretory system, e.g., the ER and Golgi (15, 29, 57). In most eukaryotes, including the protozoan L. major, intracellular sites of protein-GPI synthesis and/or GPI accumulation have not been established directly. To address this question, we developed a general assay to localize GPIs in eukaryotes. Two lectins, alpha-toxin and ConA, and a Leishmania cell line in which a protein-GPI deficiency could be induced were used to establish the conditions for the assay (see above).

ConA binds mannose-containing molecules including GPIs, GDP-mannose, and N-glycans (20, 33, 35, 41, 44, 48, 49). In contrast, alpha-toxin is highly specific for GPI-anchored proteins (12, 19, 62). The choice of Leishmania as the test cell is important because the protozoan contains both GPIs that are linked to polysaccharide (i.e., lipophosphoglycan) and GPIs that are attached to protein (e.g., gp63) (reviewed in references 21 and 26). Therefore, specificity of alpha-toxin for GPIs could be ascertained (or discredited). Equally relevant is the fact that a single L. major strain could conditionally be rendered deficient in GPIs that are found on protein but not lipophosphoglycan (40); this situation enables the selectivity of alpha-toxin in detecting GPIs to be further evaluated.

In L. major, heterologous expression of a GPI-PLCp from Trypanosoma brucei (8, 9, 23) causes a protein-GPI deficiency without affecting polysaccharide-linked GPIs (40). GPI-PLCp cleaves intermediates of protein-linked GPI biosynthesis in vivo, creating a shortage of prefabricated protein-linked GPIs. L. major stably transfected with a plasmid encoding GPI-PLCp was cultured in medium containing either 30 or 200 µg of G418/ml. The cells retained significant amounts of protein-GPIs if grown in medium containing 30 µg of G418/ml (40). However, when cultured in medium containing 200 µg of G418/ml, high expression of GPI-PLCp (from the extrachromosomal expression plasmid pX63NEO [30]) occurs, which leads to a protein-linked GPI deficiency; no deficiency of polysaccharide-linked GPIs is observed under these conditions (40). The presence of protein-GPI in cells containing the expression vector UTK (47) (i.e., pUTK/L. major) alone or pUTK-GPIPLC (i.e., pUTK-GPIPLC/L. major) was investigated with alpha-toxin. ConA binding sites were studied as a control.

For all cells cultured with 30 µg of G418/ml, both alpha-toxin and ConA detected intracellular glycans (Fig. 1A to D) in immunofluorescence assays. ConA staining zones included regions detected by alpha-toxin, in agreement with the expectation that alpha-toxin recognizes only protein-GPIs, whereas ConA detects other molecules in addition to the protein-GPIs. In protein-GPI-deficient cells (i.e., pUTK-GPIPLCp/L. major cells cultured in 200 µg of G418/ml), alpha-toxin binding sites disappeared (Fig. 1K), whereas some ConA sites persisted (Fig. 1J). In control pUTK/L. major cells cultured in 200 µg of G418/ml, both ConA and alpha-toxin sites were readily detected (Fig. 1F and G).



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  		FIG. 1. Detection of intracellular GPIs with alpha-toxin. Parasites were adapted to growth in either 30 µg (A to D) or 200 µg (E to L) of G418/ml. Cells were fixed, permeabilized, and incubated with alpha-toxin (2.5 µg/ml) for 30 min and then with ConA-Alexa Fluor 594 (5 µg/ml) for another 30 min. Anti-alpha-toxin antibody and Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG) (green) were added. ConA labeling (B, F, and J), alpha-toxin binding sites (C, G, and K), and digitally merged images (D, H, and L) are presented. The cell nucleus (n) and the kinetoplast (k) (mitochondrial DNA) were stained with DAPI (blue).

 
We surmise that a loss of intracellular alpha-toxin binding correlates with a protein-GPI deficiency in vivo (39, 40). Therefore, alpha-toxin binding sites detected under these conditions denote regions where the bulk of GPIs accumulate in L. major.

GPIs accumulate on endosomes of L. major. To identify the organelle on which protein-linked GPIs accumulated in L. major, double-labeling experiments were performed with alpha-toxin and organelle-specific markers. Alpha-toxin binding sites were not detected on the ER or Golgi (data not shown). Hence, protein-linked GPIs in Leishmania do not appear to concentrate on the Golgi or ER. To test whether alpha-toxin associated with the endolysosomal system, Leishmania cells were cultured in medium containing a low concentration of G418 (i.e., 30 µg/ml) to prevent extensive loss of protein-linked GPIs and allowed to endocytose FM4-64, a marker for the endolysosomal system (17, 55, 61). Afterwards, alpha-toxin binding sites were detected in the fixed, permeabilized cells containing FM4-64.

Binding sites for alpha-toxin coincided with endocytosed FM4-64 in both pUTK/L. major (Fig. 2A to D) and pUTK-GPIPLC/L. major (Fig. 2E to H) cells. Thus, intracellular protein-GPIs associate with the endolysosomal system of Leishmania.



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  		FIG. 2. Endocytosed FM4-64 colocalizes with intracellular GPIs. pUTK/L. major (A to D) and pUTK-GPIPLC/L. major (E to H) cells (both cultured in medium containing 30 µg of G418/ml) were exposed to FM4-64 (10 µM) for 30 min, fixed in paraformaldehyde, and permeabilized with CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} (0.05%, 4 min, 4°C). Cells were incubated with alpha-toxin (2.5 µg/ml) followed by anti-alpha-toxin antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). The positions of the nucleus (n) and the kinetoplastid (k) are revealed by DAPI staining (blue).

 
Alpha-toxin binds to Gp63 and to free GPIs from L. major. Gp63 is the major GPI-anchored protein on the plasma membrane of L. major (13, 14, 60). Alpha-toxin binds to GPI-anchored proteins (19). Yet the protocol that we developed (see above) did not detect GPIs on the plasma membrane of L. major (Fig. 1). To investigate the basis for this observation, we performed four studies. First, we checked whether alpha-toxin could bind to the plasma membrane of cells that had not been permeabilized with detergent (Fig. 3). Second, we determined whether alpha-toxin could bind to gp63 (Fig. 4A). Third, we investigated whether "free GPIs" are recognized by alpha-toxin (Fig. 4B). Fourth, we checked whether gp63 was detectable in detergent extracts of fixed, permeabilized L. major cells (Fig. 5).



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  		FIG. 3. Alpha-toxin and anti-gp63 antibody bind to the plasma membrane of nonpermeabilized Leishmania. pUTK/L. major (UTK) was allowed to endocytose FM4-64 (10 µM) for 30 min. After fixing in glutaraldehyde (0.5%, 10 min), alpha-toxin ({alpha}-toxin) (2.5 µg/ml) was added to the cells for 30 min. Subsequently, anti-alpha-toxin antibody (C) or anti-gp63 antibody (G and K) was added. Antibodies were detected (in separate experiments) with Alexa Fluor 488-conjugated goat anti-rabbit IgG. In panel J, biotinylated alpha-toxin was added, followed by streptavidin-Alexa Fluor 594. The cell nucleus (n) and the kinetoplast (k) were detected with DAPI staining (blue).

 


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  		FIG. 4. Alpha-toxin binds to free GPIs and to a GPI-anchored protein. (A) Alpha-toxin binds to gp63. Cell lysates (108 cell equivalents) were immunoprecipitated with anti-gp63 antibody (see Materials and Methods for details), separated by SDS-PAGE (107 cell equivalents per lane), and transferred to an Immobilon-P membrane, which was blotted in separate experiments with anti-gp63 antibody or alpha-toxin-biotin. Lane 1: the membrane was blotted with anti-gp63 antibody followed by alkaline phosphatase-conjugated goat anti-rabbit secondary antibody and color development. Lane 2: Gp63 was detected with biotinylated alpha-toxin followed by streptavidin-conjugated horseradish peroxidase secondary antibody and enhanced chemiluminescence. (B) Alpha toxin binds to free GPIs. Glycolipids extracted from L. major (108 cells) (lanes 1, 3, and 5) or purified phosphatidylethanolamine (12 µg) (lanes 2, 4, and 6) were resolved by HPTLC andexposed with 0.5% orcinol-H2SO4 (lanes 1 and 2). The glycolipids or phospholipids were revealed by blotting the HPTLC plates with either alpha-toxin-biotin (lanes 3 and 4) or ConA-biotin (lanes 5 and 6). Biotinylated alpha-toxin and ConA were detected with streptavidin-conjugated horseradish peroxidase and enhanced chemiluminescence. (C) PI-PLC cleavage of GPI glycolipids. Dried glycolipids extracted from L. major (108 cells) were resuspended in PI-PLC buffer and treated with PI-PLC (lanes 2 and 4) or buffer for the enzyme (lanes 1 and 3). Butanol-soluble products were resolved by HPTLC and blotted with alpha-toxin-biotin (lanes 3 and 4) followed by detection with streptavidin-conjugated horseradish peroxidase and enhanced chemiluminescence. Glycolipids exposed with 0.5% orcinol-H2SO4 are presented (lanes 1 and 2). *, GPI bound by alpha-toxin.

 


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  		FIG. 5. Effect of detergent treatment on cell surface gp63. (A) Flow chart of experimental scheme. (B) pUTK/L. major cells (107) were fixed mildly in 0.5 ml of 2% paraformaldehyde (8 min, 4°C), pelleted, and permeabilized in 0.5 ml of 0.25% Triton X-100 (5 min, 4°C). After centrifugation (14,000 x g, 5 min), the supernatant (1 ml) was saved, and the cell pellet was subjected to phase partition in Triton X-114. Proteins in the original cell pellet, supernatant, detergent, and aqueous phases of Triton X-114 partition were adsorbed to anti-gp63 antibody-protein A-Sepharose. Fifteen microliters of protein (107 cells equivalents per lane) was electrophoresed and transferred to an Immobilon-P membrane, which blotted with anti-gp63 antibody followed by detection with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody and color development. Lane 1, supernatant (S) from cell extraction with Triton X-100; lane 2, pellet (P) from fixed cells extracted with Triton X-100; lane 3, detergent (D) phase from Triton X-114 partition of pellet; lane 4, aqueous (A) phase from Triton X-114 partition of pellet.

 
To investigate whether cell surface GPIs could be detected with alpha-toxin, cells (pUTK/L. major) were first allowed to endocytose FM4-64 to mark endosomes. Subsequently, the parasites were fixed with 0.5% glutaraldehyde and stained with either alpha-toxin or anti-gp63 antibody. No detergent permeabilization of cells was used in this protocol. Alpha-toxin bound the plasma membrane of L. major (Fig. 3C and G) without associating with endosomal FM4-64 (Fig. 3D). Hence, in the absence of cell permeabilization, alpha-toxin does not have access to intracellular GPIs (Fig. 2). Similar observations were made with anti-gp63 antibody (Fig. 3E to H).

These observations indicate that glutaraldehyde fixation neither permeabilizes Leishmania nor extracts GPI-anchored proteins. Intracellular protein-GPIs can be detected only with alpha-toxin in permeabilized cells.

Alpha-toxin binds to GPI-anchored proteins (19). However, there is no direct evidence that the protein binds to gp63. To test whether alpha-toxin bound gp63, the L. major protein was immunoprecipitated from the parasites, and the immunoprecipitate was separated by SDS-PAGE. After transfer to Immobilon-P membrane, gp63 was detected with biotinylated alpha-toxin (Fig. 4A). A duplicate membrane was blotted with anti-gp63. Alpha-toxin detected immunoprecipitated gp63 with a molecular mass of about 65 kDa (Fig. 4A, lane 2). In the control experiment, the immunoprecipitated gp63 was also recognized by anti-gp63 antibody, as expected (Fig. 4A, lane 1). We conclude that alpha-toxin binds to gp63.

Intracellular alpha-toxin binding sites in L. major (Fig. 2 and 3) may represent free GPIs (22, 46, 54). However, there is no report of alpha-toxin binding to free GPIs from any organism. We therefore tested whether (or not) alpha-toxin recognized free GPIs from L. major. For this goal, glycolipids extracted from L. major were separated by HPTLC and detected by orcinol staining. GPIs among the orcinol-stained glycolipids were revealed by (i) their susceptibility to PI-PLC and (ii) alpha-toxin blotting of the HPTLC plates (Fig. 4).

Alpha-toxin binds to polar glycolipids from L. major (Fig. 4B, lane 3) but not to purified phosphatidylethanolamine (Fig. 4B, lane 4), which was detectable with orcinol (Fig. 4B, lane 2). In a control experiment, ConA bound glycolipids from L. major (Fig. 4B, lane 5) as reported previously (43). To ascertain that the polar glycolipids recognized by alpha-toxin (Fig. 4B, lanes 3 and 5) were GPIs, the glycolipids from L. major were either treated or not treated with PI-PLC before the toxin blot. PI-PLC digested the GPIs in the organic extract from L. major. Orcinol staining exposed GPIs that were lost after PI-PLC cleavage (Fig. 4C, compare lane 2 to lane 1). Without PI-PLC digestion, the GPIs were bound by alpha-toxin (Fig. 4C, lane 3). Glycolipids that bound alpha-toxin were no longer detected after PI-PLC digestion of the glycolipids (Fig. 4C, lane 4). We conclude that the glycolipids bound by alpha-toxin are GPIs.

The possibility that gp63 was extracted from the plasma membrane of L. major during detergent permeabilization of fixed cells was investigated. For this purpose, cells were fixed and then permeabilized with Triton X-100 (0.25%). After centrifugation, the pellet (i.e., the "cell ghost") and supernatant fractions were immunoprecipitated with anti-gp63 antibody and developed by Western blotting (see an outline of the protocol in the legend of Fig. 5A). Supernatant from the detergent permeabilization step contained gp63 (Fig. 5B, lane 1). Similarly, the cell ghost (i.e., the pellet) contained gp63 (Fig. 5B, lane 2). We conclude that Triton X-100 (0.25%) extracts some gp63 from fixed L. major cells. However, a significant proportion of the protein remains cell associated (Fig. 5B, lane 2).

To test whether the cell-associated gp63 from the detergent permeabilization stage contained a GPI anchor, we analyzed an aliquot of that cell pellet by Triton X-114 phase partitioning (Fig. 5B, lanes 3 and 4). GPI-anchored gp63 associates with the detergent phase after Triton X-114 phase partitioning (6, 14). The majority of the cell ghost-associated gp63 was found in the aqueous phase of the Triton X-114 phase separation (Fig. 5B, compare lanes 3 and 4). Therefore, gp63 associated with the cell ghost is not GPI anchored. This conclusion is strengthened by data presented in Fig. 6 (see below).



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  		FIG. 6. (A to D) Intracellular gp63 does not associate with endolysosomal system. pUTK-GPIPLC/L. major cells (cultured in medium containing 30 µg of G418/ml) were exposed to FM4-64 (10 µM) for 30 min (red). Cells were fixed with 2% paraformaldehyde (8 min, 4°C), permeabilized with 0.05% CHAPS (4 min, 4°C), and incubated with anti-gp63 antibody followed by detection with Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). (E to H) GPIs do not colocalize with intracellular gp63. Parasites were fixed, permeabilized (0.25% Triton X-100, 5 min, 4°C), and incubated with anti-gp63 antibody and alpha-toxin-biotin. Streptavidin-conjugated Alexa Fluor 594 (red) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies (green) were added. (I to L) Intracellular gp63 is found in a subregion of the ER. L. major cells were fixed, permeabilized (0.25% Triton X-100, 5 min, 4°C), and incubated with anti-gp63 antibody and anti-Sec61p-biotin, followed by detection with streptavidin-conjugated Alexa Fluor 594 (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). The nucleus (n) and the kinetoplast (k) were stained by DAPI (blue).

 
Intracellular gp63 is associated with the ER. We found earlier that protein-GPI-deficient L. major lacks intracellular alpha-toxin binding sites (Fig. 1K). Since gp63 is a major GPI-anchored protein in these cells, it was possible that the intracellular GPI detected by alpha-toxin was not free GPI (i.e., glycolipids containing Man{alpha}1-6Man{alpha}1-4GlcNH2-PI) (see above) but gp63 that had been internalized from the plasma membrane. To evaluate this possibility, we tested whether intracellular gp63 was predominantly associated with the endolysosomal system. FM4-64 colocalized with alpha-toxin (Fig. 2), so the dye was initially used to reveal (indirectly) alpha-toxin binding sites.

Intracellular gp63 was not coincident with endocytosed FM4-64 in double-labeling experiments (Fig. 6A to D). The implication of this result is that most intracellular gp63 is absent from the endolysosomal system. These data rule out the possibility that the intracellular alpha-toxin binding sites are derived from endocytosed gp63.

Further evidence for separation of free GPIs and gp63 was obtained by double labeling cells with alpha-toxin-biotin and anti-gp63 antibody (Fig. 6E to H). Most of the gp63 in the permeabilized cells failed to colocalize with free GPIs detected directly with biotinylated alpha-toxin (Fig. 6H). These results support earlier conclusions that intracellular gp63 does not colocalize with protein-linked GPIs (Fig. 6A to D).

We tested whether intracellular gp63 was associated with the ER, because the protein is expected to move through that organelle en route to the plasma membrane. Antibody against the ER membrane protein Sec61p was used as a marker for the ER (Fig. 6J). Membranes of the ER were detected in a tubule-like structure that was found near the cell nucleus and in anterior regions of L. major (Fig. 6J). In double-labeling experiments with anti-gp63 and biotinylated anti-Sec61p, gp63 was found in the ER but only in the anterior sections (Fig. 6L). From this data, we conclude that intracellular gp63 concentrates in a subregion of the ER.

Microscopy evidence suggested that intracellular gp63 lacked the GPI anchor (Fig. 6H). To test whether biochemical evidence would support this claim, we investigated whether all gp63 in L. major could be digested by PI-PLC. To achieve this objective, the effect of PI-PLC digestion on phase partition of gp63 into Triton X-114 was analyzed (Fig. 7). In the absence of PI-PLC digestion, gp63 was detected in both aqueous and detergent phases of the Triton X-114 separation (Fig. 7A, lanes 1 and 2). When the lysate was treated with PI-PLC, most of the gp63 in the detergent phase was no longer present (Fig. 7, compare lanes 3 and 4). That is, gp63 in the detergent phase is GPI anchored and susceptible, consequently, to PI-PLC cleavage.



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  		FIG. 7. Some cell-associated gp63 lacks GPI. (A) Parasites (108 cells) were lysed in buffer containing Triton X-114 (TX-114) (0.5%). The lysates were treated with PI-PLC (lanes 3 and 4) or buffer for the enzyme (lanes 1 and 2) and subjected to phase partition in Triton X-114. gp63 was immunoprecipitated from detergent and aqueous phases, separated by SDS-PAGE, and transferred to Immobilon-P membrane. Membranes were blotted with anti-gp63 antibody followed by alkaline phosphatase goat anti-rabbit secondary antibody and color development. Lanes 1 and 3, detergent (D) phase of Triton X-114 partition; lanes 2 and 4, aqueous (A) phase of Triton X-114 partition. (B) An aliquot of Triton X-114 aqueous phase (107 cell equivalents) was subjected to further phase partition in Triton X-114. Immunoprecipitated gp63 from detergent and aqueous phases was electrophoresed, transferred to Immobilon-P membrane, and blotted with anti-gp63 antibody followed by alkaline phosphatase-conjugated goat anti-rabbit secondary antibody and color development. Lane 1, detergent phase of Triton X-114 partition; lane 2, aqueous phase of Triton X-114 partition.

 
Since there was concern that some gp63 partitioned into the aqueous phase of the Triton X-114 separation because the detergent phase was saturated with protein, a portion of the aqueous phase was subjected to a second round of phase partitioning with Triton X-114 (Fig. 7B). gp63 in this aqueous phase partitioned again into the aqueous phase (Fig. 7B, compare lanes 1 and 2). These data indicate that L. major contains a pool of gp63 that is not GPI anchored, consistent with data from the microscopy analysis (Fig. 6H).

Killing of Leishmania by alpha-toxin. Alpha-toxin, as a protoxin, recognizes GPI-anchored proteins on the plasma membrane of live cells (19, 25). Following cleavage in a polybasic region close to the C terminus by a furin protease (18), the protoxin is activated to monomers that aggregate to form a hexameric or heptameric prepore complex (51). At physiological temperatures, a conformational change in the prepore complex leads to insertion of beta barrel regions of alpha toxin into membranes, creating lytic pores (reviewed in reference 3).

L. major contains GPI-anchored gp63 (5 x 105 molecules/cell) as a major cell surface protein (7, 10, 14). Given the specificity of alpha-toxin for GPI-anchored proteins, it seemed reasonable to test whether the toxin could lyse the parasite.

Alpha-toxin was highly effective against L. major. The 50% lethal concentration of the protein was 0.77 nM (calculated as a monomer) under our experimental conditions (Fig. 8). These results are reminiscent of the effect of alpha-toxin on Toxoplasma gondii, which also has a high concentration of GPI-anchored proteins on its plasma membrane (62).



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  		FIG. 8. Lysis of Leishmania by alpha-toxin. L. major CC-1 (untransfected) (107 cells/ml) was pelleted and resuspended in PBS supplemented with 1.0% glucose (pH 7.4) at a density of 5 x 106 cells/100 µl in 96-well plates. Purified alpha-toxin in 100 µl of PBS supplemented with 1.0% glucose (final concentration of up to 42 ng/ml) was added. After incubation at 27°C for 12 h, cell number (mean of duplicates) was obtained. Data presented are representative of three independent experiments that produced similar results.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Alpha-toxin expands the repertoire of tools for studying intracellular GPIs. GPIs can serve as membrane anchors for proteins and polysaccharides (16, 24, 53), although intracellular GPIs that are not linked to any macromolecules are also present. In vitro biosynthesis of GPIs and isolation of genes needed for GPI pathways have been accomplished (reviewed in references 15, 29, and 37). However, the subcellular location of GPIs within the secretory system remains unclear. Discovery of the location of GPIs in vivo will be facilitated by reagents that are highly specific for GPIs.

Alpha-toxin can reveal the location of GPIs within cells (Fig. 1). Combined with markers of "substations" of the secretory system (e.g., Golgi, ER, endosomes, and lysosomes), the technique enables determination of those regions within the secretory pathway where GPIs accumulate. We emphasize that the protocol does not necessarily reveal the site of synthesis; instead, it identifies regions of GPI concentration.

Leishmania is a good test case for developing the methodology for detection of intracellular GPIs because the cells contain polysaccharide-linked GPIs (e.g., lipophosphoglycan) and protein-GPIs. If alpha-toxin bound to polysaccharide-linked GPIs, no difference in the fluorescence signal would have been noted in a comparison of the protein-GPI-deficient and control cells (Fig. 1). The technique developed here is easily adapted for work with other eukaryote cells.

GPIs on L. major endosomes. In L. major, protein-linked GPIs accumulated on endosomes (Fig. 2), in agreement with results from another study. Glycoinositol phospholipids are found on megasomes (late endosomes-lysosomes) of the intracellular (amastigote) stage of Leishmania mexicana (63).

These data have two possible implications. Protein-GPIs may be synthesized in the secretory system, after which they are transported to endosomes. Alternatively, protein-GPIs may be synthesized on endosomes. We cannot distinguish between these possibilities with the data at hand.

Presence of unanchored gp63 in L. major and its localization in the ER. L. major gp63 is the major cell surface protein of promastigotes (i.e., insect stage). The protein is encoded by seven genes, six of which contain a COOH-terminal signal peptide for GPI anchoring (58). A majority of the gp63 protein in L. major is expected on the plasma membrane, anchored by a GPI. Unanchored gp63 is not retained in the parasite; it is secreted from L. major (13, 38, 40). Transport of gp63 to the plasma membrane and secretion of the protein into the extracellular environment involve translocation of the protein into the ER, because of the N-terminal signal peptide that is found on all gp63 coding sequences. Hence, one would expect that some gp63 might be detected in the secretory pathway, as previously reported in one study (60).

Our biochemical (Fig. 5B and 7A and B) and microscopic (Fig. 6F to H) analyses indicate that a proportion of intracellular gp63 in L. major does not have a GPI anchor. Unanchored gp63 accumulates in the ER (Fig. 6I to L). Two hypotheses may explain the gp63 in the ER. First, gp63 in the ER may be the pool of newly synthesized protein that will receive a GPI at a later time point. Since we could not detect GPIs in the ER, we cannot rule out the possibility that the addition of GPIs to gp63 takes place in organelles other than the ER. In support of this explanation, the optimum pH of GPI transamidase, the enzyme that adds GPI to protein, is acidic (28). GPIs accumulate at acidic organelles (i.e., endosomes) (Fig. 2). Therefore, it is conceivable that gp63 receives its GPI anchor in endosomes. This explanation will be more convincing if there was evidence that gp63 could be transported directly from the Golgi to endosomes. In vertebrates and yeast, Golgi-to-vacuole and trans-Golgi network-to-endosome protein transport have been reported (5, 11, 52, 59). In L. major, it is not known whether the pathway exists.

A second hypothesis to explain presence of gp63 in the ER is that cell surface and ER gp63 are encoded by different genes. All seven GP63 genes contain N-terminal signal peptides for entry into the ER, but only six GP63 genes are predicted to have a COOH-terminal GPI signal peptide (reviewed in reference 64). GP63-6 appears to have a COOH-terminal trans-membrane domain instead of a GPI signal peptide. (Apparent GPI anchoring of GP63-6 [58] is controversial [64].) After entry of the protein into the ER, the trans-membrane domain of Gp63-6 could retain the polypeptide in the organelle, allowing detection of the protein in our study (Fig. 6F to H). (Currently, there are no antibodies for distinguishing between different gp63s.)

Alpha-toxin for reduction of Leishmania transmission? Leishmania is transmitted to humans from the bite of a sand fly (Phlebotomine spp.). Since alpha-toxin kills promastigotes (insect stage) of Leishmania (Fig. 8), the protein could be useful for controlling transmission of the parasite from insects to humans. If phlebotomine species could be genetically engineered to express alpha-toxin in their gut, parasites ingested by the fly may be killed before a Leishmania infection is established in the insect. In this way, transmission of parasites from flies to humans may be reduced. That said, a large amount of work, including tests of vector fitness, will need to be performed before feasibility of using alpha-toxin as a biological control agent against Leishmania transmission can be ascertained. Related transmission-blocking strategies for parasite control are being pursued for some pathogens, including Plasmodium spp. (1).


   ACKNOWLEDGMENTS
 
This study was supported by National Institutes of Health grant AI53086 to K.M.-W.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Cellular Biology, The University of Georgia, 724 Biological Sciences, Athens, GA 30602. Phone: (706) 542-3355. Fax: (706) 542-4271. E-mail: mensawil{at}cb.uga.edu. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Eukaryotic Cell, March 2005, p. 556-566, Vol. 4, No. 3
1535-9778/05/$08.00+0     doi:10.1128/EC.4.3.556-566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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