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Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterized. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analyzed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant negative mutants. While TMK39 co-localized with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilized for non-redundant functions by the parasite.
Cell surface receptors mediate the response to the environment across all forms of life. In metazoan organisms and plants, transmembrane kinases (TMKs) make up one of the major classes of cell surface receptors, with humans encoding about 80 TMKs (Manning et al., 2002) and Arabidopsis thaliana encoding over 400 (Champion et al., 2004; Shiu et al., 2004). The paradigm of receptor-mediated signaling governing cellular responses to environmental cues in plants and metazoa has not been fully extended to protozoa because protozoa have a general paucity of TMKs. However, examples of protozoan TMKs are beginning to be discovered and include nine predicted TMKs in Dictyostelium discoideum (Goldberg et al., 2006), 10 potential TMKs in Trypanosoma brucei (Parsons et al., 2005), 11 putative TMKs in Plasmodium (Ward et al., 2004), 88 predicted TMKs in Monosiga brevicollis (King and Carroll, 2001; Manning et al., 2008) and over 90 novel TMKs predicted in the protozoan parasite Entamoeba histolytica (Beck et al., 2005). The significance of these proteins remains unclear, as the majority have been characterized by sequence analysis only. A more complete understanding of protozoan TMKs will help define the mechanisms that these organisms use to respond to their environment and may shed light on the evolution of eukaryotic protein kinases.
The large family of novel TMKs identified in the E. histolytica genome has proposed roles in both amebic response to the environment and immune evasion (Beck et al., 2005). Entamoeba histolytica is the causative agent of amebiasis, a disease responsible for significant morbidity and mortality worldwide (WHO/PAHO/UNESCO, 1997). The parasite’s biphasic life cycle consists of transmissible cysts and replicating trophozoites that colonize the lumen of the large intestine and occasionally invade the mucosa. Trophozoites must survey and adapt to the complex intestinal milieu and evade the immune system, but mechanisms that regulate the parasite’s ability to persist for months within its human host remain incompletely understood.
In protozoan parasites such as Giardia lamblia, Plasmodium falciparum and Trypanosoma brucei, antigenic variation, or the alteration of immunodominant surface antigens, is a common mechanism used to subvert host defenses (Adam et al., 1988; Su et al., 1995; Stockdale et al., 2008). The process requires three elements: a large gene family encoding antigenically distinct surface proteins, expression of one variant antigen at a time by a single pathogen and a mechanism to switch the expressed gene (Borst and Genest, 2006). The E. histolytica TMKs have been implicated in the process of antigenic variation due to the nature of the gene family, the course of amebic infection and observations made during transcriptional profiling studies (Beck et al., 2005). Additionally, some E. histolytica TMKs share sequence similarity with the variant-specific surface proteins (VSP) that are involved in the process of antigenic variation in Giardia lamblia (Beck et al., 2005).
It is possible that Entamoeba trophozoites undergo antigenic variation, as prolonged E. histolytica infections do occur (Haque et al., 2002) and antibody mediated protective immunity against Entamoeba is incomplete (Haque et al., 2001). However, trophozoites are known to use the process of capping, whereby antibody-antigen complexes are concentrated and released from the cell surface, as a means to avoid immune attack (Calderón et al., 1980). Additionally, amebic trophozoites directly kill and ingest host cells, providing the organism with another mechanism for immune evasion (Ravdin et al., 1980). Nonetheless, it remains possible that the unusual TMK family may be involved with the process of antigenic variation. Real time PCR analysis of TMK expression by trophozoites during growth in culture revealed temporal changes in expression levels of some TMKs (Beck et al., 2005). As antigenic variation is known to occur without immune pressure (Roberts et al., 1992), the observed changes could be indicative of an antigenic switching event, where the averaging of population data masked expression of a single TMK by each cell.
Changes in TMK expression levels could also indicate that TMKs have specialized functions, as the TMKs have also been proposed to represent a major receptor system used by the cell to sense and respond to extracellular cues. The structural organization of the E. histolytica TMKs suggests that they are type 1 integral membrane proteins, with signal-peptides, receptor-like extracellular domains and intracellular kinase domains, phylogenetically related to both S/T and Y kinases (Beck et al., 2005). When the TMKs were divided into nine sub-groups (A, B1–3, C, D1–2, E and F) based on signature motifs found within the substrate recognition regions of their kinase domains, similarity in the extracellular domains became apparent within sub-groups with respect to the size and the distribution of cysteine-rich motifs, suggesting that TMK subgroups represent functionally distinct receptor families with sub-family-specific substrates and ligands (Beck et al., 2005). However, only two TMKs have been partially characterized to date: PaTMK (TMK96, Subfamily B3) is expressed at the cell surface and functions in erythrophagocytosis (Boettner et al., 2008) and members of the B1 family of TMKs play a role in proliferation and sensitivity to serum-derived growth factors (Mehra et al., 2006).
In this study, we sought to determine whether TMKs represent a gene family that undergoes antigenic variation or are an example of a protozoan TMK family that likely represents a major cell surface receptor system. We used laser capture microdissection (LCM) and single cell microarray analysis and determined that a single ameba expresses more than one TMK. To confirm expression of some TMKs at the protein level, anti-peptide antibodies were developed against TMK39 and TMK54. These TMKs were chosen because microarray data previously indicated that the genes were highly transcribed (amongst the TMKs) in both cultured and animal passaged trophozoites (Gilchrist et al., 2006), and TMK39 was identified at an early time point in a phagosomal proteome (Okada et al., 2006). We used the anti-peptide antibodies to stain cells for flow cytometric analysis and determined that the TMKs were homogenously expressed by trophozoites within a population. The antibodies were also used to localize TMK39 and TMK54 to discrete regions of the amebic plasma membrane. We then utilized a functional genetic approach to demonstrate that TMK39 and TMK54 likely serve non-redundant cellular functions.
Entamoeba histolytica trophozoites, strain HM-1:IMSS, were grown axenically at 37°C in complete TYI-S-33 medium containing 100 U/ml of penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) (Diamond, 1961). For all experiments, trophozoites were harvested during log-phase growth by a 10 min incubation on ice. For LCM analysis, harvested trophozoites were allowed to adhere to PEN foil-coated glass slides specifically designed for laser microdissection (Leica Microsystems, Bannockburn, IL, USA) for 15 min at 37°C in TYI-S-33 media. Adherent trophozoites were sequentially fixed for 5 min in 70% and 100% ethanol followed by a few dips in xylene to completely dehydrate the samples and air-dried. Subsequently, single cells were captured from the PEN slides using the Leica AS LMD microdissection system (Leica Microsystems, Bannockburn, IL, USA). Captured cells were immediately processed as described below.
RNA was purified from a single ameba using the PicoPure™ RNA Isolation Kit (Molecular Devices, Sunnyvale, CA, USA) and the WT-Ovation™ Pico System (NuGEN, San Carlos, CA, USA) was used for cDNA synthesis and amplification. The quantity of cDNA obtained from one amplification cycle was insufficient for microarray analysis. Therefore one cycle amplified cDNA (1C) was subjected to a second cycle of amplification. Prior to microarray analysis, the linearity of the relationship between 1C and twice amplified cDNA (2C) was validated by quantative reverse transcription PCR (qRT-PCR) (Section 2.4). 2C from a single cell was used for biotinylated cRNA synthesis. After biotinylation, 2 µg of cRNA was hybridized to the E_his-1a520285 Affymetrix custom array that has been described elsewhere (Gilchrist et al., 2006). The arrays were washed and stained with streptavidin-phycoerythrin (Molecular Probes, Carlsbad, CA, USA), following the standard Affymetrix protocol for eukaryotic targets (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). The arrays were scanned with an Affymetrix Gene Chip scanner 3000l and Affymetrix® GeneChip® Operating Software (GCOS) (http://www.affymetrix.com/products/software/specific/gcos.affx) was used to determine the detection call (present, marginal, absent) for each probe set. The experiment was carried out in duplicate. Additionally, raw data from the arrays were normalized at the probe level by the gcRMA algorithm and then log2 transformed (Irizarry et al., 2003). The average log intensity values for all TMKs and for a few reference genes are listed in Supplementary Table S2. The complete microarray data was deposited in NCBI’s Gene Expression Omnibus (Barrett et al., 2005) and is accessible through GEO Series accession number GSE19064 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19064).
The re-annotated E. histolytica genome, available at http://pathema.tigr.org, (GenBank accession number AAFB00000000) was used in this analysis.
For validation of RNA amplification procedures, RNA from 106 E. histolytica trophozoites was prepared using the PicoPure™ RNA Isolation Kit and then subjected to either one or two cycles of amplification with the WT-Ovation™ Pico System. Two rounds of amplification yielded approximately 1.67 times more RNA than one round. 1C and 2C were adjusted to the same concentration and qRT-PCR was performed for 10 TMKs (TMK6, 56, 19, 60, 39, 63, 40, 65, 42 and 71) as previously described (Beck et al., 2005). See Supplementary Table S1 for primer sequences and annealing temperatures. The TMK threshold cycles (CTs) for 1C and 2C were then compared. As shown in Supplementary Fig. S1, 1C and 2C yielded similar CT values for all TMKs examined, indicating that the linearity of amplification was maintained throughout the second cycle.
For microarray validation, cDNA was prepared from a single cell as described above. The 2× amplified cDNA was diluted 1:100 with H2O and qRT-PCR was carried out using iQSYBRGreen super mix (Bio-Rad, Hercules, CA, USA) and previously developed methods (Beck et al., 2005). Supplementary Table S1 lists primer sequences and annealing temperatures. Two “present” and two “absent” transcripts were selected for validation, and as a positive control cDNA was prepared from 106 trophozoites.
Peptides corresponding to amino acids (aa) 491–506 of TMK39 and 242–254 of TMK54 were synthesized, conjugated to Keyhole Limpet Hemocyanin and used to immunize New Zealand White rabbits. This work was contracted to Covance Research Products Inc.; formal animal ethics approval was obtained and animal treatment was in accordance with all applicable laws and regulations. The resultant serum was affinity purified using immobilized peptide and dialyzed against PBS. Resulting anti-TMK39 and anti-TMK54 antibodies were stored at −80°C until u se. Antibodies against TMK96 (PaTMK) and the heavy subunit of the Gal/GalNAc lectin (Hgl) have been previously described ( Petri et al., 1989; Boettner et al., 2008). Negative control, anti-Ft, an antibody which is directed against a Francisella tularensis protein, was a kind gift from Nicole Ark and Barbara Mann at the University of Virginia, USA. Polyclonal anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal anti-V5 (Sigma) antibodies were commercially available. For Western blotting, polyclonal antibodies were used at a concentration of 5 µg/ml, whereas monoclonal anti-V5 was used at 1µg/ml. For confocal microscopy and flow cytometry antibody concentrations were doubled.
Harvested trophozoites (HM-1:IMSS or induced HM-1:IMSS transfectants) were washed in PBS and lysed at a concentration of 104 amebae/µL (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and 0.02 mM E-64 (Sigma, St. Louis, MO, USA)). Cell lysate, immunoprecipitation or fractionated cellular sub-fractions (below) were resolved in 10% SDS-PAGE gels, transferred to polyvinylidene fluoride membrane using standard methods and membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TTBS) for 1 h at room temperature (RT). If noted, the membrane was cut into strips or primary antibodies (5 µg/ml) were pre-incubated with the indicated amount of unconjugated peptide for 1 h at RT prior to use. Primary antibodies diluted in TTBS were incubated with blocked membranes for 1 h at RT, membranes were washed with TTBS (5 × 5 min) and exposed to secondary antibody (anti-rabbit:AP or anti-mouse:AP) at a concentration recommended by the manufacturer (Sigma, St. Louis, MO, USA) for 1 h at RT. Finally, membranes were washed five times in TTBS and bands were visualized on film using the enhanced chemiluminescent (ECL) kit (Roche, Indianapolis, IN, USA).
Late-log phase E. histolytica trophozoites were harvested, washed twice with PBS and fixed with 3.7% paraformaldehyde (PFA) in PBS for 30 mins at RT. If indicated, cells were permeabilized for 1 min with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) in PBS and non-specific binding was blocked by incubation with 20% goat serum and 5% BSA for 1 h at 37°C. To assess TMK levels, permeabilized HM-1:IMSS trophozoites were stained for 1 h at 37°C with anti-TMK39, anti-TMK54, anti-PaTMK (TMK-96), anti-Hgl (as a positive control) or anti-Ft (as a negative control).
To assess lectin levels, both non-permeabilized (cell surface Hgl) and permeabilized (total Hgl) transfected trophozoites were stained with anti-Hgl for 1 h at 37°C. In both instances, Cy™3-conjugated Goat Anti-Rabbit IgG (Jackson Immuno Research, West Grove, PA, USA) was used as the secondary antibody at a 1:200 dilution in blocking buffer. As a control, cells were stained with secondary antibody only (no primary antibody). After 1 h incubation at 37°C with the secondary antibody, samples were washed three times in PBS, resuspended in 200 µl of PBS and analyzed using a FACSCalibur (BD Biosciences) on channel FL2. In all instances, an intact ameba gate was set prior to data collection (using side scatter (SSC) and forward scatter (FSC) and 10,000 gated events were collected for each sample. FlowJo software (http://www.treestar.com/flowjo/) was used for data analysis. All experiments were carried out three times or more and representative overlaid FL2 histograms are shown.
Cellular fractionation was carried out as previously described (Aley et al., 1980). Briefly, 108 trophozoites were harvested, washed twice with 19 mM potassium phosphate buffer, pH 7.2, and 0.27 M NaCl (PD). Cells were re-suspended to 2 × 107 amebae/ml in PD + 10 mM MgCl2 and mixed with an equal volume of 1 mg/ml concanavalin A in the same buffer. After 5 min at RT, cells were centrifuged at 50 g for 1 min and the supernatant (containing excess conA) was discarded. The pellet was re-suspended in 12 ml of a hypotonic buffer containing 10 mM Tris-HCl, pH 7.5, 2 mM PMSF (Tris buffer) and 1 mM MgCl2. After a 10 min swell, the cells were homogenized using 18–20 strokes of a glass Dounce homogenizer. A two-step gradient consisting of 0.5 M mannitol (8 ml) over 0.58 M sucrose (4 ml), both in Tris buffer, was prepared (gradient 1). The homogenate was layered on top and then centrifuged at 250 g for 30 min. Large plasma membrane fragments formed a pellet at the bottom of gradient 1. The material remaining above gradient 1 was spun at 40,000 g for 1 h to separate soluble cytoplasmic components (supernatant) from internal membranes (pellet). The plasma membrane pellet from the bottom of gradient 1 was re-suspended in 1 ml of Tris buffer + 1 M α-methyl mannoside and iced for 40 min with occasional mixing. The mixture was diluted into 3 vol. of Tris buffer and homogenized with 80 strokes of a Dounce homogenizer. The homogenate was layered onto 20% sucrose in Tris buffer (gradient 2) and centrifuged at 250 g for 30 min. Vesiculated plasma membranes that remained above gradient 2 were collected and concentrated by centrifugation at 40,000 g for 1 h. The resulting pellet was re-suspended in Tris buffer and served as the plasma membrane fraction.
The three fractions used for analysis (soluble, internal membranes, plasma membranes) were adjusted to equal volumes and analyzed by Western blotting. As TMK39 and Hgl are similar in size (respectively, 127 kDa and 170 kDa), membrane panels were first probed with anti-TMK antibodies and developed, then stripped with ReBlot Plus Strong Antibody Stripping solution (Millipore, Billerica, MA, USA) and re-probed with anti-Hgl antibodies.
Entamoeba histolytica trophozoites (HM-1:IMSS) in TYI-S-33 medium were allowed to adhere to glass coverslips in a 24-well plate for 1 h at 37°C at a concentration of 5.0 × 105 trophozoites/well. Adherent amebae were washed with warm PBS and fixed with 3.7% PFA for 30 min at RT. Non-specific binding was blocked with 20% goat serum and 5% BSA (Sigma, St. Louis, MO, USA) in PBS (1 h at 37°C). Cells were stained for 1 h at 37°C with anti-TMK antibodies diluted in blocking buffer. If indicated, primary antibodies were pre-incubated with 300 nM unconjugated peptide for 1 h at RT. Cells were then washed three times with PBS and Cy3-conjugated goat anti-rabbit secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA) were added at a 1:200 dilution (in blocking buffer) for 1 h at 37°C. After three washes, coverslips were mounted to slides with Fluoromount-G (Southern BioTech, Birmingham, AL, USA). A Zeiss LSM 510 laser-scanning microscope was used to visualize cells and final images were analyzed using LSM Image Browser software (Carl Zeiss, Inc., Thornwood, NY, USA).
For expression of truncated proteins containing V5 and 6× His tags, the indicated regions of TMK39 and TMK54 were PCR amplified with the primers: 39F -CACC ATG TTT CTT TTA TTT ACA ATC CTC, 39R - AAT AAT AAT AAG AAT AAT CAC AAT CAG, 54F - CACC ATG TTG CTT CTT TTT TCA CTT ATT TCA, 54R - ACC AAG AAA TAT TAA AAT AGA TAA TAT AG. These fragments were cloned into the Gateway pENTR™/SD/D-TOPO® (Invitrogen) plasmid, sequence verified and Gateway® LR Clonase™ II Enzyme Mix (Invitrogen) was used, according to manufacturer’s instructions, to transfer the truncated TMK fragments into the Gateway® pET-DEST42 vector in-frame with the C-terminal epitope tags. Truncated TMK fragments and tags were then PCR amplified from the pET-DEST42 vector with N-terminal KpnI and C-terminal BamHI restriction sites using the primers: K39F - CTA CTG GGT ACC ATG TTT CTT TTA TTT ACA ATC CTC, K54F - CTA CTG GGT ACC ATG TTG CTT CTT TTT TCA CTT ATT TCA, BamHisR - ATA ATG GGA TCC TCA ATG GTG ATG GTG ATG ATG. Resulting PCR products were cloned into the KpnI and BamHI sites of the digested and gel purified pEhHYG-tetR-O-CAT vector (Hamann et al., 1997). Final constructs were sequence verified and the parental pEhHYG-tetR-O-CAT vector was used as a control.
The GenElute™ HP Plasmid Maxiprep Kit (Sigma, St. Louis, MO, USA) was used to prepare plasmid DNA and DNA was quantified using the NanoDrop™ 2000 (Thermo Fisher, Wilmington, DE, USA). A known quantity of DNA was precipitated using standard methods and re-suspended to a concentration of 200 µg/ml in supplemented (5.7 mM cysteine, 25 mM HEPES and 0.6 mM ascorbic acid) and filter-sterilized Medium 199 (M199S) (Invitrogen, Carlsbad, CA, USA), that had been adjusted to pH 7.0. One hundred µl of the DNA (20 µg) was mixed with 15 µl of Attractene or SuperFect (Qiagen, Valencia, CA, USA) and incubated as per the manufacturer’s instructions to allow formation of transfection complexes. Log-phase trophozoites were then harvested on ice, washed in M199S and re-suspended to a concentration of 5.0 × 105 amebae/ml in M199S supplemented with 15% heat-inactivated bovine serum. Processed amebae (0.9 ml) were added to transfection complexes and incubated for 3 h at 37°C. After the incubation period, amebae were added to 25 cm2 tissue culture flasks containing complete TYI-S-33 medium supplemented with 100 U/ml of penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). After 18 h at 37°C, transfected cells were selected using 15 µg/ml hygromycin (Invitrogen, Carlsbad, CA, USA). Debris from dead cells was removed and fresh media added beginning 4–5 days post-selection. Approximately 2 weeks after selection, transfectants obtained log-phase growth. Following 24 h of induction with 10 µg/ml of tetracycline, expression was verified by Western blotting using a monoclonal anti-V5 antibody (Sigma) as described.
Transfected cells were induced for 24 h and lysed on ice at a concentration of 107 amebae/ml in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and 0.02 mM E-64 (Sigma). Cellular debris was removed by centrifugation at 9,500 g for 10 min at 4°C. V5-agarose was washed in PBS five times and 20 µl of the washed agarose was added to 100 µl of cleared lysate. The mixture was incubated for 1.5 h at 4°C on a shaker. Following the incubation period, the resin was washed three times in lysis buffer diluted 1:1 with PBS and twice in PBS. Twenty µl PBS and 5 µl 5× SDS-PAGE sample buffer were added to the agarose; the samples were heated to 95°C for 10 min and analyzed via Western blot.
Transfected (parental vector, t-39 or t-54) and non-transfected HM-1:IMSS trophozoites were harvested during log-phase growth and 10,000 cells were seeded into 15 ml of TYI-S33 media that contained 10 µg/ml of tetracycline. Parasite numbers were recorded every 24 h for 4 days and expression of the protein was verified each day, in parallel. At least two independently transfected clones were tested, each sample was assayed in triplicate and results represent the mean of three or more experiments.
Phagocytosis assays were carried out as described below, using 2 × 106 E. histolytica trophozoites and 2 × 107 carboxylate-modified 2.0 µm fluorescent yellow-green beads (Sigma, St. Louis, MO, USA). Following PFA fixation, amebae were permeabilized with 0.2% Triton X 100 in PBS for 1 min if indicated and paraformaldehyde was neutralized with 50 mM NH4Cl. Non-specific binding was then blocked by incubation (1 h at 37° C) with 10% goat serum in PBS (blocking buffer). TMKs were detected by incubation for 1 h at 37°C with anti-TMK antibodies diluted to 15 µg/ml in blocking buffer. Three PBS washes were performed and R-PE-conjugated goat anti-rabbit secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA) were added at a 1:200 dilution for 1 h at 37° C. Following the incubation, samples were washed with PBS three times, re-suspended in 50 µl PBS and filtered through a 70 µm nylon cell strainer (BD Falcon, Bedford, MA, USA). Where indicated the procedure was carried out in the absence of amebae (stained beads) or in the absence of anti-TMK antibodies (secondary only). At least 5,000 images were collected using the Amnis Imagestream imaging cytometer (Amnis Corporation; Seattle, WA, USA) and ImageStream Data Exploration and Analysis Software (IDEAS) was used for data analysis. Prior to data analysis, spectral compensation was performed using “stained” beads and stained cells. Raw image files from the same experiment were all compensated with the same matrix and all compensated image files from the same experiment were opened with the same template. In each template, gating was performed to generate a population of single, in-focus, bead-positive cell images (usually yielding 500–1,000 images per sample) and masking was used to identify beads and the brightest 20% of antibody staining. Within the template, the Bright Detail Similarity (BDS) feature was used to calculate the extent of correlation between the two masks and thus quantify the extent of co-localization between ingested beads and TMKs. BDS scores ≥ 3 are considered co-localized.
Ficoll-Paque™ Plus (GE Healthcare, UK) isolated Jurkat cells or packed erythrocytes were suspended in 0.1% BSA in PBS at a concentration of 5 × 106 /ml and incubated with 5 µM carboxyfluorescein succinimidyl e ster (CFSE) for 10 min at 37°C. FBS was used to quench unbound dye and the cells were washed three times with RPMI media. Jurkat cells were then killed using UV irradiation, whereas erythrocytes were Calcium-treated by incubation at 37°C for 48 h in HEPES buffer containing 2.5 mM CaCl2. Both methods have been described elsewhere (Bratosin et al., 2001; Teixeira and Huston, 2008).
Phagocytosis was assayed by flow cytometry as previously described (Huston et al., 2003). Particles used for ingestion assays included fluorescent green 2.0 µm carboxylate-modified latex beads (Sigma, St. Louis, MO, USA), CFSE-labeled apoptotic Jurkat cells and Ca2+ treated red blood cells. Amebae were induced with 10 µg/ml of tetracycline for 24 h. Particles were then mixed with amebae at a 5:1 ratio, centrifuged for 5 min at 200 g and incubated at 37°C for 30 min. D-galactose (110 mM) in ice-cold PBS was used to wash away non-ingested material and cells were fixed with 3.7% PFA. Samples were washed, re-suspended in PBS and analyzed using a FACSCalibur (BD Biosciences) on channel FL1. SSC and FSC were used to distinguish amebae from non-ingested particles and a live cell gate was established prior to data collection with 10,000 gated events collected for each sample. The mean fluorescence intensity (MFI) was calculated for each sample, background fluorescence was subtracted and data was plotted as a percentage of control MFI. Cell types were assayed in duplicate (at minimum) and the experiments were repeated at least three times. Data represents the mean of all experiments and error bars represent the S.D.
Pinocytosis was assayed in a similar manner to phagocytosis, however 1 mg/ml FITC-dextran (Sigma) in PBS was incubated with amebae instead of a particle. Incubation times and procedures were otherwise the same.
LCM was used in conjunction with microarray analysis to examine TMK gene expression at the single cell level. RNA isolated from a laser-captured E. histolytica trophozoite was subjected to two cycles of amplification and analyzed via microarray (Supplementary Fig. S1). Affymetrix® GCOS was then used to generate detection calls for each TMK probe set. Multiple TMK transcripts were detected as present within a single cell (Table 1). The experiment was carried out in duplicate and TMK transcripts identified within both cells are underlined in Table 1. Average log intensity values for TMKs and reference genes are listed in Supplementary Table S2. Multiple TMK genes were expressed in both cells, and one cell expressed detectable levels of multiple members of TMK sub-groups A, B1, D1 and E. Microarray results were validated by qRT-PCR conducted on RNA isolated from an independently laser-captured trophozoite (data not shown). The presence of multiple TMK transcripts within a single cell indicated that these genes do not undergo antigenic variation at the level of transcription.
To enable examination of TMK expression at the protein level, polyclonal antibodies directed against unique peptides within the extracellular domains of TMK39 (sub-family C) and TMK54 (sub-family E) were generated. Peptides were chosen for immunization by first manually identifying hydrophilic and Cysteine-free stretches of sequence in each extracellular domain and then using the Basic Local Alignment Search Tool (BLAST) to check the sequences against the E. histolytica Genome Project Database and ensure specificity. Resultant antibodies were deemed specific as both recognized single bands of the predicted size in E. histolytica lysate and both bands disappeared when the antibodies were pre-incubated with increasing amounts of the corresponding unconjugated peptide (Fig. 1A). An anti-peptide antibody against PaTMK (TMK96, sub-family B3) has previously been developed and was also used in the following studies (Boettner et al., 2008).
Expression of PaTMK and sub-family B1 TMKs has been examined at the protein level (Mehra et al., 2006; Boettner et al., 2008) however studies have been limited to Western blotting and microscopy. Consequently, it is unknown whether TMKs are heterogeneously expressed by trophozoites within a population. To examine expression of TMKs at the population level, we labeled permeabilized trophozoites with anti-TMK39, anti-TMK54 or anti-PaTMK, and analyzed the samples by flow cytometry. In these experiments, trophozoites were harvested from the same flask, immediately fixed with 3.7% PFA and then stained. Flow cytometric analysis of the stained samples revealed homogenous expression of each TMK by more than 95% of cells within the population, compared with the anti-Ft negative control (Fig. 1B). The absence of distinct sub-populations strongly suggests that single amebae express multiple TMKs at the protein level, further discounts the notion of antigenic variation, and points instead to the possibility of non-redundant function among TMK family members.
TMKs generally possess predicted signal peptides of approximately 20 aa and single-pass transmembrane domains. Specific antibodies against PaTMK and cross-reactive antibodies against B1 sub-family members have localized the corresponding proteins to punctate regions of the plasma membrane (Mehra e t al., 2006; Boettner et al., 2008). As the cellular localizations of TMK39 and TMK54 are unknown, cellular fractionation and confocal microscopy were used to localize the two proteins in log-phase trophozoites. When an established method (Aley et al., 1980) was used to separate soluble, internal membrane and plasma membrane components, TMK39 and TMK54 were identified in both membrane fractions by Western blotting (Fig. 2A). As the proteins both contain canonical signal peptides and membrane spanning regions, this was not surprising.
However, when TMK39 and TMK54 were localized using confocal microscopy, the pattern of plasma membrane staining in non-permeabilized cells was surprisingly different, with TMK39 in membrane microdomains (Fig. 2B). This difference was noticeable but less dramatic when the cells were permeabilized. In both instances, pre-incubation of the antibody (10 µg/ml) and corresponding peptide (300 nM) competitively inhibited the staining (Fig. 2B). The accumulation of TMK39 in discrete regions of the plasma membrane is reminiscent of membrane microdomains, such as aggregated lipid rafts and cavaolae that are thought to be centers of cell signaling in metazoan organisms (Parton and Hancock, 2004; Pike, 2006; Pani and Singh, 2009), as well as E. histolytica (Laughlin et al., 2004). In contrast, the even distribution of TMK54 throughout the plasma membrane mirrored the localization of the E. histolytica Gal/GalNAc lectin (Petri et al., 1987). In permeabilized cells, both proteins were occasionally found associated with internal vesicles, however TMK54 was more consistently associated with some type of large intracellular compartment (Fig. 2B). While the biological significance of the proteins awaits further investigation, the distinct localization patterns observed implied that TMK39 and TMK54 served non-redundant functions.
The kinase-containing intracellular regions of TMK39 and TMK54 were replaced with V5 and poly-histidine tags, and a tetracycline-inducible E. histolytica expression vector (Hamann et al., 1997) was used to over-express the truncated proteins, t-39 and t-54 respectively, in trophozoites (Fig. 3A). Parental vectors were transfected in parallel and served as a control for any unexpected effects of the induction of the tetO vector in the experiments. Each construct was used to generate at least two independently transfected clones. To confirm expression of the truncated proteins, cells were induced with 10 µg/ml of tetracycline for 24 h and cellular lysate was subjected to immunoprecipitation using anti-V5 agarose. The anti-peptide antibodies described above were used to probe Western blots of the immunoprecipitations, and both t-39 (Fig. 3B) and t-54 (Fig. 3C) cells expressed proteins of the expected size.
Importantly, wild type and truncated proteins appeared to interact, as endogenous TMK39 and TMK54 co-immunoprecipitated with the corresponding truncated protein (Fig. 3B and C). Receptor kinases are generally activated by ligand-induced dimerization followed by trans-autophosphorylation of kinase domains (Lemmon and Schlessinger, 1994; Heldin, 1995). Consequently, over-expression of dominant-negative receptors that lack cytoplasmic kinase domains has long been used to study the biological relevance of receptor kinases (Ueno et al., 1991). Such truncated receptors bind ligand, fail to propagate a downstream signal, and can inhibit wild type receptors through ligand-induced heterodimerization of the truncated and wild type proteins. These mutants therefore allowed us to address the biological functions of TMK39 and TMK54.
As shown in Fig. 4, cells induced to over-express t-54 had a severe growth defect during the first 24 h of induction but recovered to a near normal growth rate by 48 h (as indicated by the slope of the line in Fig. 4). This was in contrast to t-39 cells, which had no defect. It is unclear how the t-54 cells were able to compensate for their growth defect.
To assess major cell surface changes in the mutant cell lines, flow cytometry was used to measure expression of the 170-kDa heavy subunit of the parasite’s Hgl. After 24 h of induction, total levels of Hgl were comparable in t-39, t-54 and control cells (Fig. 5B), but the levels of surface expressed Hgl were significantly (P < 0.05) lower in t-54 cells (Fig. 5A).
Aside from this study, there is no published information available on the biological role of TMK54. However, TMK39 was identified at an early time point in an amebic phagosomal proteome, and has therefore been implicated in the process of phagocytosis (Okada et al., 2006). To address the potential role of TMK39 in phagocytosis, we first assessed the extent of co-localization between TMKs and ingested beads using multispectral imaging flow cytometry. Anti-peptide antibodies were used to stain trophozoites that had been allowed to ingest 2 µm carboxylate-modified fluorescent beads and samples were imaged using the Amnis Imagestream imaging cytometer. Beads were stained in an identical manner to bead-containing cells to ensure that the anti-peptide antibodies and beads did not directly interact. While there was no evidence of non-specific binding of antibodies to beads, or of (as a control) co-localization between TMK54 and ingested particles (Fig. 6A), TMK39 did appear to co-localize with ingested beads (Fig. 6A). Amnis IDEAS software was used to calculate the BDS score of single, in-focus, bead-positive cell images and the results are plotted in Fig. 6B as mean BDS +/− S.D. BDS scores are indicative of co-localization between fluorescence in two channels: in this instance between ingested beads and TMKs. The mean BDS scores for permeabilized cells stained with TMK39 approached three (the accepted value for co-localized images) and TMK39 had higher BDS scores (P < 0.05), indicating a greater extent of co-localization.
As TMK54 did not appear in any phagosomal proteome (Marion et al., 2005; Okada et al., 2006; Boettner et al., 2008) or co-localize with ingested beads, and as t-54 cells had striking defects in growth and surface expressed Hgl levels, the ability of t-54 cells to phagocytose was not assessed. However, flow cytometry was u sed to measure the ability of t-39 cells to ingest carboxylate-modified fluorescent beads, CFSE labeled apoptotic Jurkat cells and Ca2+ treated erythrocytes. Uptake of FITC-labeled dextran was also measured as a marker of fluid phase pinocytosis. As shown in Fig. 7A and B, t-39 cells were significantly impaired in their ability to ingest carboxylate-modified beads and apoptotic Jurkat cells. The defect was specific, as t-39 cells were unimpaired in their ability to uptake FITC-dextran and Ca2+ treated erythrocytes.
The most important conclusion from these studies is that single E. histolytica trophozoites express multiple members of a large TMK family and utilize the TMKs for non-redundant functions. While large families of TMKs have been considered hallmarks of multi-cellularity, the life cycle of E. histolytica provides clues as to why the protist would require an extensive network of cell surface signaling molecules. In the complex intestinal microenvironment, the organism must compete with bacteria for nutrients and space, sense stressors to regulate developmental changes between cyst and trophozoite, subvert host defenses, ingest bacteria and control its invasive behavior. Upon invasion, trophozoites continue to face a battery of challenges that require the ability to chemotax, adhere, kill and ingest human cells, and obtain sufficient nutrients. Survival of E. histolytica within its human host must require a profound ability to sense and respond to environmental challenges and utilization of the extensive TMK network may therefore be critical.
Single cell microarray analysis demonstrated expression of multiple members of TMK sub-families A, B1, D1 and E. Expression of multiple sub-family members by a single cell raises the possibility that the TMK sub-families function in a complex manner similar to ErbB receptors in mammalian cells. ErbB family members form homo- and hetero-dimers, bind to different ligands and can be transactivated by other proteins (Linggi and Carpenter, 2006). It will be important to keep in mind the potential for such complexity while initiating preliminary studies on TMKs.
A limitation of this analysis was the minute quantity of RNA obtained from a single cell. Previous reports indicate that trophozoite populations in culture or isolated from mice cumulatively express 65–80% of E. histolytica genes (Gilchrist et al., 2006). However, only 15–20% of genes were detected as expressed within a single cell. While the averaging of population data in previous studies may have partially contributed to such a discrepancy, it is more likely that false-negative calls were generated in this study, as single cell microarray analysis is an inherently insensitive technique (Esumi et al., 2008), and members of the TMK gene family have been generally shown to be expressed at low to medium levels in other studies (expression levels in prior studies are accessible through NCBI’s Gene Expression Omnibus GEO Series accession numbers GSE8484, GSE13023, GSE6648, GSE6650). Moreover, TMK54 was detected as present by Affymetrix® GCOS (in both cells), but TMK39 and PaTMK were both GCOS absent in this study. In contrast, more than 95% of trophozoites within a population expressed each TMK at the protein level. Consequently, we believe that the single cell TMK transcriptome described in this study should be considered a minimal estimate.
Additional similarities between metazoan and Entamoeba TMKs are likely to exist. For example, the widely studied metazoan TMKs are activated by ligand-induced dimerization (Lemmon and Schlessinger, 1994; Heldin, 1995) and typically contain extracellular furin-like and/or epidermal growth factor- (EGF) like moieties. Furin-like domains are thought to be involved with the aggregation of metazoan receptor tyrosine kinases and EGF-like moieties contribute to protein-protein interactions. Although the pairing of TMK and EGF or furin-like domains is a rare occurrence in protozoa, many TMKs (including both TMK39 and PaTMK) possess cysteine-rich extracellular domains containing furin-like and/or EGF-like moieties. We observed hetero-dimerization between wild type and truncated receptors in this study, which may indicate that TMKs are subject to the same ligand-induced dimerization events as their metazoan counterparts.
Without identification of receptor ligand or kinase substrate, it is not possible to definitively ascribe functions to any of the TMKs that have been studied thus far. However, we have discovered a variety of clues that can help us begin to understand the function of these proteins. The striking difference in the surface localization patterns of TMK39 and TMK54 was the first indication that these proteins served non-redundant functions. The uniform cell surface staining of TMK54 was similar to that of the heterotrimeric Gal/GalNAc lectin that mediates adhesion, cytotoxicity, phagocytosis and complement resistance (Petri et al., 2002). It is currently unknown how the Gal/GalNAc lectin orchestrates such a wide variety of events but sequence similarity between the short cytoplasmic tail of Hgl and the cytoplasmic tails of β2 and β7-integrins is considered to play a key role (Vines et al., 1998). Tyrosine phosphorylation of β-integrins stimulates their translocation to the cell surface (Naccache et al., 1994). Interestingly, t-54 cells expressed less Hgl on their surface compared with both t-39 and control cells, indicating that TMK54 may regulate Hgl surface expression. Additionally, t-54 cells had a striking growth defect during the first 24 h of t-54 protein induction, indicating that TMK54 may represent a major growth factor receptor. Cross-talk between growth factor receptors and integrins is also known to affect surface integrin levels (Somanath et al., 2009). Members of the B1 family of TMKs have also been found to impact cellular proliferation but any impact on surface Hgl expression has not been described (Mehra et al., 2006). Future studies in our laboratory will address the biological role of TMK54 directly and examine the relationship between TMK54 and the Gal/GalNAc lectin.
In contrast to TMK54, TMK39 was localized to punctate regions of the plasma membrane, in a pattern reminiscent of membrane microdomains such as lipid rafts. As expected from its prior identification as a member of the phagosomal proteome (Okada et al., 2006), TMK39 was found to co-localize with ingested beads at the surface and to a greater extent within cells. Additionally, t-39 cells had a specific defect in their ability to ingest carboxylate modified beads and apoptotic lymphocytes, but not Ca2+ treated erythrocytes. PaTMK has been previously shown to play a role in uptake of Ca2+ treated red blood cells (Boettner et al., 2008), providing additional evidence that TMKs serve non-redundant cellular functions. Considering previous studies that identified TMK39 as a component of the phagosomal proteome at early time points and the phenotype of t-39 cells, it is tempting to speculate that TMK39 may function as a scavenger receptor and mediate the internalization of polyanionic macromolecules. Although phosphatidyl serine (PS) is a critical “eat me” signal recognized by phagocytes (Grimsley and Ravichandran, 2003), it is unlikely that TMK39 recognizes the molecule because recognition of exposed PS on the surface of aged or Ca2+ treated erythrocytes is known to impact uptake of red blood cells by E. histolytica (Boettner et al., 2005). Alternative ‘eat-me’ signals, such as modified low density lipoprotein (LDL), are recognized by scavenger receptors that mediate the uptake of bacteria and apoptotic corpses in other systems (Grimsley and Ravichandran, 2003). TMK39 shares 30% identity with the Drosophila scavenger receptor Eater across the first 200 aa of the proteins. Eater binds modified LDL and mediates uptake of bacteria, and the first 200 aa of Eater are known to facilitate binding to polyanionic ligands (Kocks et al., 2005). Consequently, modified LDL is a candidate recognition signal for TMK39 and for E. histolytica phagocytosis of apoptotic lymphocytes.
In plants and metazoa, TMKs are known to regulate a myriad of cellular processes including cellular proliferation, survival, differentiation, migration, metabolism and host defense. This study suggests that TMKs are likely to mediate a similarly diverse and essential set of processes for this E. histolytica.
Laser capture microdissection (LCM) to examine gene expression within a single Entamoeba histolytica trophozoite. (A) LCM was used to capture a single ameba. (B) Amebic RNA was amplified using one or two cycles of the WT-Ovation™ Pico System and resultant cDNA was amplified using quantitative PCR (qPCR) for 10 transmembrane kinases (TMKs). qPCR results were plotted as the normalized threshold cycle (CT) value obtained for the indicated TMK after two rounds of amplification versus one round of amplification. qPCR results remained consistent after two rounds of RNA amplification.
This work was supported by NIH grant 5RO1 AI026649 (to W.A. Petri) and Grant-in-Aid for Scientific Research (to S.Hamano).
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