2.1. Cultivation of E. histolytica and LCM
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.
2.2. RNA isolation, amplification and microarray hybridization
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
2.3. Genome analysis and datasets
The re-annotated E. histolytica
genome, available at http://pathema.tigr.org
, (GenBank accession number AAFB00000000
) was used in this analysis.
2.4. qRT-PCR for validation of amplification and microarray
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 H2
O 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
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.
2.6. SDS-PAGE gels and Western blotting
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).
2.7. Flow cytometry
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.
2.9. Confocal microscopy
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).
2.10. Inducible expression vectors
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 Kpn
I and C-terminal Bam
HI 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, Bam
HisR - ATA ATG GGA TCC TCA ATG GTG ATG GTG ATG ATG. Resulting PCR products were cloned into the Kpn
I and Bam
HI 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.
2.11. Transfection of E. histolytica trophozoites
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.
2.13. Growth curves
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.
2.14. Amnis Imagestream data collection and analysis
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.
2.15. Fluorescent labeling and killing of cells
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
2.16. Phagocytosis assays
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.
2.17. Pinocytosis assay
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.