Identification of genes homologous to the Igl subunit of the Gal/GalNAc lectin.
The genes were identified in the 7X assembly available from The Institute for Genomic Research (TIGR) and Sanger sequencing centers (http://www.tigr.org/tdb/e2k1/eha1
) by searching the database for homologs of Igl1. Genes with high sequence similarity to Igl1 were used to search the database and identify additional family members. Additionally, the Sanger assembly was translated in all six reading frames, and genes were identified by sequence similarity to known genes in the National Center for Biotechnology Information (NCBI) database. These sequences were then screened for genes containing sequences for three or more CXXC motifs, or kinase domains. Genes containing sequences for CXXC motifs but not kinase domains, transmembrane domains, or signal peptides were eliminated from the data set.
Identification of other virulence genes and control genes.
Genes were identified by sequence similarity to genes for amoebapores, cysteine proteinases, and the Gal/GalNAc lectin Igl, Lgl, and Hgl subunits. Additionally, genes were identified by examination of the translated Sanger assembly, which had been annotated to known genes in the NCBI database. Phagocytosis genes and control genes were similarly identified. BspA genes were identified in the translated Sanger assembly and then identified by sequence similarity in the TIGR assembly.
Phylogenetic analysis of the TMK proteins.
A 260-amino-acid alignment of the kinase domains of the TMK proteins was made to Hanks's kinase alignment (Protein Kinase Resource [http://pkr.sdsc.edu/html/pk_classification/pk_catalytic/pk_hanks_class.html
]) using CLUSTALX (20
). One representative per family, called the query panel of kinases, was employed (http://pkr.sdsc.edu/html/pk_classification/pk_catalytic/query_panel.html
). The alignment was manually optimized using Genedoc (39
), and then sequences were analyzed using the PHYLIP v3.6 package (15
) and bootstrapped using Seqboot, Protdist, Neighbor, and Consense. A subset of the sequences were then bootstrapped using Seqboot, Protpars, and Consense. The TMKs were broken into groups based on signature motifs found in the kinase domains and aligned using CLUSTALX and with manual adjustments using Genedoc.
Probes for microarray analysis.
Oligonucleotide probes typically ranging from 50 to 60 bases, and optimized for standard hybridization conditions, were designed using Array Designer 2.0 software (Premier Biosoft International, Palo Alto, CA). The selected probes were then analyzed by BLAST against the 7X assembly of the E. histolytica
genome at both TIGR and Sanger. Probes were redesigned if they contained more than 75% sequence similarity with other target sequences or had a continuous stretch of complementary sequence exceeding 15 bases (28
). In some cases it was not possible to design gene-specific probes. The actin probe was predicted to hybridize to several actin genes, the Jacob probe to all three Jacob genes, the EHCP1/2 probe to genes for both E. histolytica
cysteine proteases 1 and 2 (EHCP1 and EHCP2), the Hgl family probes to all five Hgl genes but not homolog Sp1, and the Hgl1/5 probe to Hgl1 and Hgl5 genes. The Hgl1/5, Hgl2, Hgl3, and Hgl4 probes were more than 75% similar.
The oligoarray had probes to genes for amoebapores A, B, and C and homologs (32
), BspA homologs (25
), actin, intergenic regions, L37a from mouse and human, chitin synthase (10
), chitinase (11
), Jacob (16
), Jessie1-3 (65
), EHCPs (6
), EhRabs (53
), Vps26, Vps35, glycerate dehydrogenase (3
), methionine gamma-lyase (62
), phosphoglycerate dehydrogenase (2
), Ebp1 and Ebp2 (55
), L10 (7
), ribosomal gene Sa, indigoidine synthase homolog (50
), Hgl1-5 (35
), Igl1 and Igl2 (8
), Lgl1-6 (36
), Sp1 (an Hgl homolog), ferredoxin (26
), Ariel1 (34
), an HMW1 homolog (19
), serine-rich E. histolytica
), TMK genes, and other hypothetical surface genes (Table S1 in the supplemental material). TMK genes for which we did not generate a specific probe are shown in Table S2. Jacob is an amebic cyst wall glycoprotein expressed during encystation (16
). EHCP1 and EHCP2 are highly homologous cysteine proteinases (6
). Sp1 is a homolog of Hgl, recently identified in the TIGR database (B. Mann, personal communication).
Probe synthesis and microarray printing.
A 200-nmol quantity of each probe (typically ranging from 50 to 60 bases optimized for standard hybridization conditions) was synthesized on an ABI 3900 DNA synthesizer (Applied Biosystems, Foster City, CA). The probe oligonucleotides were dissolved in 50% dimethyl sulfoxide at a concentration of 0.25 mg/ml and arranged in 96-well microtiter plates. The panel of probes, including control (housekeeping) oligonucleotides, was printed with two spot replicates on Corning UltraGAPS coated slides using an Affymetrix 417 arrayer (Affymetrix, Santa Clara, CA). Slide quality control was analyzed by hybridizing two randomly selected slides per batch (up to 40 slides/batch) with Cy3-labeled universal oligonucleotide probes. The hybridized slides were then washed and scanned with a ScanArray 4000 scanner (PerkinElmer Life Sciences Inc., Boston, MA). If the spotting quality standards were met, the batch was deemed satisfactory for analysis.
Trophozoites of E. histolytica
strain HM1:1MSS were grown axenically at 37°C in TYI-S-33 medium (12
) with 100 U/ml of penicillin and 100 μg/ml of streptomycin sulfate (Invitrogen, Carlsbad, CA). For growth curve analysis, amebae were grown until they became nonadherent but still viable (144 h), seeded into T25 flasks with 300,000 ameba per flask (Corning Life Sciences, Corning, NY), and grown for 12 to 144 h.
Human erythrocytes were isolated using Mono-Poly resolving medium (ICN Biomedicals, Aurora, OH) according to the manufacturer's directions. Erythrocytes were washed twice in 10 mM HEPES (pH 7.0), 140 mM sodium chloride, and 0.1% bovine serum albumin and then resuspended in the same buffer until use. One million log-phase trophozoites were grown in 50 ml of TYI-S-33 medium for 24 h in the presence or absence of 24 million erythrocytes per ml of medium.
Isolation of RNA.
Amebae were lysed with 2 ml of buffer RLT containing β-mercaptoethanol (the first component of the RNeasy kit from Qiagen, Valencia, CA). Samples were processed immediately or flash-frozen in liquid nitrogen and stored at −80°C until processing using QIAshredders, followed by the RNeasy mini kit, including all optional steps and a 5-min incubation with buffer RWI. Samples were treated on the columns with RNase-free DNase from Qiagen according to the manufacturer's directions (Qiagen). Samples were analyzed for residual DNA contamination by PCR using primers for Jacob (conditions are described below). Samples that contained residual DNA were retreated with DNase I (Roche, Indianapolis, IN) for 1 h at 37°C in a 100-μl total volume with 10 μl of 10× DNase I buffer (100 μM Tris [pH 7.5], 25 mM MgCl2 and 5 mM CaCl2) and 3 μl DNase I, repurified on RNeasy columns and rescreened for residual DNA contamination. The Agilent BioAnalyzer (Agilent Technologies, Palo Alto, CA) was used to assess RNA quality. The results were inspected to ensure that both ribosomal peaks were intact and that no degradation had occurred. Acceptable 260/280 ratios ranged from 1.8 to 2.1.
Sample labeling, hybridization, and scanning.
DNA oligoarray assay of gene expression used cohybridization of two fluorescently labeled cDNA targets, prepared from different samples. For routine oligoarray expression analysis, a previously described (24
) indirect labeling procedure was used. Approximately 10 μg of RNA per sample and random hexamers were used for synthesis of cDNA containing amino-allyl-labeled nucleotides. The newly synthesized cDNA was then labeled by a covalent coupling of an appropriate cyanine fluor (CyDye postlabeling reactive dye pack; Amersham Biosciences Corp., Piscataway, NJ). In a typical oligoarray assay, the cDNA of one preparation (control) was labeled with Cy5, while the second cDNA (experiment) was labeled with Cy3. Both reactions were purified with a QIAquick PCR purification kit (Qiagen) for removal of the uncoupled dye. The labeling efficiencies of the purified target preparations were examined by spectrophotometry, as well as by calculations of the mass of cDNA and Cy5 or Cy3 dye incorporation. The nucleotide-to-dye molecular ratios were considered suitable for oligoarray experiments with a ratio of less then 50 nucleotides/dye molecule. Both targets were equalized based on the total amount of dyes incorporated before hybridization (24
). The samples were mixed, dried by speed vac, and dissolved in hybridization buffer solution (50% formamide, 5x SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate] and 0.1% sodium dodecyl sulfate [SDS]). The cDNA-containing hybridization solution was then denatured, applied to the oligoarray (prehybridized in 5x SSC, 0.1% SDS and 1% bovine serum albumin), and hybridized at 42°C for 18 h. Following 5-min washes in 2× SSC-0.1% SDS and in 0.1× SSC, the slide was scanned using a ScanArray 4000 scanner (PerkinElmer, Wellesley, MA). Both Cy5 and Cy3 images of one experiment were analyzed with QuantArray 3.0 microarray analysis software. Normalization to median between both channels was used.
RT-PCR primer design.
Real-time PCR (RT-PCR) primers (Table ) were designed using Beacon Designer 2.0 (Premier Biosoft International, Palo Alto, CA). RT-PCR primers and oligoarray probes were designed independently; thus, the PCR fragment and the oligoarray probe represented different regions of the same gene. Each primer was analyzed against the TIGR E. histolytica database, and any primer that had significant sequence similarity to multiple genes was rejected. Thus, both the forward and reverse primers were specific for one gene, except actin, Jacob, and Hgl, which detected all family members and/or alleles in the genome. Optimal annealing conditions (determined by gradient PCR) were used to ensure specificity, and any PCR primer pair that produced more than one melt peak was discarded. PCR products that produced single melt peaks were analyzed by gel electrophoresis in 1.5% agarose-Tris-borate-EDTA, and if multiple bands were observed, the primer pair was discarded. Finally, all PCR products were sequenced using the forward amplification primer to verify specificity.
Real-time PCR validation of oligoarray results.
RNA was reverse transcribed using iQsuperscript (Bio-Rad, Hercules, CA) according to the manufacturer's directions. cDNA levels were measured using RiboGreen (Molecular Probes, Eugene, OR) with DNA of known quantity as a standard in a SPECTRAmax Gemini EM fluorescent plate reader, according to the manufacturer's directions. Before proceeding to analysis of cDNA samples, a no-reverse-transcriptase control PCR was done using primers to the cyst-specific gene Jacob to verify that there was no residual DNA contamination of the samples. All samples were analyzed in duplicate, and all time points were analyzed in triplicate. Each time point was thus represented in six wells during the real-time PCR assays, and the six wells were averaged after normalization to the RNA polymerase II gene's average (47
). Two sequentially performed growth curves were analyzed in the real-time PCR assays to ensure reproducibility. All real-time PCR assays were quantitative to allow direct comparison of gene expression levels.
A PCR master mix consisted of 1,100 μl of iQSYBRGreen super mix (Bio-Rad, Hercules, CA), 1,100 μl of distilled H2O, 88 μl of forward primer (50 pmol/μl), and 88 μl of reverse primer (50 pmol/μl). To each well containing 2 μl of cDNA was added 25 μl of master mix. Duplicate assays were performed on each sample. Each assay included standards, no-DNA-control wells, and no-RT-control wells. The cycling conditions were 95°C for 5 min; 30 cycles of 95°C for 30 s, annealing for 30 s (see Table for annealing temperatures), and 72°C for 30 s; and 1 cycle of 72°C for 2 min 30 s followed by a 90-step melt curve increasing 0.2°C with a 5-s hold.
Production of anti-ΔTMK96 rabbit serum production.
The kinase region of Tmk96 (ΔTMK96) was PCR amplified with the primers 5′-CAATTTAGAGAAGGAATTCCT-3′ (5′ primer) and 5′-TCACATTAATTGAAGATGTTTTAAAACAACA-3′ (3′ primer). This 1,000-bp fragment was cloned into TOPO NT/T7 (Invitrogen), in frame with an amino-terminal six-His tag via TA cloning. Bacteria were grown at 37°C to an optical density at 600 nm of 0.5 and induced with isopropyl-β-d-thiogalactopyranoside for 4 h, and the recombinant protein was purified with nickel agarose beads (Qiagen). Antibodies were raised to this purified recombinant protein by a 90-day protocol including three inoculations of New Zealand White rabbits with recombinant ΔTMK96 (Covance, Princeton, NJ), and the antibodies were purified from serum with a protein A column.
Western blots using ΔTMK96.
Soluble proteins were extracted from amebae by harvesting 5 × 107 trophozoites by incubation on ice for 10 min, followed by centrifugation (200 × g at 4°C for 5 min). The amebae were lysed in 10 mM sodium phosphate buffer with protease inhibitor cocktail I (Sigma, St. Louis, MO) per the manufacturer's directions. Membranes were then cleared by centrifugation (100,000 × g at 4°C for 1 h). Whole-cell lysates were prepared by sonication of 106 amebae in three 5-min pulses on ice. Large intact particles were eliminated by centrifugation (20,000 × g at 4°C for 30 min). All samples were then separated on 10% polyacrylamide gels and then electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Nonspecific binding was blocked by preincubation with Tris-buffered saline with 5% bovine serum albumin and 0.05% Tween 20 (TBST). In order to detect proteins on the blot, either anti-ΔTMK96 rabbit serum (a dilution of 1:5,000) or preimmune serum (1:2,500) was added in TBST for 1 h at room temperature. Interactions were detected by the addition of peroxidase-conjugated goat anti-rabbit IgG (Sigma) and development with ECL (Amersham) per the manufacturer's directions.
E. histolytica trophozoites (106) were bound to glass coverslips in a 24-well plate for 30 min at 37°C in TYI-S-33 medium. Adherent amebae were washed twice in phosphate-buffered saline (PBS) and fixed in 3% paraformaldehyde for 30 min at room temperature. To make the plasma membrane permeable, amebae were treated with 0.2% Triton X-100 in PBS for 1 min. Nonspecific binding was blocked by incubation with 20% goat serum and 5% bovine serum albumin (Sigma) in PBS for 1 h at 37°C. After incubation with either the anti-ΔTMK96 rabbit polyclonal antibody (200 μg/ml) or anti-Gal/GalNAc lectin antibody (6 μg/ml) for 1 h at 37°C, the coverslips were washed twice before Cy3-conjugated goat anti-rabbit secondary antibodies (Jackson Laboratories, Bar Harbor, ME) were added at a 1:160 dilution for 1 h at 37°C. The coverslips were washed twice and mounted on slides with Gel/Mount (Biomeda, Foster City, CA). Confocal images were visualized using a Zeiss LSM 510 laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY).