Strains and growth conditions. E. histolytica
strain HM1:IMSS clone 6 was maintained and grown in TYI-S-33 medium (10a
) containing 125 μl of 250 U ml−1
benzyl penicillin and 0.25 mg ml−1
streptomycin per 100 ml of medium. Neomycin (Sigma) was added at 10 μg ml−1
for maintaining transgenic cell lines.
Escherichia coli strains (BL21 and DH5α) were maintained in Luria broth containing 100 μg ml−1 ampicillin or 30 μg ml−1 kanamycin as indicated.
Isolation of EhDLP1 gene and cloning into different vectors.
The EhDLP1 gene was amplified from E. histolytica HM1:IMSS genomic DNA by PCR using the following primers: forward, 5′ GACTATGAAAAGTCTTATTCCAGTT 3′, and reverse, 5′ GACGTTAATTAACTTTGATTGTAAC 3′. The amplicon (2,047 bp) was cloned into the pGEM-T Easy vector (Promega) and subcloned into the NcoI/SalI sites of the pET-30(a) vector (Novagen). BL21(DE3) cells were transformed with the resulting construct and used for the production of recombinant EhDLP1 (rEhDLP1) protein.
The EhDLP1 gene was also cloned upstream of the green fluorescent protein (GFP) gene in the pEh-NEO-GFP vector (13
). The primers used were as follows: forward, 5′ CCCGGTACCATGAAAAGTCTTATTCCA 3′, and reverse, 5′ GGGGGTACCTTAATTAACTTTGATTGT 3′.
In order to generate the lysine mutant of EhDLP1 protein, site-directed mutagenesis was carried out according to the instructions of the mutagenesis kit manufacturer (Stratagene). The primers used for mutagenesis were as follows: forward, 5′ GGGTCTCAAAGTGCTGGTGCATCATCTGTATTAGAAAG 3′, and reverse, 5′ CTTTCTAATACAGATGATGCACCAGCACTTTGAGACCC 3′.
All constructs were verified by nucleotide sequencing.
Expression and purification of rEhDLP1 from E. coli.
E. coli strain BL21(DE3) was transformed with the constructs expressing EhDLP1 or the K38A mutant of EhDLP1 (hereinafter referred to as EhDLP1-K38A) in order to produce the recombinant proteins by inducing the cultures with IPTG (isopropyl-β-d-thiogalactopyranoside; 0.1 mM for EhDLP1 and 0.4 mM for EhDLP1-K38A) for 3 h at 37°C with aeration.
The expressed proteins were purified using Ni2+-nitrilotriacetic acid (NTA) affinity chromatography, as the recombinant proteins were each carrying a His tag at the amino terminus. Briefly, the induced bacterial cells were harvested at 6,000 rpm for 5 min at 4°C. The cells were lysed by incubation of the pellet in lysis buffer (1× phosphate-buffered saline [PBS] in 20 mM imidazole) containing 200 μg/ml lysozyme, 1 mM dithiothreitol (DTT), and a protease inhibitor cocktail, followed by sonication. The recombinant proteins were further recovered from the pellets by solubilization in 0.2% Sarkosyl solution (0.2% N-lauryl sarcosine solution containing 25 mM triethanolamine, 20 mM imidazole, and 1 mM EDTA [pH 8.0], supplemented with 0.1% 0.1 M CaCl2 and Triton X-100) for 1 h at 4°C. The supernatant containing the desired protein was loaded onto Ni2+-NTA beads, and the proteins were purified per the instructions of the bead manufacturer (Amersham). The fractions were analyzed by SDS-10% PAGE. Fractions containing purified fusion protein were pooled and dialyzed against HCB 300 (20 mM HEPES [pH 7.2], 2 mM EGTA, 2 mM MgCl2, 1 mM DTT, and 300 mM NaCl) containing 10% glycerol. The protein samples obtained were further concentrated with an Amicon concentrator (with a cutoff at a molecular weight of 30,000) and stored as small aliquots at −80°C.
The purified antigen (EhDLP1, as described above) was dialyzed against PBS. Rabbits were immunized subcutaneously four times at multiple sites with 100 μg of protein per injection and an interval of 3 weeks between each injection. The first dose of the protein was emulsified with complete Freund's adjuvant, while incomplete Freund's adjuvant was used for the subsequent booster immunization. Sera from immunized rabbits were collected 2 weeks after the last booster and stored in aliquots at −80°C.
Subcellular localization of EhDLP1 in Entamoeba lysate.
To separate the nuclear fraction from the cytoplasm and the membrane fraction, the protocol described by Dey et al. (10
) was followed. Briefly, 107
cells growing in log phase were harvested at 280 × g
for 7 min at 4°C and resuspended in 2 ml lysis buffer (10 mM HEPES [pH 7.5], 1.5 mM MgCl2
, 10 mM KCl, 0.5 mM DTT, 0.2% Nonidet P-40 detergent, and protease inhibitors). The suspension was incubated on ice for 15 min and then centrifuged at 3,000 × g
for 10 min at 4°C. The pellet thus obtained contained the nuclear fraction, and the supernatant was ultracentrifuged at 100,000 × g
for 30 min at 4°C to obtain the cytoplasmic and membrane fractions. The nuclear pellet was resuspended in 50 μl of the lysis buffer, and the protein content of each fraction was estimated by the bicinchoninic acid assay.
GTP binding and hydrolysis assays.
GTP binding assays were performed in two separate ways: (i) covalent cross-linking of [α-32P]GTP to the purified rEhDLP1 or mutant protein under UV light exposure and (ii) use of a filter binding assay. To perform the UV cross-linking assay, a 2-μg sample of purified rEhDLP1 or mutant protein in buffer containing 20 mM HEPES (pH 7.2), 2 mM MgCl2, 1 mM DTT, 10 μCi [α-32P]GTP, 150 mM NaCl, and 10% glycerol was incubated on ice for 10 min and then exposed to UV light irradiation (wavelength, 254 nm) at a distance of 5 cm for 30 min at 4°C with a system from Stratagene. After termination of UV exposure, 0.8 μl of 100 mM dGTP and 20 μg of bovine serum albumin (BSA) were added to the reaction mixtures. Proteins were precipitated by incubation in 10% trichloroacetic acid for 45 min at 4°C and washed with acetone. These proteins were separated by SDS-PAGE, and the labeled proteins were visualized by using a PhosphorImager.
Alternatively, rEhDLP1 or the mutant (2 μg) was incubated in buffer containing 20 mM HEPES (pH 7.2), 2 mM MgCl2, 1 mM DTT, 150 mM NaCl, and 1 μCi [α-32P]GTP for 20 min at 4°C. The samples were loaded onto a nitrocellulose membrane (0.45-μm pore size) in a filter dot blot apparatus under a vacuum. The filter was rapidly washed once with 250 μl of cold buffer. The dried filter was imaged using a PhosphorImager, and spots were quantified using MultiImage software.
GTP hydrolysis by rEhDLP1 or the mutant was monitored as the hydrolysis of [γ-32P]GTP by the purified proteins. One microgram of purified protein was incubated in 1× GTPase buffer (20 mM HEPES [pH 7.4], 2 mM MgCl2, 150 mM NaCl, 1 mM DTT) containing 1.0 μCi of [γ-32P]GTP in a 20-μl reaction volume for 15 min at room temperature. The reaction was terminated by the addition of 1× SDS sample buffer, and the reactants were resolved by polyethyleneimine thin-layer chromatography (TLC) using 0.75 M KH2PO4 (pH 3.75) as the solvent phase. The TLC plates were then exposed to a PhosphorImager or an X-ray film.
Velocity sedimentation assay.
Three to five micrograms of purified protein dialyzed against high-salt-concentration buffer HCB 300 was used for each 100-μl total reaction volume. The final salt concentration was then varied to either 150 or 50 mM NaCl by using HCB 0, which contains 0 mM NaCl. Three to five micrograms of rEhDLP1 or the mutant protein was added to buffers with different salt concentrations in the ultracentrifuge tubes. These tubes were then incubated for 10 min at room temperature and centrifuged at 100,000 × g for 10 min. The supernatants containing unassembled proteins were then carefully collected and analyzed by SDS-PAGE after precipitation with trichloroacetic acid. The pellet containing the assembled protein oligomers was also resuspended in SDS-PAGE sample buffer and analyzed by SDS-PAGE.
Lipid binding assay.
Membrane binding of rEhDLP1 was checked by monitoring the ability of the protein to make tubes upon incubation with phosphatidylcholine (PC) liposomes. To prepare PC liposomes, synthetic PC (Avanti Polar Lipids) was resuspended at a concentration of 3 mg/ml in chloroform and then dried using nitrogen gas with constant swirling. The dried lipids form a thin layer on the surface of the glass tube. These dried lipids were then resuspended in HCB 100 (20 mM HEPES [pH 7.2], 2 mM MgCl2, 2 mM EGTA, 1 mM DTT, and 100 mM NaCl) to a final concentration of 3 mg/ml. The glass tube containing the lipid suspension was subjected to a vortex in a water sonicator five to six times until the solution became turbid.
Protein-lipid tubes were monitored by incubating the target protein (rEhDLP1) with PC liposomes in a ratio of 1:1 with 0.1- to 0.25-mg/ml concentrations of both protein and lipid for 3 to 4 h at room temperature. In one of the reactions, the tubes were incubated with 1 mM GTP for 30 min at 37°C prior to the preparation of a sample for electron microscopy.
All the samples prepared as described above (liposomes alone, lipid tubes, and the lipid tubes incubated with GTP) were examined by transmission electron microscopy at the All India Institute of Medical Science (New Delhi). Samples were diluted to 0.1 mg/ml, adsorbed to 400-mesh carbon-coated electron microscopy grids, and negatively stained with 2% uranyl acetate before being air dried. Electron micrographs were obtained using a Philips 400 or CM120 electron microscope at 100 kV and recorded at a defocus level of 1.15 mm.
E. histolytica resuspended in warm incomplete TYI-33 medium (TYI-S-33 without serum) was transferred onto prewarmed, acetone-cleaned coverslips in a petri dish and allowed to adhere for 10 min at 37°C. The culture medium was removed, and coverslips were fixed with 3.7% prewarmed paraformaldehyde (PFA) for 30 min. After fixation, the cells were permeabilized with 1% Triton-PBS for 1 min. The cells were then washed with PBS and quenched for 30 min in PBS containing 50 mM NH4Cl. The coverslips were blocked with 1% BSA-PBS for 30 min and then incubated with primary antibody at 37°C for 1 h. These coverslips were then washed with PBS followed by 1% BSA-PBS before being incubated with secondary antibody for 30 min at 37°C. The following antibody dilutions were used: anti-EhDLP1, 1:50; anti-GFP, 1:200; and anti-rabbit Alexa 488, 1:200. To stain DNA, DNA-specific dye Hoechst 33342 was used. The fixed cells were incubated with 20 μg/ml Hoechst 33342 for 10 min at room temperature. The cells were then washed three times with PBS. The preparations were further washed with PBS and mounted onto glass slides by using DABCO [1,4-diazbicyclo(2,2,2)octane; Sigma].
The fluorescent slides were then examined with an LSM 510 confocal laser scanning microscope (Zeiss, Germany) equipped with a 63× objective. Alexa green-labeled samples were excited at 488 nm by using an argon laser, and UV excitation was used to view Hoechst staining. Pictures were processed using an offline version of LSM 510 software (Zeiss).
Different E. histolytica cells (with constructs expressing GFP, EhDLP1-GFP, and EhDLP1-K38A-GFP) were grown in the presence of 30 μg ml−1 G418, and the cells were counted by a hemocytometer at the times indicated in Fig. . Cell viability was determined by microscopy using a trypan blue dye exclusion test.
FIG. 8. Properties of cells overexpressing the WT and mutant EhDLP1 proteins. (A) The proliferation of E. histolytica cells expressing the indicated proteins is depicted. All transfectants were grown at 30 μg ml−1, and the growth was measured (more ...) RITC-dextran uptake.
Pinocytosis was studied by observing the uptake of rhodamine isothiocyanate (RITC)-dextran as described before (13
). The mid-log-phase cells were harvested, washed, and resuspended in fresh medium. The washed cells were then incubated with RITC-dextran (2 mg ml−1
; Sigma) for 30 min at 36°C, harvested, and washed with PBS. The cells were then resuspended in PBS containing 0.1% Triton X-100. The amount of endocytosed intracellular rhodamine was determined by measuring total fluorescence using a Cary fluorescence spectrophotometer.
The ability of E. histolytica
to kill target cells was studied using Chinese hamster ovary (CHO) cells as target cells. The destruction of a monolayer of CHO cells was assayed as described earlier (13
). Briefly, trophozoites (2 × 105
suspended in Dulbecco's modified Eagle medium [DMEM] without fetal calf serum) were added in triplicate to wells containing a confluent monolayer of CHO cells (2 × 105
) prewashed with DMEM to remove traces of fetal calf serum, and the wells were incubated for 60 min at 37°C in an atmosphere of 95% air and 5% CO2
. The reaction was stopped by chilling the reaction mixture for 10 min, and the wells were then washed three times with cold PBS. The monolayer was fixed with 4% PFA for 10 min and stained with methylene blue (0.1% in 0.1 M borate buffer, pH 8.7). The excess stain was washed away with 0.01 M borate buffer, and the incorporated dye was extracted by adding 1.0 ml of 0.1 M HCl at 37°C for 30 min. The color was read in a spectrophotometer at 660 nm after appropriate dilutions with 0.1 M HCl. The destruction of cells was expressed in relation to the amount of dye extracted from the control monolayer of CHO cells.
Analysis of EhDLP1 expression in stressed cells by Northern blotting.
Heat shock was administered by transferring proliferating E. histolytica cells into a water bath maintained at 42°C for 1 h. The cells were then chilled, and RNA was extracted. Cells were exposed to oxidative stress by being grown in 10 ml of complete TYI-S-33 medium in a 50-ml tissue culture flask for 1 h at 36°C with gentle shaking at 40 rpm. To achieve serum starvation conditions, the medium was replaced with TYI-33 medium containing 0.5% adult bovine serum for 24 h, and RNA was extracted.
Total RNA was purified using TRIzol reagent according to the instructions of the manufacturer (Invitrogen). RNA samples (30 μg) were resolved in formaldehyde agarose in a solution of gel running buffer (0.1 M MOPS [morpholinepropanesulfonic acid; pH 7.0], 40 mM sodium acetate, 5 mM EDTA [pH 8.0]) and 37% formaldehyde at 4 V/cm. The RNA was transferred onto GeneScreen Plus nylon membranes (NEN). Hybridization and washing conditions for RNA blots were per the manufacturer's protocol.
Sequence alignment and phylogenetic construction.
Amino acid composition determination, restriction enzyme site analyses of DNA sequences, and multiple-sequence alignments (with CLUSTAL W) were performed using the BioEdit sequence alignment editor (version 7.0; Tom Hall). Secondary structure analysis of the protein sequence was carried out using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/
), NPS (9
), and JPRED (8
). Common predictions made by all three methods are reported.
Phylogenetic analysis of the extracted sequences from different organisms along with the E. histolytica sequence was done with the PHYLIP 3.67 package and the unweighted-pair group method using average linkages (UPGMA). The analysis was done with a bootstrap value of 100.
Standard molecular techniques, such as Western blot analysis, SDS-PAGE, Northern analysis, and protein estimation, were performed as described previously (24