Escherichia colistrains and growth conditions
The source of purified LLO was E. coli strain BL21(DE3) (Novagen, Madison, Wis) with plasmid pET29B (Novagen) carrying cloned hly gene, kindly provided by Dr. Higgins . The bacteria were grown in LB broth (Sterbios, Warsaw, Poland), supplemented with 30 μg/ml kanamycin (Merck, Darmstadt, Germany) at 37°C with 120 rpm shaking. Bacteria were cultured until the optical density reached 1.5. To induce LLO-His synthesis, IPTG (Sigma, Taufkirchen, Germany) was added to a final concentration of 1 mM.
Eukaryotic cell isolation and growth conditions
The target Eukaryotic cells were: SRBC - sheep red blood cells (Biomed, Warsaw, Poland), human acute T cell leukaemia cell line (Jurkat, ATCC TIB 152), and PBMC isolated from humans or mice.
Jurkat cells were grown in RPMI 1640 culture medium (Gibco, Gaithersburg, MD, USA) with 10% of inactivated NCS containing 100 U of penicillin and 0.1 mg of streptomycin per 1 ml or in DMEM medium (Sigma) with 10% of inactivated NCS. Serum contained 150 mg/dl cholesterol, and the final cholesterol concentration in the culture medium was 15 μg/ml (39 μM).
PBMC were cultured, after isolation, in vitro for 2–7 days before a cytotoxicity experiment. For human peripheral blood experiments in vitro, patients gave their personal consent and the procedure was accepted by the Bioethical Commission at the Medical Center of Postgraduate Education, Warsaw (Feb 27, 2008).
Isolation of PBMC was performed by an overlayer of peripheral blood diluted with PBS (1:1), on 5 ml of Histopaque 1077 (Sigma) preparation in a test tube and centrifugation for 30 min at 540 × g. The interlayer of PBMCs consisting of lymphocytes and monocytes was separated and diluted with 10 ml of culture medium, centrifuged for 5 min at 200 × g and the washed cells suspended in fresh culture medium were counted in a Burker camera. The concentration of the cell suspension was corrected to a final value of 2 × 106/ml before culturing. The cultures were performed in Falcon vessels (dishes) or in 6-well flat bottom plates in RPMI 1640 with 10% NCS and antibiotics, and half of the medium volume was changed twice weekly.
For some experiments cell cultures were supplemented with the following compounds to modify the LLO activity: cholesterol (Sigma), NCS containing 150 μg/ml cholesterol (390 μM), and MβCD (Sigma).
The affinity chromatography of LLO
Ten ml of E. coli suspension (approximately 108/ml) grown in the presence of 1 mM IPTG was centrifuged at 4400 × g for 10 min at 4°C, and the supernatant was removed. The pelleted cells were resuspended in 1 ml of sterile column buffer (50 mM Tris, pH 8, 300 mM NaCl, 50 mM imidazole) and were ruptured by sonication in 3 pulses of 30 s each. Then, the bacterial fragments were removed by centrifugation (7000 × g, 15 min). The supernatant was collected, diluted with column buffer and applied onto the affinity column (His-Bind Ni Column, Novagen) previously equilibrated with the column buffer. The column was washed with the same buffer, and then LLO was desorbed with the elution buffer (50 mM MES, pH 6, 300 mM NaCl, 250 mM imidazole). The collected fraction was subsequently dialysed to remove imidazole (50 mM MES, pH 6, 300 mM NaCl, 5 mM cysteine HCl). Protease inhibitors: fluoro 4-(2-aminoethylo)-benzenesulphonyl.HCl (AEBSF, Sigma), EDTA (Sigma), and glycerol (Merck) were added to a final concentration of 1 mM, 10 mM, and 15% (v/v), respectively. Protein concentrations were assayed with NanoOrange® Protein Quantitation Kit (Molecular Probes) using an infinite M200 PRO reader (TECAN, Goring on Thames, UK).
Western blot analysis of LLO preparations
Electrophoretically resolved proteins (SDS-PAGE) were transferred onto a nitrocellulose membrane using the BIO-RAD Trans-Blot system, according to a protocol recommended by the manufacturer. The membrane was blocked using 5% skimmed milk in Tris-buffered saline buffer (TBS), pH 7.6. Primary rabbit polyclonal anti-LLO antibody (Abcam, Cambridge, MA, USA) and secondary goat polyclonal antibody (Abcam) conjugated with alkaline phosphatase were used at 1:1500 and 1:10,000 dilutions, respectively. The membrane was developed using the NBT/BCIP reagent (Merck).
Assay of haemolytic activity
Haemolytic activity was assayed using SRBC. The erythrocytes were washed three times with PBS and suspended to a final concentration of 20% (v/v) in PBS (pH 7.4). The LLO preparation was diluted by adding 10 μl of its preparation to a final volume of 1 ml of 2% SRBC suspension in PBS. The prepared solution was incubated for 30 min at 37°C and then centrifuged for 3 min at 150 × g. The released haemoglobin was measured spectrophotometrically at λ = 410 nm. Haemolytic Units (HU) were calculated after setting 0 HU as the activity of a negative control, and 100 HU for total haemolysis, observed in samples of erythrocytes lysed with 0.01% SDS.
Jurkat cells were resuspended in fresh RPMI medium without any additives (control), supplemented with 26 μM cholesterol, with 5 mM MβCD or with both these compounds. The cells were incubated for 1 h at 37°C, washed with PBS three times, counted and disrupted by sonication. Membrane cholesterol was extracted with isopropanol and quantified by a cholesterol assay kit (R-Biopharm, Darmstadt, Germany).
The activities of the LLO preparations were tested on Jurkat cells or PBMC. The Jurkat cell suspension in a culture medium (1 × 106/ml) of 20 μl, was mixed with 30 μl of LLO preparation diluted with physiological salt solution at different proportions, and immediately incubated for 30 min at room temperature (20–22°C). The time of the incubation was assumed to be the same as for the haemolytic assays. The final cell concentration was 0.4 × 106/ml. The effect of the LLO preparation was titrated in an assay volume of 50 μl containing cell suspension (20 μl) and 30 μl of physiological salt solution containing the LLO preparation. The LLO preparation content investigated was: 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, and 50% of the original LLO concentration (1.5 ng/μl) with activity set at 3 HU/μl. After cell sample incubation, physiological saline containing PI (1 μg/ml) was added before acquisition by a cytometer. PI penetrates the cell membrane of damaged cells and binds to the cell nucleus. In the fluorescence quadrant readings of forward scatter (FSC)/PI fluorescence, the PI unstained events indicate the percentage of living cells. In the FACSCalibur BD cytometer, 5000 events were collected from each sample and analysed by Cell Quest software (Becton Dickinson, Franklin Lakes, NJ, US). Experiments were repeated at least three times.
The flow cytometry results were presented as median values and percentile values P25 and P75. Statistical analysis of flow cytometric experiments was in the global linear model (GLM) after logit transformation of values. The post hoc test of Tukey was used. Results of haemolytic assays were presented as mean values ± standard deviation (SD). Analysis of variance was performed by ANOVA. Statistically significant values of p < 0.05 were assumed. Statistical software SAS 9.2 was used.