All chemicals were purchased from Sigma (Poole, Dorset, UK), unless otherwise stated.
Acanthamoeba cultures
Two isolates of Acanthamoeba were used in the present study: 1) A. castellanii (ATCC 50494) belonging to T1 genotype was isolated from an AGE patient, and 2) A. castellanii (ATCC 50492)belonging to T4 genotype was isolated from a keratitis patient. For simplicity, the former is referred to as the AGE isolate and the latter is referred to as the AK isolate. Both isolates were grown in tissue culture flasks in PYG medium [proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v) and glucose 1.5% (w/v)] at 30°C and the medium was refreshed 17–20 h prior to experiments as previously described [26]. This resulted in >95% Acanthamoeba in the trophozoite forms.
Human brain microvascular endothelial cell cultures
The primary brain microvascular endothelial cells were isolated from human tissue and purified by fluorescent activated cell sorting (FACS). The purified cells were tested for endothelial characteristics, such as expression of endothelial markers, F-VIII, carbonic anhydrase IV and uptake of acetylated low density lipoprotein (AcLDL), indicating their endothelial origin and expression of gamma-glutamyl transpeptidase, indicating the brain origin (27). HBMEC were grown in RPMI-1640 containing 10% foetal bovine serum, 10% NuSerum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/ml), streptomycin (100 U/ml), non-essential amino acids and vitamins at 37°C, 5% CO2 as previously described [27, 28].
Cytotoxicity assays
To examine the cytopathic potential of Acanthamoeba isolates, cytotoxicity assays were performed as previously described [26]. Briefly, HBMEC were grown to confluent monolayers in 24-well plates. Acanthamoeba (5 × 105 amoebae/well) were incubated with HBMEC monolayers in serum free medium (RPMI 1640 containing 2 mM glutamine, 1 mM pyruate and non-essential amino acids) in plates incubated at 37°C in 5% CO2. The HBMEC monolayers were observed periodically under a phase-contrast microscope for cytopathic effects for up to 24 h. After 24 h, the supernatants (termed conditioned medium, CM) were collected, and the monolayers were fixed and stained with hematoxylin to visualize HBMEC monolayer disruptions. In addition, supernatants were examined for HBMEC cytotoxicity by measuring lactate dehydrogenase (LDH) release (cytotoxicity detection kit; Roche Applied Science, Lewes, East Sussex, UK). Briefly, CM of co-cultures of Acanthamoeba and HBMEC were assessed for the presence of LDH, the release of which is considered as an estimate of cell death. The percentage LDH release was calculated as follows: [LDH activity in experimental sample (measured by optical density at 590 nm) - LDH activity in control samples/total LDH activity release - LDH activity in control samples × 100 = % cytotoxicity). Control samples were obtained from HBMEC or Acanthamoeba incubated alone. Total LDH activity release was determined by total HBMEC lysis with 1% Triton X-100 for 30 min at 37°C.
To determine the cytopathic effects of extracellular proteases released from intact Acanthamoeba, CM was produced by inoculating Acanthamoeba in serum free medium described above for 24 h. Next day, Acanthamoeba free supernatants, i.e., CM was collected by centrifugation and used for cytotoxicity assays as described above.
Some cytotoxicity assays were performed in the presence or absence of broad spectrum matrix metalloprotease (MMP) inhibitors, i.e., GM 6001 and GM 1489 (Merck Biosciences Ltd. Nottingham, England) or phenylmethanesulfonyl fluoride (PMSF), an irreversible inhibitor of serine proteases. Briefly, the protease inhibitors (PMSF, 2 mM final conc.; GM 6001 and GM 1489, 5 μM final conc.) were added to CM for 30 min and then mixtures were added to HBMEC monolayers.
Zymography
Zymographic assays were performed to visualize and begin to characterize Acanthamoeba extracellular proteases as previously described [29]. Briefly, CM was mixed (1:1) with sample buffer (containing 4% sodium dodecyl sulfate (SDS) but without β-mercaptoethanol) and electrophoresed on SDS-polyacrylamide gels (SDS-PAGE) containing gelatin (1 mg/mL). After electrophoresis, gels were soaked in 2.5% Triton X-100 (w/v) for 60 min to remove the SDS. Finally, the gels were incubated in a developing buffer (50 mM Tris-HCl, pH 7.5, containing 10 mM CaCl2) at 37°C overnight, rinsed, and stained with Coomassie brilliant blue. Areas of gelatin digestion indicating protease activities are seen as non-staining regions in the gel. In some experiments, samples were pre-treated with PMSF (2 mM final concentration), or 1, 10-phenanthroline (10 mM final concentration) for 30 min. Because 1, 10-phenanthroline is a reversible inhibitor, it was also included in the developing buffer.
To determine the optimum temperature for Acanthamoeba protease activities, gels were incubated at 4°C, 10°C, 20°C, 30°C, 37°C, 42°C, 50°C and 65°C. For the determination of optimum pH, gels were incubated at pH ranging from 3 – 13. For pH 3 and 4, developing buffers were prepared using citrate-phosphate buffer (70 mM sodium citrate and 60 mM sodium phosphate) containing 10 mM CaCl2; for pH 5 and 6, developing buffers were prepared using N- [2-morpholino] ethanesulfonic acid (MES) containing 10 mM CaCl2; and for pH 7 – 13, developing buffers were prepared using 50 mM Tris containing 10 mM CaCl2.
In some experiments, we used extracellular matrix (ECM) proteins including collagen I (obtained from bovine achilles tendon; 1 mg/mL final conc.) dissolved in buffer A (50 nM TES {N-tris [hydroxymethyl]methyl-2-aminoethane-sulfonic acid}, pH 7.4 plus 0.36 mM CaCl2), collagen III (calf skin; 1 mg/mL final conc.) dissolved in buffer B (distilled H2O, pH 3 using acetic acid), elastin (bovine neck ligament; 1 mg/mL final conc.) dissolved in buffer C (200 mM Tris, pH 8.8) and plasminogen (rabbit plasma; 1 mg/mL final conc.) dissolved in buffer D (20 mM lysine, pH 7) as substrate for Acanthamoeba proteases. In addition we used casein (bovine milk; 1 mg/mL final conc.) dissolved in buffer E (60 mM Tris, 90 mM NaCl, pH7.5) and haemoglobin (bovine erthyrocytes; 1 mg/mL final conc.) dissolved in distilled H2O with gentle heat as substrate for Acanthamoeba proteases. All substrates were dissolved according to manufacturer's instructions (i.e., Sigma Labs).
Ammonium sulphate precipitation
In an attempt to purify extracellular proteases of Acanthamoeba, (ca. 500 mL), CM was produced and proteins precipitated by adding ammonium sulphate (0.7 g/mL) slowly to the CM while stirring at 4°C to achieve 70% saturation. The mixture was stirred for an additional 25 min. The precipitate was recovered by centrifugation (10,000 × g for 10 min at 4°C) and dissolved in 500 μl of 0.02 M ammonium acetate buffer, pH 6.5. The concentrated proteins were dialysed against HPLC grade H2O overnight to remove residual ammonium sulphate.
Ion-exchange chromatography
The dialysed precipitated proteins were applied to a DEAE-Sepharose fast flow beads column (16 cm length, 2 cm diameter) (Amersham Biosciences). The column was equiliberated with 100 mL of 0.02 M ammonium acetate buffer, pH 6.5 at a flow rate of 4 mL per min. Using a fast performance liquid chromatography system (Biorad, Hemel Hempstead, UK), proteins were eluted in 0.02 M ammonium acetate with a 0 to 1.0 M NaCl gradient. Proteins in the eluted fractions (6 mL) were freeze dried for long term storage. The lyophilised fractions were reconstituted in 1 mL of 50 mM Tris-HCl, pH 6.8 and tested for protease activities using zymographic assays.