Amoeba and cell culture
A. castellanii (American Type Culture Collection, ATCC 30011), which was originally isolated from a keratitis patient, was cultured axenically in peptone-yeast-glucose (PYG) medium at 26 °C, and trophozoites were harvested in the logarithmic growth phase. Human larynx epidermoid carcinoma cells (HEp-2) purchased from ATCC were cultured in EMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA), 100 U/mL penicillin and 100 μg/mL streptomycin. Human corneal epithelial cells (HCECs) purchased from Bioleaf (Shanghai, China) were grown in DMEM (Gibco, USA) with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Both cells were cultured in a 37 °C incubator with 5% CO2.
Encystation assays were performed as described previously  with slight modifications. Briefly, trophozoites from post-logarithmic growth phase cultures were treated with phosphate-buffered saline (PBS; containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) with 50 mM MgCl2 and 10% glucose in culture plates at 30 °C (5 × 105 trophozoites per mL of medium) for 72 h. The treated materials were collected at 24 h, 48 h and 72 h for RNA extraction, respectively.
Transmission electron microscopy
Transmission electron microscopy was performed to observe the morphology of cysts. The cyst pellets were fixed in 3% glutaraldehyde and 4% formaldehyde in phosphate-buffered saline (0.1 M, pH 7.2) at 4°C for 72 h, washed three times in 0.1 M PBS (pH 7.2), postfixed with 1% osmium tetroxide in PBS at 4 °C for 2 h, washed three times in 0.1 M PBS (pH 7.2), dehydrated in an ascending ethanol series and embedded with epoxy resin 618. Ultrathin sections were cut on an LKB Ultratome control unit, stained with 3% uranyl acetate and lead citrate, and observed under a Tecnai G2 Spirit TWIN transmission electron microscope (FEI, Czech Republic).
Cloning of AcCP genes and expression level analysis
The CP genes of Acanthamoeba were cloned from the cDNA of trophozoites using the primers listed in the Additional file 1: Table S1 by polymerase chain reaction (PCR). PCR was performed in a 9902 Veriti 96-well Thermal Cycler (Applied Biosystems, USA) (94 °C for 3 min; 35 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 1 min; followed by 72 °C for 7 min). The amplified PCR products were purified and ligated into a pMD19-T vector (Takara, Japan), and the nucleotide sequences were obtained by automated sequencing.
A.castellanii mRNA was extracted from trophozoites and cysts using RNeasy® Plus Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesised using a PrimeScript® 1st strand cDNA synthesis kit (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was carried out in a final reaction volume of 20 μL according to the manufacturer’s recommendations on an ABI 7500 Real-time PCR system (Applied Biosystems, USA). Reactions were performed in a 96-well plate with TB Green Premix Ex Taq II (Takara, Japan) to analyse the expression level of AcCPs. The primers for AcCPs genes and the GAPDH internal reference are listed in Additional file 1: Table S2. The amplification cycling conditions were as follows: 30 s at 95 °C and 40 cycles of 5 s at 95 °C and 35 s at 60 °C. Each experiment was performed at least three times.
Expression, purification and refolding of recombinant AcCP3 protein
The correct plasmids containing AcCP3 were amplified with primers containing HindIII and BamHI restriction sites, and the PCR products were ligated into the pQE-30 expression vector (Qiagen, Germany). The sequences of all constructs were confirmed on both strands and analysed with Vector NTI software (Invitrogen, Waltham, USA). Plasmids were transformed into M15 (pREP4) cells (Qiagen, Germany) for protein expression. The selected clones were cultured in Luria-Bertani broth containing 100 μg/mL ampicillin and recombinant AcCP3 (rAcCP3) expression was induced using 1 mM isopropyl-β-d-thiogalactoside for 3 h at 37 °C. The recombinant rAcCP3 protein was purified using a QIA Express kit in accordance with the manufacturer’s instructions. The purity and mass of protein were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Since the N-terminal peptide sequence in recombinant CPs can inhibit the hydrolytic activity of the protease, it was necessary for the recombinant AcCP3 to be refolded to obtain the mature peptide with hydrolytic activity . Refolding of the purified recombinant protein was performed as described previously [11, 21]. In brief, purified rAcCP3 (2 mg) was slowly added to 100 mL refolding buffer, containing 100 mM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetracetic acid (EDTA), 250 mM L-arginine, 5 mM reduced glutathione (GST) and 1 mM oxidised glutathione. The protein was gently stirred at 4°C overnight and then dialysed against 10 mM Tris-HCl (pH 7.5). The obtained rAcCP3 was further processed as described previously . In brief, sodium acetate buffers with pH 4.0 to 7.0 were used to analyse the optimal pH condition required for obtaining the fully matured rAcCP3 enzyme. The concentration of matured rAcCP3 enzyme was measured using a protein assay (Bio-Rad, USA).
Analysis of biological characteristics of AcCP3
To assess the role of AcCP3 in Acanthamoeba pathogenesis, the reactivation of the physiological properties of trophozoites was performed using HEp-2 cell monolayers as described previously [23, 24]. In brief, HEp-2 cells were cultured in 75 cm2 tissue culture flasks (Corning, USA) at 37 °C under sterile conditions until the monolayer covered the bottom of the flask completely, at which point the supernatant was removed. Trophozoites (106) suspended in 25 mL physiological 0.9% NaCl were inoculated onto the monolayer three times consecutively. Cocultures of amoebae and HEp-2 cells were incubated at 26 °C until the monolayer was completely lysed. Reactivated trophozoites were collected and total mRNA was extracted. qRT-PCR was performed to analyse the expression level of AcCP3. In addition, MBP was used as a virulence protein in Acanthamoeba . 18S rDNA was used as internal reference . The primers for AcCP3, MBP and 18S rDNA are listed in Additional file 1: Table S2.
To determine the effect of AcCP3 on host proteins, haemoglobin (from human blood), collagen (from human placenta) and albumin (from bovine serum) were purchased from Sigma-Aldrich (USA). Each protein (2 mg/mL) was incubated with matured rAcCP3 (100 nM) in 50 mM sodium acetate (pH 4.0 or pH 7.0) with 1 mM GST for 3 h at 37 °C. The reactions were terminated by adding reducing sample buffer and the degradation activity of rAcCP3 was analysed by SDS-PAGE.
The protease activities of trophozoite crude proteins were analysed using Novex™ 10% Zymogram Plus (Gelatin, China) Protein Gels (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions, and 1.5 μg crude protein extract was added to each lane. Various inhibitors for different proteases were used. Crude protein extracts treated and untreated with inhibitors (1 h before electrophoresis) were analysed with zymography. The final concentrations of inhibitors were as follows : for serine proteases, 5 mmol/L phenylmethylsulphonyl fluoride (PMSF); for metalloproteases, 2 mmol/L EDTA; for CPs, 40, 60, 80 and 100 mmol/L N-ethylmaleimide (NEM).
AcCP3 gene silencing
siRNA targeting the catalytic domain of AcCP3 was synthesised by RiboBio Co. (China) and based on the cDNA sequence. The sequence of the forward strand was 5'-AGUACAUCAUCAACAACAA-3'. Trophozoites were plated at a density of 5 × 104 cells in 48-well plates, cultured overnight and then transfected with siRNA (15 μg/mL) for 12 h using SuperFectin™ In Vitro siRNA Transfection Reagent (Pufei, China). As a control, a negative siRNA provided by RiboBio Co. (China) was also applied to cultured trophozoites. Untreated trophozoites and transfection reagent-treated trophozoites were also processed. After the transfection, the differentially treated trophozoites were harvested to determine the efficacy of the knockdown by examining the expression level of AcCP3 with qRT-PCR and then for the cytopathic tests.
Effect of AcCP3 knockdown in trophozoites on Acanthamoeba-mediated cytotoxicity
To determine the effects of reduced AcCP3 expression on Acanthamoeba-mediated HCECs death, cytotoxicity assays were performed as previously described . Confluent HCEC monolayers in 12-well culture plates (Corning, USA) were incubated with differentially treated trophozoites (ratio 1:2) at 37 °C in a 5% CO2 atmosphere for 24 h. Four different experimental groups were included: group 1, normal cultured confluent HCECs; group 2, HCECs co-cultured with normal trophozoites; group 3, HCECs co-cultured with negative control siRNA transfected trophozoites; group 4, HCECs co-cultured with AcCP3-knockdown trophozoites. The cytopathic effects for the different groups of trophozoites were observed using light microscopy (Olympus, Japan). The trophozoites were detached with cold 2 mM EDTA-PBS buffer, chilled on ice for 20 min and then harvested. In addition, HCECs were cultured in fresh DMEM medium with CCK-8 reagent for 2 h, and the cytotoxicity was determined by measuring dehydrogenase release (Cell Counting Kit-8, DOJINDO, Japan). The absorbance of each well was measured at 450 nm using a Model 680 Microplate Reader (Bio-Rad, USA). To investigate the signalling pathways in HCECs activated by Acanthamoeba, four groups of differentially treated HCECs were harvested after co-culturing with amoeba for 24 h. qRT-PCR was performed to analyse the expression level of the Ras gene (NM_004985.4). The forward strand was 5'-AGGAAGCAAGTAGTAATTGATGGA-3'; the reverse strand was 5'-GCCTGTTTTGTGTCTACTGTTCT-3'. Human GAPDH used as an internal reference, with forward strand 5'-TCACCACCATGGAGAAGGC-3' and reverse strand 5'-GCTAAGCAGTTGGTGGTGCA-3'.
Western blotting assays
The activation states of Raf, Erk and p53 in differentially treated HCECs were determined using Western blotting assays as previously described . Briefly, four groups of differentially treated HCECs were harvested for Western blotting after coculturing with amoeba for 24 h. The cells were lysed in 100 μL lysis buffer (1 mL lysis buffer containing 20 μL phosphatase inhibitors, 20 μL protease inhibitor cocktail, 100 μL PBS, 5 μL NP-40 and 855 μL ddH2O). The cell lysate (20 μg) was resolved by SDS-PAGE and transferred to PVDF membranes (Roche, Switzerland). We used the following primary antibodies purchased from Cell Signaling Technology (USA): rabbit anti-p-Raf (Ser338), rabbit anti-Raf, rabbit anti-p-Erk (Thr202/Tyr204), rabbit anti-Erk, mouse anti-p-p53 (Ser15) and mouse anti-p53. HRP-labelled goat anti-mouse and goat anti-rabbit secondary antibodies (Abcam, UK) and rabbit polyclonal to β-actin (Abcam, UK) were detected with Tanon™ High-sig ECL Western Blotting Substrate (Tanon, China), observed with an ECL detection system (Tanon, China), and the scanned images were quantified using Image-Pro Plus 4.5.1 software (Media Cybernetics, USA).
The results of qRT-PCR were calculated using the 2−△△Ct method. Statistical analyses were performed using GraphPad Prism software (San Diego, CA, USA). Significance was calculated by one-way analysis of variance followed by a Tukey test or Student’s t-test. Data were expressed as mean ± SD and at least three independent experiments were performed for each experiment. A P value < 0.05 was considered significant in all analyses.