INTRODUCTION
Proton-translocating inorganic pyrophosphatases (H+-PPases) are extremely hydrophobic integral membrane proteins that utilize the energy released by hydrolysis of pyrophosphate (PPi), which possesses a high-energy phosphoanhydride bond, to transport H+ across the biological membranes against the electrochemical potential gradient (Maeshima, Reference Maeshima2000; Belogurov and Lahti, Reference Belogurov and Lahti2002; Serrano et al. Reference Serrano, Pérez-Castiñeira and Baltscheffsky2004; Gaxiola et al. Reference Gaxiola, Palmgren and Schumacher2007). The first H+-PPase was discovered in membranes isolated from the photosynthetic bacterium Rodospirillum rubrum (Baltscheffsky et al. Reference Baltscheffsky, von Stedingk, Heldt and Klingenberg1966). It was subsequently found in homogenates and in higher plant vacuoles (V-H+-PPases) as a proton pump (Karlsson, Reference Karlsson1975) and was later detected in acidocalcisomes of parasitic protozoa (Scott et al. Reference Scott, de Souza, Benchimol, Zhong, Lu, Moreno and Docampo1998). Although this enzyme was long considered to occur only in plants and some photosynthetic bacteria (Drozdowicz et al. Reference Drozdowicz, Shaw, Nishi, Striepen, Liwinski, Roos and Rea2003), it has since been identified in a wide range of organisms including prokaryote extremophiles, fungi, some algae and protozoa (Maeshima, Reference Maeshima2000; Drozdowicz and Rea, Reference Drozdowicz and Rea2001). In plants, V-H+-PPases are present in the vacuole membrane (tonoplast) and also in the plasma membrane (Rea and Poole, Reference Rea and Poole1993; Long et al. Reference Long, Williams, Nelson and Hall1995; Robinson et al. Reference Robinson, Haschke, Hinz, Hoh, Maeshima and Marty1996). In protozoa, V-H+-PPase is an integral membrane-associated protein that has been located in the acidocalcisomes and also in the Golgi apparatus, plasma membrane, digestive vacuoles and in a microneme maturation vacuolar compartment of trypanosomatids (Harper et al. Reference Harper, Huynh, Coppens, Parussini, Moreno and Carruthers2006).
The first indication of the existence of the diversity and functional heterogeneity of V-H+-PPases was found in the plant Arabidopsis thaliana, and two distinct enzyme categories were observed: AVP1 and AVP2 (Arabidopsis vacuolar H+-pyrophosphatase). Phylogenetic analysis of these and V-H+-PPases showed that AVP2, rather than being an isoform of AVP1, is representative of a novel category of AVP2-like (type II) V-PPases that coexist with AVP1-like (type I) V-H+-PPases in plants and also in apicomplexan protists such as the malarial parasites (Drozdowicz et al. Reference Drozdowicz, Kissinger and Rea2000). There is a clear evidence for the wide occurrence of V-H+-PPase genes in hymenostomatid, peritrich and hypotrich ciliates (Pérez-Castiñeira et al. Reference Pérez-Castiñeira, Alvar, Ruiz-Pérez and Serrano2002); however, until recently the presence of V-H+-PPase activity has only been demonstrated in the scuticociliate parasite of the turbot Philasterides dicentrarchi (Mallo et al. Reference Mallo, Lamas, Piazzon and Leiro2015).
More specifically, we report the results of a study showing, for the first time, the existence of two isoforms of H+-PPase in P. dicentrarchi. One isoform of the enzyme was found in flat cortical sacs, designated ‘alveolar sacs’ in Ciliophora, and its gene expression was modulated by salt concentration.
MATERIALS AND METHODS
Parasites and experimental animals
Specimens of P. dicentrarchi (isolates B1, C1, D2, D3, I1, S1, P1; Iglesias et al. Reference Iglesias, Paramá, Álvarez, Leiro, Fernández and Sanmartín2001; Budiño et al. Reference Budiño, Lamas, Pata, Arranz, Sanmartín and Leiro2011) were collected under aseptic conditions from peritoneal fluid obtained from experimentally infected turbot, Scophthalmus maximus, as previously described (Paramá et al. Reference Paramá, Iglesias, Álvarez, Leiro, Aja and Sanmartín2003). The ciliates were cultured at 21 °C in complete sterile L-15 medium, as previously described (Iglesias et al. Reference Iglesias, Paramá, Álvarez, Leiro, Aja and Sanmartín2003). In order to maintain the virulence of the ciliates, fish were experimentally infected every 6 months by intraperitoneal (ip) injection of 200 µL of sterile physiological saline containing 5 × 105 trophozoites, and the ciliates were recovered from ascitic fluid and maintained in culture as described above.
Turbot of approximately 50 g body weight were obtained from a local fish farm. The fish were held in 250-L tanks with aerated recirculating sea water maintained at 14 °C. They were subjected to a photoperiod of 12L:12D and fed daily with commercial pellets (Skretting, Burgos, Spain). The fish were acclimatized to laboratory conditions for 2 weeks before the experiments were started.
Eight to 10-week-old Institute for Cancer Research (ICR) (Swiss) CD-1 mice initially supplied by Charles River Laboratories (USA) were bred and maintained in the Central Animal Facility of the University of Santiago de Compostela (Spain) following the criteria of protection, control, care and welfare of animals and the legislative requirements relating to the use of animals for experimentation (EU Directive 86/609/EEC), the Declaration of Helsinki, and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The Institutional Animal Care and Use Committee of the University of Santiago de Compostela approved all experimental protocols.
Polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), real-time quantitative reverse transcriptase PCR (qRT-PCR)
P. dicentrarchi DNA was purified with DNAesy Blood and Tissue Kit (Qiagen) by following the manufacturer's instructions. DNA was analysed to estimate its quality, purity and concentration by A260 measurement in a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, USA.).
The P. dicentrarchi trophozoites were incubated for 24 h in culture medium supplemented with different concentrations of NaCl: 4, 8 and 37‰. Total RNA was isolated from the trophozoites with a NucleoSpin RNA isolation kit (Macherey-Nagel, Düren, Germany) by following the manufacturer's instructions. After purification of the RNA, the quality, purity and concentration were measured in a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, USA). The reaction mixture used for cDNA synthesis (25 µL reaction mixture−1) contained 1·25 µ m random hexamer primers (Promega), 250 µ m of each deoxynucleoside triphosphate (dNTP), 10 mm dithiothreitol (DTT), 20 U of RNase inhibitor, 2·5 mm MgCl2, 200 U of MMLV (Moloney murine leukemia virus reverse transcriptase (Promega) in 30 mm Tris and 20 mm KCl (pH 8·3) and 2 µg of sample RNA. PCR (for DNA and cDNA amplification) was performed with gene-specific primers for the H+PPase gene: forward/reverse primer pair (FPiPh/RPiPh) 5′-CGGGACCAGAGGTATCTTTTA-3′/5′-ATTGATGTCAACGCCCCCTT-3′; and forward/ reverse primer pair (F1qPiPh/R1qPiPh) 5′-GCCTACGAAATGGTCGAAGA-3′/5′-GCATCGGTGTATTGTCCAGA-3′ for real-time qRT-PCR. In parallel, real-time qRT-PCR was performed with β-tubulin as a reference gene, by including the primers for the β-tubulin gene (forward/reverse primer pair, 5′-ACCGGGGAATCTTAAACAGG-3′/5′-GCCACCTTATCCGTCCACTA-3′). The Primer 3Plus program was used, with default parameters, to design and optimize the primer sets. The PCR mixtures (25 µL) contained PCR reaction buffer (10 mm Tris-HCl, 50 mm KCl, 1·5 mm MgCl2, pH 9·0), 0·2 mm of each deoxynucleoside triphosphate (dNTPs, Roche), 0·4 µ m of each primer, 3 units of recombinant Taq polymerase (NZY Taq DNA polymerase, NZYTech, Portugal) and 50 ng of genomic DNA or 2μL of cDNA. The reactions were run in an automatic thermocycler (Biometra, Germany) as follows: initial denaturing at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 57 °C for 45 s and 72 °C for 1 min; and finally a 7 min extension phase at 72 °C. Quantitative PCR mixtures (10 µL) contained 5 µL Maxima SYBR green qPCR Master Mix (Thermo Scientific), 300 nm of the primer pair, 1 µL of cDNA and RNase-DNase-free water. Quantitative PCR was developed at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s, ending with a melting-curve analysis at 95 °C for 15 s, 55 °C for 15 s and 95 °C for 15 s. The specificity and size of PCR products were confirmed by 4% agarose gel electrophoresis. All quantitative PCRs were performed in an Eco RT-PCR system (Illumina). Relative quantification of gene expression was determined by the 2−ΔΔCt method (Livak & Schmittgen, Reference Livak and Schmittgen2001) applied with software conforming to minimum information for publication of qRT-PCR experiments (MIQE) guidelines (Bustin et al. Reference Bustin, Benes, Garson, Hellemans, Huggett, Kubista, Mueller, Nolan, Pfaffl, Shipley, Vandesompele and Wittwer2009).
Production of recombinant H+-PPase of P. dicentrarchi in yeast cells
The P. dicentrarchi RNA was purified using a NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions, and cDNA synthesis was performed as indicated in the previous section. The PCR was carried out with gene-specific primers designed from a partial sequence of the H+-PPase of P. dicentrarchi (Mallo et al. Reference Mallo, Lamas, Piazzon and Leiro2015) (forward/reverse primer pair 5′-AAAGAAGAAGGGGTACCTTTGGATAAAAGAattgatgtcaacgccccctt-3′/5′- TGGGACGCTCGACGGATCAGCGGCCGCTTAGTGGTGGTGGTGGTGGTGgggaccagaggtatctttta-3′). These primers were designed and optimized using the Saccharomyces Genome Database (http://www.yeastgenome.org/) and by including a hybridization region with the yeast YEpFLAG-1 (Eastman Kodak Company) plasmid and a poly His region (lower case letters correspond with the gene annealing zone). The PCR reaction was initially developed at 95 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1·5 min and 72 °C for 2 min and finally, a 7-min extension phase at 72 °C. The PCR products were purified using Gene Jet PCR Purification Kit (Fermentas, Life Sciences) according to the manufacturer's instructions.
Purified PCR products were cloned in YEpFLAG-1 (Eastman Kodak Company) yeast expression vector, a plasmid carrying a N-5′-phosphoribosylanthranilate isomerase (TRP1) gene that completes the auxotrophy for the tryptophan for the host yeast (López-López et al. Reference López-López, Fuciños, Pastrana, Rúa, Cerdán and González-Siso2010).
Linearized plasmid YEpFLAG-1 was digested with EcoRI and SalI (Takara) and used to transform Saccharomyces cerevisiae cells (strain BJ 3505) by the lithium acetate procedure (Ito et al. Reference Ito, Fukuda, Murata and Kimura1983). The procedure involves co-transformation of yeast cells with the linearized empty plasmid and the PCR-generated DNA fragment so that a recombination process occurs within the cell, yielding a plasmid bearing the desired insert. Positive colonies were selected using complete medium without tryptophan (CM-Trp) containing glucose (20 g L−1), Yeast Nitrogen Base medium without amino acids (Sigma-Aldrich), supplemented with adenine (40 mg L−1) and amino acids (histidine, leucine, tyrosine, 40 mg L−1 each; arginine, methionine, threonine 10 mg L−1 each; isoleucine and phenylalanine 60 mg L−1 each and lysine 40 mg L−1).
Plasmid DNA was then extracted with Easy Yeast Plasmid Isolation Kit (Clonetech) according to the manufacturer's instructions. The purified and cloned DNA fragment was subjected to sequencing analysis (Sistemas Genómicos, Spain).
Recombinant protein of H+-PPase of P. dicentrarchi was purified from transformed S. cerevisiae cultures, which had been incubated for 72 h in modified Yeast Peptone High Stability Expression Medium containing 1% glucose, 3% glycerol, 1% yeast extract and 8% peptone, at 30 °C, in Erlenmeyer flasks filled with 20% volume of culture medium (López-López et al. Reference López-López, Fuciños, Pastrana, Rúa, Cerdán and González-Siso2010). As the inoculum, a suitable volume of a pre-culture was added to obtain an initial OD600 of 0·1. The cell suspension was centrifuged at 7500 g for 15 min and the cleared supernatant was purified by immobilized metal affinity chromatography on a pre-charged Ni-Sepharose Histrap column (ÄKTAprime plus, GE Healthcare Life Sciences). The column was initially equilibrated with 25 mL of binding buffer (20 mm sodium phosphate, 0·5 M NaCl, 20 mm imidazole, pH 7·4). One hundred mL of culture medium was then passed through the column, and the protein bound to the column was finally eluted in 10 mL of elution buffer (20 mm sodium phosphate, 0·5 M NaCl, 250 mm imidazole, pH 7·4) (Mallo et al. Reference Mallo, Lamas, Piazzon and Leiro2015). The fractions thus obtained were analysed by 12·5% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and dialyzed overnight in 2 L of bidistilled water. The dialyzed sample was concentrated in an Amicon Ultra centrifugal filter device (Millipore, USA) with a 10-kDa cut-off membrane. The final protein concentration was calculated by the Bio-Rad Protein Assay, which is based on the Bradford assay (Bradford, Reference Bradford1976).
Peptide synthesis
A peptide of 17 amino acid long corresponding to domain HKAAVIGDTIGDPLKDT (peptide HK) of the P. dicentrarchi H+-PPase was synthesized and conjugated to keyhole-limpet haemocyanin (KLH), a carrier protein, to ensure maximum immunogenicity (ProteoGenix, France). A cysteine amino acid was added to two sequences to enable conjugation to KLH. The peptide was synthesized and conjugated to KLH by coupling with sulpho-SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) reagent to yield 10–20 mg of product >85% purity, which was lyophilized and stored at −20 °C until use.
Immunization and serum extraction
A group of five ICR (Swiss) CD-1 mice were immunized by i.p. injection with 200 µL per mouse of a 1:1 (v/v) mixture of Freund complete adjuvant (Sigma-Aldrich) and a solution containing 500 µg of purified recombinant H+-PPase (rH+-PPase) or with 400 µg of KLH conjugated to the synthetic peptide HK in phosphate buffered saline (PBS). The same dose of purified protein and peptide was prepared in Freund's incomplete adjuvant and injected i.p in mice 15 and 30 days after the first immunization. The mice were bled via the retrobulbar venous plexus 7 days after the second immunization (Piazzon et al. Reference Piazzon, Lamas and Leiro2011). In order to obtain the polyclonal anti-rH+-PPase (PABrPPase) or anti-HK (PABHK) antisera, the blood was left to coagulate overnight at 4° C before the serum was separated by centrifugation (2000 × g for 10 min), mixed 1:1 with glycerol and stored at −20 °C until use. In some experiments, a commercial rabbit polyclonal serum against KLH-conjugated synthetic peptide derived from the AVP1 isoform (UniProt P311414) of Arabidopsis thaliana V-PPase (PABAVP1, Agrisera, Sweden) was also used.
Western-blot analysis
Ciliate membrane-associated proteins (MAPs) were extracted by phase separation in Triton X-114 solution (Bordier, Reference Bordier1981), by a previously described method (Mallo et al. Reference Mallo, Lamas and Leiro2013). Specifically, 107 cells were resuspended in 1 mL of ice-cold 10 mm Tris–HCl buffer, pH 7·5, to which 1 mL of ice-cold extraction buffer (300 mm NaCl, 20 mm Tris–HCl, pH 7·5, 2% Triton X-114) was subsequently added. The cytoskeletal elements were eliminated by centrifugation at 16 000 × g for 10 min at 4 °C. The supernatant was then transferred to 1·5 mL Eppendorf tubes, which were heated for 5 min at 37 °C. At the end of this period, the solution became cloudy due to condensation of detergent micelles. The sample was then placed in 0·5 mL Eppendorf tubes (200 µL tube−1) containing 300 µL of sucrose cushion (6% sucrose, 150 mm NaCl, 10 mm Tris–HCl, pH 7·5, 0·06% Triton X-114). The detergent and aqueous phases were separated by centrifugation at 300 g for 4 min at room temperature (RT). The resulting supernatants lying on the sucrose cushion of each tube were carefully removed and mixed in new 1·5 mL Eppendorf tubes. The extraction process was repeated by adding sufficient Triton X-114 to the aqueous mixture to yield a final concentration of 0·5%. The mixture was re-heated at 37 °C for 5 min. Once micellar condensation had taken place, the mixture was distributed among the original Eppendorf tubes containing the sucrose cushion and the detergent phase that was separated in the first extraction. The tubes were then recentrifuged at 300 g for 4 min at RT. The resulting supernatant was discarded and the proteins contained in the detergent phase were precipitated (by adding 9 volumes of cold acetone), resuspended (by vortexing) and finally incubated for 30 min on ice. The precipitated membrane proteins were then collected by centrifugation, at 16 000 g for 15 min at 4 °C, and dried in a speed vacuum concentrator (MiVac, GeneVac, UK). Finally, the extracts obtained were resuspended in 10 mm Tris–HCl, pH 7·5, and stored at −80 °C until use. The protein concentration of preparation was determined by the Bradford assay.
Samples from MAPs were separated under non-reducing conditions on linear SDS-PAGE 12·5% gels (Piazzón et al. Reference Piazzón, Lamas, Castro, Budiño, Cabaleiro, Sanmartín and Leiro2008). On completion of the electrophoresis, the gels were stained with Thermo Scientific GelCode Blue Safe Protein Stain (Thermo Fisher, USA) for qualitative determination of the protein bands. At the same time, a gel was immunoblotted at 15 V for 35 min to Immobilon-P transfer membranes (0·45 µm; Millipore, USA) in a trans-blot s.d. transfer cell (Bio-Rad, USA) with transference buffer (48 mm Tris, 29 mm glycine, 0·037% SDS and 20% methanol, pH 9·2). The membrane was washed with Tris buffer saline (TBS; 50 mm Tris, 0·15 M NaCl, pH 7·4) and immediately stained with Ponceau S to verify transfer. After membrane destaining with bidistilled water, a blocking solution consisting of TBS containing 0·2% Tween 20 and 3% bovine serum albumin (BSA) was added, and the membrane was incubated for 1·5 h at RT. The membrane was then washed in TBS and incubated overnight with PABHK and PABrPPase, diluted 1/100, at 4 °C. The membrane was washed again with TBS and incubated with rabbit anti-mouse IgG (Dakopatts; dilution 1:6000) for 1 h at RT. After the membrane was washed 5 times for 5 min with TBS, it was incubated for 1 min with enhanced luminol-based chemiluminiscent substrate (Pierce ECL Western Blotting Substrate, Thermo Scientific, USA), before being visualized and photographed with a FlourChem® FC2 imaging system (Alpha Innotech, USA).
Immunofluorescence, Immunoelectron microscopy, fluorescent enzyme linked immunoassay (FELISA), and fluorescent staining with pH-sensitive dye
For immunolocalization of H+-PPase isoforms, an immunofluorescence assay was performed following the previously described protocol (Mallo et al. Reference Mallo, Lamas, Piazzon and Leiro2015). Briefly, 5 × 106 ciliates were centrifuged at 750 × g for 5 min, washed twice with Dulbecco's phosphate buffered saline (DPBS, Sigma Aldrich) and fixed for 5 min in a solution of 4% formaldehyde in DPBS. The ciliates were then washed twice with DPBS, resuspended in a solution containing 0·1% Triton X-100 (PBT) for 3 min and then washed twice with DPBS. They were then incubated with 1% BSA for 30 min. After this blocking step, the ciliates were incubated at 4 °C overnight with a solution containing a 1:100 dilution of anti- PABrPPase, PABHK or PABAVP1. The ciliates were then washed 3 times with DPBS, before being incubated for 1 h at RT, with a 1:100 dilution of fluorescein isotiocyanate (FITC) conjugated rabbit/goat anti-mouse/rabbit IgG-FITC antibody (Sigma). After three washes in DPBS, the samples were double-stained with 0·8 mg mL−1 4′, 6-diamidine-2-phenylindole (DAPI; Sigma-Aldrich) in DPBS for 15 min at RT (Paramá et al. Reference Paramá, Castro, Lamas, Sanmartín, Santamarina and Leiro2007). After another three washes in DPBS, the samples were mounted in PBS-glycerol (1:1) and visualized by fluorescence microscopy (Zeiss Axioplan, Germany) and/or confocal microscopy (Leica TCS-SP2, LEICA Microsystems Heidelberg GmbH, Mannheim, Germany).
For immunoelectron microscopy, an aliquot of 5 × 106 ciliates obtained from cultures in exponential growth phase was centrifuged at 750 × g for 5 min and washed in two changes of Sörensen buffer (SB; 0·1 M sodium/potassium phosphate buffer, pH 7·3) at RT. The resulting pellet was fixed for 60 min in 4% paraformaldehyde and 0·1% glutaraldehyde in SB at 4 °C. The fixed samples were washed in two changes of SB (10 min each) and incubated with 0·02 M glycine in SB for 10 min at RT. The ciliates were dehydrated in series of pre-cooled ethanol solutions (30, 50, 70, 80, 96 and 100% of 10 min each). The dehydrated pellet was placed in a 2:1 mixture of ethanol and resin (LR White uncatalyzed, Santa Cruz Biotechnology, USA; Philimonenko et al. Reference Philimonenko, Janácek and Hozák2002) for 20 min and then in pure resin for 2 h. Samples were infiltrated overnight with fresh resin at 4 °C. The resin was replaced the next day and the mixture was allowed to polymerize at 65 °C in vacuum for 48 h. Thin sections (80 nm thick) were cut, with a diamond knife, on a Reichert Ultracut E (Leica Microsystems AG, Germany). Thin sections were collected on 300 mesh nickel grids (Sigma-Aldrich) and were blocked by preincubation with 10% normal goat serum (NGS) in PBS-10% albumin and 0·1% Tween-20 (PBTB) for 30 min at RT. The sections were incubated for 1 h with PABHK in PBTB at 1:100 dilution, washed in PBS-albumin, and incubated with 10 nm gold-labelled goat anti-rabbit IgG (Sigma) at 1:50 dilution for 60 min. Finally, the sections were washed in distilled water, stained with uranyl acetate (1% in distilled water) and lead citrate (4·5 mm in 4·5 mm NaOH), and observed under a JEOL-JEM-2010 transmission electron microscope operated at 120 kV (JEOL, Japan). Negative controls were included to establish the specificity of the polyclonal antibodies used in these immunoassays. Some sections were processed with an unrelated antibody or were incubated in the presence of the secondary antibody only. No positive staining was observed in these controls.
For quantification of the expression of the alveolar H+-PPase, we used a FELISA. One μg of MAPs (isolated from trophozoites incubated for 24 h in culture media with different saline concentrations: 4, 8 and 37‰) was added to 100 µL of carbonate-bicarbonate buffer (pH 9·6) in 1 × 8 well ELISA-strips (high binding, Greiner Bio-One, Germany) and incubated overnight at 4 °C. The wells were then washed three times with TBS, blocked 1 h with TBS containing 0·2% Tween 20 (TBS-T1), 5% non-fat dry milk, incubated for 30 min at RT in a microplate shaker for ELISA (Stuart, UK) at 750 rpm with 1:100 dilution (in TBS-T1 containing 1% non-fat dry milk) of PABHK, and washed five times with TBS containing 0·05% Tween 20. Bound anti-mouse antibodies were detected with FITC-conjugated rabbit anti-mouse Ig (Sigma) at 1:1000 dilution in TBS-T1, and incubated for 30 min with shaking. After five washes in TBS, 100 µL of TBS was added to each well, and fluorescence was measured in a microplate fluorescence reader (Bio-Tek Instruments, USA) with an excitation wavelength of 490 nm, emission wavelength of 525 nm and a sensitivity of 70%. The results were expressed in arbitrary fluorescence units.
For identification of the acidic compartments on trophozoites of P. dicentrarchi we used a fluorescent stain assay with the pH-sensitive dye Lysotracker Red DND-99. An aliquot of 5 × 105 ciliates was centrifuged at 700 × g for 5 min, washed twice with PBS and stained for 10 min with 75 nm Lysotracker Red DND-99 (Lifetechnologies) solution. The ciliates were then observed in a fluorescence microscopy with an excitation filter (BP 546 nm), dichroic mirror (FT 580 nm) and emission filter (LP 590 nm).
Bioinformatic and statistical analysis
The amino acid sequences obtained for the H+-PPase gene were aligned using the Clustal Omega multiple sequence alignment program (Sievers et al. Reference Sievers, Wilm, Dineen, Gibson, Karplus, Li, López, McWilliam, Remmert, Söding, Thompson and Higgins2011). Genetic distances were calculated in order to quantify sequence divergence between isolates by use of Kimura's (Reference Kimura1980) two-parameter model, as implemented in MEGA version 6.0 (Tamura et al. Reference Tamura, Stecher, Peterson, Filipski and Kumar2013). Phylogenetic trees were constructed with the MEGA programme, by applying the neighbour-joining (NJ) method to the Kimura two-parameter correction model (Kimura, Reference Kimura1980) by bootstraping with 1000 replicates (Felsenstein, Reference Felsenstein1985).
The results are expressed as means ± standard error of the mean (s.e.m.). The data were examined by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test for multiple comparisons, and differences were considered significant at α = 0·05.
RESULTS
Cellular localization of H+-PPase of P. dicentrarchi in acidic compartments
A cDNA fragment encoding 169 aa located between positions 305 and 474 of the amino acid sequence of the H+-PPase (GenBank accession AHH28243) containing domain HKAVIGDTIGDPLKDTS was cloned in a yeast expression vector YepFlag-1. The mouse anti-rH+-PPase polyclonal antibodies (PABrPPase), generated after immunization with the recombinant protein fragment, produced intense fluorescent staining of vacuoles located on the basal part of trophozoites and in alveolar sacs located under the plasma membrane (Fig. 1A).
(A, B) Detection by immunofluorescence and confocal microscopy of H+-PPase in a ciliate trophozoite by using a mouse antibody anti-recombinant H+-PPase expressed in the yeast Saccharomyces cerevisiae (PABrPPase) and a secondary polyclonal antibody anti-mouse Ig conjugated with FITC. After the immunofluorescence assay, trophozoites were counterstained with DAPI to identify the macronucleus (M). (A) Arrows indicate immunolocalization of H+-PPase in the vacuoles and arrowheads indicate the presence of specific immunostaining appearing as a dotted line coinciding with the alveolar sacs. (B) Lysotracker Red DND-99 staining of acidic compartments of P. dicentrarchi showing fluorescent labelling of the posterior vacuoles (arrows) and the alveolar sacs (arrowheads). Scale bars = 10 µm. Abbreviations: DAPI, 4′, 6-diamidine-2-phenylindole; FITC, fluorescein isotiocyanate.
Incubation of P. dicentrarchi trophozoites with the pH sensitive dye Lysotracker Red DND-99 produced intense staining in the vacuoles and in the alveolar sacs (Fig. 1B).
Immunohistochemical pattern of the H+-PPase revealed by polyclonal antibodies (PABHK)
Indirect immunofluorescence studies with a mouse polyclonal antibody generated against a KLH-conjugated synthetic peptide of the conserved amino acid domain HK (PABHK; Fig. 2A) and the polyclonal antibody anti-AVP1 (PABAVP1), a KLH-conjugated synthetic peptide derived from Arabidopsis thaliana V-PPase (Fig. 2B), revealed a unique labelling pattern on the surface of the parasite. Immunoelectron microscopic analysis with PABHK revealed specific labelling in the membranes of the alveolar sacs (Fig. 2C and D).
Detection by immunofluorescence and confocal microscopy of a H+-PPase isoform with a mouse polyclonal antibody generated to conserved amino acid domain HK (PABHK) (A) and with the commercial anti-AVP1 polyclonal antisera corresponding to AVP1 isoform of A. thaliana (PABAVP1) (B). A pattern of discontinuous fluorescence can be seen on the surface of trophozoites (arrows). (C, D) Immunoelectron microscopic localization of the H+-PPase isoform using PABHK and which shows specific staining (arrows) on the membrane of the alveolar sacs (as).
Sequence characteristics of H+-PPase isoforms
To investigate the possible existence of H+-PPase isoforms in the posterior vacuoles and in the alveolar sacs of P. dicentrarchi, we amplified a fragment of this gene by PCR and also amplified several cDNA from RNA using the FPiPh/RPiPh primer pair. Figure 3A shows the results of the nucleotide sequence amplified by PCR corresponding to a partial open reading frame (ORF) of H+-PPase gene and its amino acid translation. Analysis of the DNA fragment amplified with primers FPiPh/RPiPh on 4% agarose gel revealed a single band of 558 nucleotides; however, when the cDNA generated from total RNA was amplified with the same primers, two bands were obtained, one of identical size to that obtained after DNA amplification (558 nucleotides) and a second band of 495 nucleotides (Fig. 3A). Sequencing of the two bands obtained by RT-PCR showed that the nucleotide sequence of the larger fragment corresponded exactly to the sequence obtained from genomic DNA by PCR. Sequencing of the minor band showed the disappearance of a 63 nucleotide fragment that was present in the largest band. After translation of both fragments to aa, the lower band produced a protein containing the complete domain HKAAVIGDTIGDPLKDTS, while the largest band generated a protein with this fragmented domain plus an internal sequence of 21 amino acids (Fig. 3A).
(A) Nucleotide sequence belonging to a gene region of the H+-PPase of P. dicentrarchi together with its corresponding translation into amino acids, containing a sequence motif recognized by the PABHK (black box). Agarose gel (4%) containing the PCR and RT-PCR products obtained using DNA and RNA as templates and the primers FPiPh/RPiPh is shown in the lower part of the figure. Two bands were observed when cDNA was used as a template, corresponding respectively to isoform 1 (the largest), and isoform 2 (the smallest). Isoform 2 contains the complete motif recognized by PABHK.. (B) Western blot with PABrPPase (lane 1) and PABHK (line 2) on ciliate MAPs of trophozoites subjected to SDS-PAGE under non-reducing conditions and which recognizes a single band of approximately 62 kD (arrow). Mw: molecular weight markers. Abbreviations: MAPs, membrane-associated proteins; PABrPPase, polyclonal anti-rH+-PPase; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR.
Polyclonal antibodies generated in mice after immunization with the synthetic peptide corresponding to the HK domain (PABHK) and with the recombinant fragment of H+-PPase (PABrPPase) recognized a single protein band of approximately 62 kD on MAPs in Western blots (Fig. 3B).
Phylogenetic analysis of H+-PPases in several strains of P. dicentrarchi
To determine the degree of protein conservation between isolates of P. dicentrarchi, we used PCR, with the FPiPh/RPiPh primer pair, to amplify the DNA of seven isolates. After obtaining the nucleotide sequence of each isolate and its translation into aa, we used the Clustal Omega program for multiple alignment of the amino acid sequences. The results showed a very high degree of conservation between the aa sequences of the isolates analysed (Fig. 4A), with a very small genetic distance between isolates. Construction of the phylogenetic tree by the NJ method revealed five isolates with 100% homology (I1, B1, D3, P1 and S1 isolates) and that D2 and C1 constituted two phylogenetically different groups (Fig. 4B).
(A) Multiple sequence alignment (Clustal Omega) of aa sequences obtained from a partial ORF of the gene of H+-PPase from several isolates (B1, D2, D3, C1, I1, S1 and P1) of P. dicentrarchi. The boxes highlighted in bold indicate the motif recognized by the PABHK antibody. (B) Phylogenetic comparison of P. dicentrarchi H+-PPase isolates. Aligned aa sequences were subjected to phylogenic analysis by the NJ method. The numbers at the nodes represent bootstrap values out of 1000 resampled values in the NJ analysis with the Kimura two-parameter correction model. Abbreviations: NJ, neighbour joining; ORF open reading frame.
Effect of salt concentration on expression of H+-PPase
The results of assays of the expression levels of RNA corresponding to the H+-PPase of P. dicentrarchi trophozoites cultivated in saline medium containing different concentrations of NaCl (between 4, 8 and 37‰, are shown in Fig. 5A. Relative mRNA levels (quantified by qPCR) of H+-PPase remain unchanged at NaCl concentrations between 8 and 37‰; however, when the medium contained a low concentration of NaCl (e.g. 4‰), expression of H+-PPase increased significantly relative to levels obtained in ciliates incubated at concentrations of NaCl between 8 and 37‰ (Fig. 5A).
(A) Relative gene expression levels of P. dicentrarchi H+-PPase, determined by real-time qRT-PCR in trophozoites incubated for 24 h in medium containing different concentrations of NaCl (4, 8 and 37‰). Gene expression was normalized to the reference gene (P. dicentrarchi β-tubulin) and normalized data are expressed in AU. (B) Expression levels of the H+-PPase containing the HK domain quantified by FELISA immunoassay with the PABHK antisera. Expression was measured on ciliate MAPs of trophozoites incubated for 24 h in medium containing NaCl at 4, 8 and 37‰ and is shown as fluorescence units. Values shown are means ± standard error (s.e.) from five assays. *P < 0·01 relative to ciliates incubated in the medium with 37‰ NaCl. Abbreviations: AU, arbitrary units; FELISA, fluorescent enzyme linked immunoassay; MAPs, membrane-associated proteins; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.
The effect of salinity on the expression of the H+-PPase containing the HK domain was analysed by a FELISA immunoassay under the same conditions of salinity as in the previous experiment with the PABHK antiserum. The levels of the enzyme containing the HK domain were highest in the ciliates incubated with the lowest concentration of salt (4‰) (Fig. 5B).
DISCUSSION
H+-PPases are enzymes that translocate H+ across a membrane by using potential energy liberated on hydrolysis of the phosphoanhydride bond of inorganic phosphate (Rea and Poole, Reference Rea and Poole1993). They are widely distributed among land plants and have been found in several protozoan parasites including the scuticociliate parasite of turbot, P. dicentrarchi (Mallo et al. Reference Mallo, Lamas, Piazzon and Leiro2015). In eukaryotes, H+-PPases are associated with certain acidic compartments of the endomembrane system, namely the vacuole and lysosomes of plant cells and the acidocalcisomes of trypanosomatides and apicomplexan protozoans (Scott and Docampo, Reference Scott and Docampo2000; Pérez-Castiñeira et al. Reference Pérez-Castiñeira, Alvar, Ruiz-Pérez and Serrano2002; Docampo et al. Reference Docampo, de Souza, Miranda, Rohloff and Moreno2005). The functions of acidocalcisomes include cation storage, Ca2+ homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation (Moreno and Docampo, Reference Moreno and Docampo2009). In parasitic protozoans, acidocalcisomes also interact with other organelles such as the contractile vacuole and other vacuoles associated with the endosomal/lysosomal pathway (Moreno and Docampo, Reference Moreno and Docampo2009; Docampo et al. Reference Docampo, Ulrich and Moreno2010). In ciliates, such as Paramecium caudatum, acidification of phagocytic vacuoles occurs through fusion of nonlysosomal vesicles, known as acidosomes, which accumulate neutral red as well as acridine orange, indicating the acidic content of the vacuoles (Allen and Fok, Reference Allen and Fok1983). Sequencing of the whole genome of several species of ciliates enables identification of genes encoding the V-ATPase, a proton pump that drives H+ across membranes and that is crucial for acidifying food vacuoles (Plattner, Reference Plattner2010); however, although there is some evidence for the existence of H+-PPases in ciliates, as several sequences are deposited in nucleotide databases (e.g. Tetrahymena thermophila, GenBank accession XM_001011583; Tetrahymena pyriformis, GenBank accession AJ251772), little is known about the occurrence of membrane-bound H+-PPases and their physiological role in these protozoa (Pérez-Castiñeira et al. Reference Pérez-Castiñeira, Gómez-García, López-Marqués, Losada and Serrano2001). Acidocalcisomes and some of their transport activities have not yet been characterized in ciliates (Plattner et al. Reference Plattner, Sehring, Mohamed, Miranda, De Souza, Billington, Genazzani and Ladenburger2012). The present study clearly showed that the H+-PPase in P. dicentrarchi is located in vacuoles and alveolar sacs and that both of these structures are acidic cellular components (as indicated by staining with the pH sensitive dye Lysotracker Red DND 99).
Subcellular location of members of the H+-PPase family is mainly in endocellular membranes (vacuolar tonoplast) and acidocalcisomal membranes of eukaryotes (algae, plants and protozoa) (Maeshima, Reference Maeshima2000; Drozdowicz and Rea, Reference Drozdowicz and Rea2001; Docampo et al. Reference Docampo, de Souza, Miranda, Rohloff and Moreno2005) and plasma membrane invaginations of both bacteria and archaea (Baltscheffsky et al. Reference Baltscheffsky, Schultz and Baltscheffsky1999; Serrano et al. Reference Serrano, Pérez-Castiñeira and Baltscheffsky2004). However, evidence for a differential subcellular localization of the AVP1 (vacuole) and AVP2 (Golgi complex and lysosomes) isoforms has only been reported in plant cells (Rea et al. Reference Rea, Kim, Sarafian, Poole, Davies and Sanders1992; Mitsuda et al. Reference Mitsuda, Enami, Nakata, Takeyasu and Sato2001). Indirect immunofluorescence microscopy with polyclonal antibodies, to investigate the subcellular localization of V-H+-PPase in Plasmodium falciparum, indicated that VP1 is present within the vacuolar membrane and, possibly, in food vacuoles (Luo et al. Reference Luo, Marchesini, Moreno and Docampo1999; MacIntosh et al. Reference MacIntosh, Drozdowicz, Laroiya, Rea and Vaidya2001). The proton pumps V-H+-PPase and V-H+-ATPase also appear to be colocalized in acidic organelles such as acidocalciomes and food vacuoles in malarian parasites (Marchesini et al. Reference Marchesini, Luo, Rodrigues, Moreno and Docampo2000; Moriyama et al. Reference Moriyama, Hayashi, Yatsushiro and Yamamoto2003; Saliba et al. Reference Saliba, Allen, Zissis, Bray, Ward and Kirk2003). In the present study, we used immunofluorescence and immunoelectron microscopy to demonstrate that the PABHK sera, which recognizes the highly conserved domain HKAAVIDTIGDPKDT, generates specific labelling only in the alveolar sacs of ciliates, which suggests that this domain is not found in the H+-PPase vacuoles. Thus, immunostaining with PABHK may indicate the possible existence of two isoforms of H+-PPase in P. dicentrarchi.
The existence of multiple isoforms of H+-PPases has been demonstrated in plants (Venter et al. Reference Venter, Gronewald and Botha2006). Thus, at least two genes encoding H+-PPases have been detected in the rice (Oryza sativa L.) genome (Sakakibara et al. Reference Sakakibara, Kobayashi and Kasamo1996), three isoforms have been found in tobacco (Lerchl et al. Reference Lerchl, Kónig, Zrenner and Sonnewald1995), two in red beet (Beta vulgaris L.) (Kim et al. Reference Kim, Kim and Rea1994), two in barley (Hordeum vulgare L.) (Fukuda et al. Reference Fukuda, Chiba, Maeda, Nakamura, Maeshima and Tanaka2004), two in grapevine (Vitis vinífera L.) (Venter et al. Reference Venter, Gronewald and Botha2006) and two in cacao (Theobroma cacao L.) (Motamayor et al. Reference Motamayor, Mockaitis, Schmutz, Haiminen, Livingstone, Cornejo, Findley, Zheng, Utro, Royaert, Saski, Jenkins, Podicheti, Zhao, Scheffler, Stack, Feltus, Mustiga, Amores, Phillips, Marelli, May, Shapiro, Ma, Bustamante, Schnell, Main, Gilbert, Parida and Kuhn2013). All of these show high homology within the coding region but differ strongly in the untranslated regions, and their expression is probably regulated in different ways (Maeshima, Reference Maeshima2000). Plants have two phylogenetically distinct V-H+-PPases that can be classified into two subclasses: AVP1, which depends on cytosolic K+ for its activity and is moderately sensitive to inhibition by Ca2+; and AVP2, which is K+-independent but extremely Ca+2-sensitive (Sarafian et al. Reference Sarafian, Kim, Poole and Rea1992; Drozdowicz et al. Reference Drozdowicz, Kissinger and Rea2000; Gaxiola et al. Reference Gaxiola, Palmgren and Schumacher2007). Two genes encoding VP1 and VP2 have been identified in the malarian parasite P. falciparum (MacIntosh et al. Reference MacIntosh, Drozdowicz, Laroiya, Rea and Vaidya2001), and the sequence of isoform 2 corresponding to the himenostomatid ciliate Tetrahymena pyriformis is also available (GenBank accession AJ251471).
There is an almost-complete conservation between AVP1/AVP2 of the amino acid sequences recognized by polyclonal antibody PABHK (HKAAVIGDTIGPLK motif), further justifying the suggestion that these antibodies are universal reagents for detecting V-PPase polypeptides (Drozdowicz and Rea, Reference Drozdowicz and Rea2001). We have previously shown that the P. dicentrarchi H+-PPase has a motif in common with the polyclonal antibody PABHK specific to AVP1 (Mallo et al. Reference Mallo, Lamas, Piazzon and Leiro2015). In the present study, we found that this motif is encoded by a gene containing an intercalated nucleotide sequence that can be transcribed into two mRNA molecules, generating two isoforms: one isoform produces a protein with the fragmented motif and the other produces a protein containing the complete motif. The total size of the proteins produced by gene isoforms varies between 62 and 64 kD (the 2 kD difference is the estimated size of the peptide intercalated between the motif HK); however, these would probably be overlooked by SDS-PAGE or Western blot analysis because of the limited ability of these techniques to separate proteins of very similar molecular size (Dauly et al. Reference Dauly, Perlman, Costello and McComb2006). The hypothesis proposed here to explain the presence of both H+-PPase isoforms is based on the differential recognition of the H+-PPase in the alveolar sacs by PABHK and also on the presence of two amino acid sequences in the cDNAs generated by RT-PCR with the FPiPh/RPiPh primers. Presence of the isoform containing the complete HK motif in the H+-PPase of the alveolar sacs may be explained by a splicing mechanism. Isoform 1 of H+-PPase present in the vacuoles does not undergo this process, generating a protein with a fragmented motif that would not be recognized by PABHK. There are some examples of genes that generate isoforms transcribed from alternate promoter sites within the gene which may mediate cell signalling and induce translocation to various cellular localizations (Saitoh et al. Reference Saitoh, Murata, Odagiri, Itoh, Itoh, Misaka and Kubo2002). Furthermore, it is well-known that splicing regulation can be modulated by several sequence elements in both exons and introns that either activate (exonic splicing enhancer, ESE; introning splicing enhancer, ISE) or repress (exonic splicing silencer, ESS; intronic splicing silencer, ISS) (Poulos et al. Reference Poulos, Batra, Charizanis and Swanson2011). However, introns are removed and exons are added during the splicing process, and therefore introns should not appear in the final mature mRNA. The results of the present study suggest that the mRNA fragment eliminated by splicing during processing of H+-PPase mRNA is also a coding region. The existence of introns with features of both introns and protein-coding exons and referred to as exitrons (exonic introns) has recently been described in Arabidopsis and humans (Marquez et al. Reference Marquez, Höpfler, Ayatollahi and Barta2015). This unusual alternative splicing event may be a novel source of alternative transcripts and protein isoforms for genes annotated as intronless and may explain the generation of the two H+-PPase isoforms in P. dicentrarchi.
Description of nucleotide sequences of H+-PPase genes from plants, bacteria and archaea highlighted an unusually high degree of sequence conservation (Serrano et al. Reference Serrano, Pérez-Castiñeira, Baltscheffsky and Baltscheffsky2007). In the present study, we also found a high level of sequence conservation of the H+-PPase gene among several isolates of P. dicentrarchi, which could also be used in conjunction with other highly conserved genes, such as β-tubulin, to detect intraspecific genetic variation within populations of scuticociliates that infect cultured turbot (Budiño et al. Reference Budiño, Lamas, Pata, Arranz, Sanmartín and Leiro2011).
In plants, it is known that efficient exclusion of excess Na+ from the cytoplasm and accumulation of vacuolar Na+ are the most important steps involved in maintaining ion homeostasis within cells, and both tonoplast and plasma membrane Na+/H+ antiporters exclude Na+ from the cytosol driven by the H+-motive force generated by the plasma membrane H+-ATPase and H+-PPase (Silva and Gerós, Reference Silva and Gerós2009). Algal and plant H+-PPases are induced under anoxia, chilling and salt-induced stress (Carystinos et al. Reference Carystinos, McDonald, Monroy, Dhindsa and Poole1995; Fukuda et al. Reference Fukuda, Chiba, Maeda, Nakamura, Maeshima and Tanaka2004), and overexpression of the vacuolar H+-PPase isoform AVP1 in the model plant Arabidopsis has been claimed to confer increased saline and drought tolerance (Gaxiola et al. Reference Gaxiola, Li, Undurraga, Dang, Allen, Alper and Fink2001). As euryhaline organisms, ciliates are well adapted to variations in salinity and tolerate salinity levels as low as 4‰ or as high as 62‰ (i.e. 27‰ higher than seawater) (Hu, Reference Hu2014). We found that P. dicentrarchi is able to respond to saline-induced stress by modifying expression of the H+-PPase isoforms located in the alveolar sacs, indicating a potential role of these structures in salt tolerance in marine scuticociliates.
In conclusion, the H+-PPase of P. dicentrarchi is located in the membranes of the phagocytic vacuole and alveolar sacs, thus promoting acidification of these cellular compartments. Specifically, an isoform of ionic pump H+-PPase containing a highly conserved amino acid motif generated by splicing is located in the alveolar sacs and is recognized by a polyclonal antibody, PABHK. Gene and protein expression of this isoform are regulated by changes in salinity, which suggests that the isoform must play an important physiological role in the adaptive responses of these marine ciliates in maintaining both intracellular pH homeostasis and osmoregulation.
FINANCIAL SUPPORT
This study was financially supported by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R, the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL), and by the Xunta de Galicia (Spain) under grant agreement GPC2014/069. All authors benefitted from these grants.
