Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects

This is the accepted manuscript of the following article: Mallo, N., Lamas, J., de Felipe, A.P., Sueiro, R.A., Fontenla, F. & Leiro, J.M. (2016). Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects. Experimental Parasitolology, 169, 59-68. doi: 10.1016/j.exppara.2016.07.012. © <Ano> Elsevier B.V. This manuscript version is made available under the CC-BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by-ncnd/4.0/) Elsevier Editorial System(tm) for Experimental Parasitology Manuscript Draft Manuscript Number: EP-15-466R1 Title: Role of H+-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: physiological effects Article Type: Research Paper Keywords: Philasterides dicentrarchi; intracellular pH; calcium; ATP; H+- PPase; osmoregulation Corresponding Author: Dr. Jose Manuel Leiro Vidal, Ph.D. Corresponding Author's Institution: University of Santiago de Compostela First Author: Natalia Mallo Order of Authors: Natalia Mallo; Jesús Lamas; Ana Paula De Felipe; Rosa Ana Sueiro; Francisco Fontenla; Jose Manuel Leiro Vidal, Ph.D. Abstract: The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anfizoic and marine euryhaline ciliate P. dicentarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca2+ and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H+-PPase) that pumps H+ through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca2+ storage). We evaluated the effects of Ca2+ and ATP on H +-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca2+ metabolism regulators led to alkalinization and a decrease in H+-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca2+ accumulation and downregulation of H+-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment. Graphical Abstract Highlights - The existence of acidocalcisome-like structures in a ciliate parasite is proposed - ATP and Ca2+ regulate the enzymatic activity of H +-pyrophosphatase - The regulation of intracellular pH is key to the survival of the parasite - Disruption of pH homeostasis it can be a chemotherapeutic target Highlights (for review) Role of H+-pyrophosphatase activity in the regulation 1 of intracellular pH in a scuticociliate parasite of 2 turbot: physiological effects 3 4 Natalia Malloa, Jesús Lamasb, Ana-Paula DeFelipea, Rosa-Ana Sueiroa,b, 5 Francisco Fontenlab, José-Manuel Leiroa,* 6 7 aLaboratorio de Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de 8 Santiago de Compostela, Santiago de Compostela, Spain. 9 bDepartamento de Biología Celular y Ecología e Instituto de Acuicultura, Facultad de Biología, 10 Universidad de Santiago de Compostela, Santiago de Compostela, Spain. 11 12 13 Short title: Intracellular pH homeostasis and osmoregulation in a scuticociliate parasite 14 15 16 *Manuscript Click here to view linked References 2 17 18 *Corresponding author: 19 José M. Leiro, Laboratorio de Parasitología, Instituto de Investigación y Análisis Alimentarios, c/ 20 Constantino Candeira s/n, 15782, Santiago de Compostela (A Coruña), Spain; Tel: 21 34981563100; Fax: 34881816070; E-mail: josemanuel.lei[email protected] 22 Abstract 23 The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose 24 causal agent is the anfizoic and marine euryhaline ciliate P. dicentarchi. Several 25 protozoans possess acidic organelles that contain high concentrations of pyrophosphate 26 (PPi), Ca2+ and other elements with essential roles in vesicular trafficking, pH 27 homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H+-28 PPase) that pumps H+ through the membranes of vacuolar and alveolar sacs. These 29 compartments share common features with the acidocalcisomes described in other 30 parasitic protozoa (e.g. acid content and Ca2+ storage). We evaluated the effects of Ca2+ 31 and ATP on H +-PPase activity in this ciliate and analyzed their role in maintaining 32 intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic 33 molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular 34 compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi 35 and Ca2+ metabolism regulators led to alkalinization and a decrease in H+-PPase 36 expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca2+ 37 accumulation and downregulation of H+-PPase expression. We conclude that the 38 regulation of the acidification of intracellular compartments may be essential for 39 3 maintaining the intracellular pH homeostasis necessary for survival of ciliates and their 40 adaptation to salt stress, which they will presumably face during the endoparasitic 41 phase, in which the salinity levels are lower than in their natural environment. 42 43 Keywords: Philasterides dicentrarchi; intracellular pH; calcium; ATP; H+-PPase; 44 osmoregulation. 45 46 47 1. Introduction 48 Philasterides dicentrarchi, a marine euryhaline scuticociliate, is a facultative 49 parasite initially described in cultured European seabass Dicentrarchus labrax 50 (Dragesco et al., 1995) and which causes serious mortalities in cultured turbot 51 Scophthalmus maximus (Iglesias et al., 2001). Like other ciliates, this species displays a 52 high capacity to adapt to changes in environmental osmolarity (Kaneshiro et al., 1969). 53 Maintenance of cell volume is a fundamental mechanism of cellular homeostatic that 54 must have arisen very early on in the evolution of cells (Maroulis et al., 2003). Parasitic 55 protozoans have developed several mechanisms to adapt to osmotic stress and possess 56 two organelles with an essential role in these adaptations: the acidic organelles 57 acidocalcisomes and the contractile vacuole complex (Rohloff and Docampo, 2008). 58 The main function of acidocalcisomes is storage of H+ and phosphorus, for use in 59 pyrophosphate (PPi) and polyphosphate (PolyP) metabolism, osmoregulation and 60 maintenance of pH and Ca2+ homeostasis (Docampo and Moreno, 2001; Moreno and 61 Docampo, 2009; Docampo and Moreno 2011; Docampo et al., 2013; Li et al., 2014). 62 Acidocalcisomes share some properties with plant vacuoles and have several pumps in 63 their membranes. Two of these pumps (V-H+-PPase and VH+-ATPase) are H+ 64 4 translocases (Hannaert et al., 2003), which participate in the conservation of acidic 65 conditions required for Ca2+ retention (García et al.,1998; Bonansea et al., 2012). 66 Several pumps such as H+-PPase, H+-ATPase, Na+/H+ and Ca2+/H+ exchangers and 67 Ca2+-ATPase participate in or interfere with pH maintenance (Luo et al., 2001; 68 Rodrigues et al., 2002; Saliba et al., 2003; Docampo et al., 2005). 69 In Paramecium, staining of acid compartments with pH sensitive fluorescent 70 dyes has revealed the distribution of several acidic vesicles in the cellular cytoplasm and 71 some acidosomes close to the cytostome (Wassmer et al., 2009). The presence of 72 granules containing Ca2+ and Mg2PPi has been reported in another ciliate, Tetrahymena 73 pyriformis (Heinonen, 2003). The possibility that ciliates possess acidocalcisomes is 74 suggested by the fact that they contain many proteins that are activated by Ca2+, 75 particularly in the cortex and cilia (Kim et al., 2002; Kissmehl et al., 2006); however, 76 these proteins have not yet been characterized in Philasterides. 77 In ciliates, the alveolar sacs are distributed along the inner side of the membrane, 78 which is covered by locomotive cilia (Lynn and Corliss, 1991). As the entire cell is 79 coated by cilia, substantial amounts of Ca2+ are required for ciliary motility and for 80 intraand extracellular signalling. The Ca2+ of alveolar sacs of ciliates, which have been 81 identified as Ca2+ stores, is actively imported inside the alveoli, in an ATP and Mg2+-82 dependent process (Stelly et al., 1995; Sahoo et al., 2004; Plattner, 2014; 2015). In P. 83 dicentrarchi, the alveolar sacs are of variable size, possibly reflecting changes in 84 osmotic or ionic concentration across the membrane in response to environmental 85 conditions (Paramá et al., 2006); the membranes of vacuoles and alveolar sacs of 86 trophozoites possess H+-PPase, a enzyme typically present in the acidocalcisomes of 87 protozoan parasites (Mallo et al., 2015; 2016a). 88 5 In this study, we investigated the potential presence of acidocalcisome-like 89 organelles in the scuticociliate P. dicentrarchi and analyzed the role of adenosine 90 triphosphate (ATP) and Ca2+ on intracellular acidification, H+ translocating activity and 91 H+-PPase expression. We also evaluated the effect of osmolarity on the levels of 92 expression of this enzyme, assessing the physiological implication of acidic intracellular 93 compartments in osmoregulation of the ciliate. 94 95 96 97 2. Material and Methods 98 2.1. Experimental animals and parasites 99 Specimens of turbot, Scophthalmus maximus, of approximately 50 g body 100 weight were obtained from a fish farm in Galicia. The fish were placed in 250L tanks 101 with recirculating seawater at 17-18ºC under a photoperiod of 12h light/dark, and they 102 were fed daily with commercial pellets (Skretting, Burgos, Spain). Before starting the 103 experiments, fish were acclimated to the laboratory conditions for 2 weeks. 104 ICR CD-1 mice (Swiss) of age 8-10 weeks were purchased from Charles River 105 Laboratories (U.S.A.) for use in the experiments. All experiments were carried out 106 following the European Regulations on Animal Protection (Directive 86/609), the 107 Declaration of Helsinki and/ or the Guide of Care and Use of Laboratory Animals 108 adopted by the US National Institutes of Health (NIH). All experimental protocols were 109 approved by the Institutional Animal Care and Use Committee of the University of 110 Santiago de Compostela. 111 We obtained naturally infected turbot, showing signs of scuticociliatosis, from a 112 fish farm in Galicia (NW Spain). Specimens of the ciliate P. dicentrarchi (isolate I1; 113 12 0.1% Triton X-100 (PBT) for 3 min and then washed twice with DPBS. Ciliates were then 262 incubated with 1% bovine serum albumin (BSA) for 30 min. After blocking, ciliates were 263 incubated at 4ºC overnight with a solution containing 1:100 dilutions of anti-rH+-PPase form 264 recombinant yeast antibody. Then, ciliates were washed 3 times with DPBS followed by 1 h 265 incubation, at room temperature; with a 1:100 dilution of FITC conjugated rabbit anti-mouse 266 IgG-FITC antibody (Sigma). After three in DPBS, the samples were double stained with 0.8 267 mg/mL 4', 6-diamidine-2-phenylindole (DAPI; Sigma-Aldrich) in DPBS for 15 min at room 268 temperature (Paramá et al., 2007). After three washes with DPBS samples were mounted in 269 PBS-glycerol (1:1) and visualized by confocal microscopy (Leica TCS-SP2, LEICA 270 Microsystems Heidelberg GmbH, Mannheim, Germany). 271 2.9. SDS-PAGE electrophoresis and Western blot 272 Enriched vesicle fractions (EVF) of the ciliates cultured for 6 hours with 273 different treatments (no treatment, 1m MATP or 0.8mM CaCl2) were obtained from 274 2.5×105 trophozoites for each preparation. Ciliates were centrifuged and washed twice 275 in PBS and once with assay buffer (100 mM KCl, 0.4 M glycerol, 1 mM Tris–EGTA 276 and 5 mM Tris–HCL, pH 8.0) containing 1 mM PMSF and 1 μg/mL leupeptin. The cell 277 pellet was homogenized in a Potter S homogenizer (Braum Biotech, USA) until lysis 278 was greater than 90% (generally 30 s). The mixture was resuspended in 5 mL of assay 279 buffer and centrifuged once at 750 g for 5 min (to remove unbroken cells). The resulting 280 supernatant was centrifuged at 15 000 g for 10 min, and the pellet was resuspended in 281 PBS with loading buffer (without DTT) (Mallo et al., 2015). Samples were separated by 282 SDS-PAGE in 12.5% linear gels under non-reducing conditions. After electrophoresis, 283 the gels were stained with Coomassie blue (Thermo Scientific Protein GelCode Blue 284 Safe Stain; Thermo Fisher, USA) to determine the concentration of protein in each 285 sample (Piazzón et al., 2008). 286 13 Simultaneously, one of the gels was transferred to a polyvinylidene fluoride 287 (PVDF) membrane, at 15V for 35 min (0.45 m, Millipore, USA) on a Trans-Blot SD 288 transfer cell (Bio-Rad, USA. UU) embedded in transfer buffer containing 48 mM Tris, 289 29 mM glycine, 0.037% SDS and 20% methanol, pH 9.2. Membranes were stained with 290 Ponceau S to verify transfer and incubated for 1.5 h at room temperature in Tris Buffer 291 Saline (TBS; 50 mM Tris, 0.15 M NaCl, pH 7.4) containing 0.2% Tween 20 and 3% 292 BSA. The membranes were then washed with TBS and incubated overnight at 4 °C with 293 anti-H+-PPase form recombinant yeast antibody at a ratio of 1:100. The membranes were 294 incubated for 1 hour at room temperature with the secondary antibody (goat anti-mouse 295 Ig antibody, 1:1000, Dakopatts) and visualized with a chemiluminescent substrate based 296 on luminol (ECL Western Blotting Substrate Pierce, Thermo Scientific, USA). 297 Membranes were photographed with a FlourChem® FC2 imaging system (Alpha 298 Innotech, USA). Band intensity was analyzed using the Total Lab image master 299 program (Mallo et al., 2015). 300 301 2.10. Extraction of total RNA, reverse transcription and real-time polymerase chain 302 reaction (qPCR) 303 Trophozoites were incubated for 2 hours with the different treatments (4, 8 and 304 37‰ NaCl, 1mM ATP 1mM and 0.8 mM CaCl2). The total RNA from 107 cells/sample 305 was isolated, treated with DNAse I and used to generate cDNA, as already described. 306 The qPCR reaction was performed with a reaction mixture already containing the assay 307 buffer and dNTPs, Maxima SYBR Green qPCR Master Mix (Thermo Scientific). The 308 primer pair for the genes under study was used at a final concentration of 300 nM, and 309 1µl of cDNA was added per well. The volume was completed with RNase free distilled 310 H2O to a final reaction volume of 10 µL/well. The mixtures were heated at 95 °C for 5 311 14 min, followed by 40 cycles of 10s at 95 °C and 30s at 60 °C. At the end of the process, 312 melting curve analysis was carried out at 95 °C for 15s, 55 °C for 15 s and 95 °C for 15 313 s. The specificity and the size of the PCR products obtained were confirmed by agarose 314 gel electrophoresis at 2%. All reactions were carried out in a real time PCR system, Eco 315 Real-time PCR system (Illumina). The relative quantification of gene expression was 316 determined by the 2-ΔΔCq method (Livak and Schmittgen, 2001), and the programme was 317 used in accordance with minimum information guidelines for publishing real-time 318 quantitative PCR experiments (Bustin et al., 2009). The following primer sequences of 319 H+-PPase gene were used: forward/reverse, 5’-GCCTACGAAATGGTCGAAGA-3’/5’-320 GCATCGGTGTATTGTCCAGA-3’. Gene expression was normalized with the β-321 tubulin reference gene from P. dicentrarchi (forward/reverse primer sequence, 5′-322 ACCGGGGAATCTTAAACAGG-3′/5′-GCCACCTTATCCGTCCACTA-3′) and the 323 normalized data were expressed in relative arbitrary units. The values show the mean  324 the standard error (SE) of three trials. 325 The primer pairs were designed and optimized with the Primer 3 Plus 326 programme (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi) with a 327 Tm of 60 °C. 328 329 2.11. Statistical analysis 330 Results shown in the figures are expressed as means ± standard error. Significant 331 differences (P = 0.05) were determined by analysis of variance (ANOVA) followed by 332 Tukey – Kramer multiple comparisons test. 333 334 3. Results 335 336 15 3.1. Presence of Ca2+ and H+-PPase in acidic intracellular compartments 337 Staining with the pH sensitive fluorescent dyes acridine orange and Lysotracker 338 Red DND-99 (Fig 1, A1 and A2) revealed intracellular acidic compartments in DIG-339 permeabilized P. dicentrarchi trophozoites. Some important differences were observed 340 in the staining patterns of ciliates from the same culture batch. Some trophozoites 341 showed intense red fluorescent staining (acid pH) in the alveolar sacs, while others 342 showed only weak green florescent staining (alkaline pH), which was most evident in 343 the endocytic vacuoles (Fig. 1, A1). In the preparations stained only with acridine 344 orange, some DIG-permeabilized trophozoites stained various colours 345 (green/yellow/orange-red), indicating varying levels of pH in the endocytic vacuoles, 346 ranging from alkaline (green fluorescence) to acidic (orange/red fluorescence) (Fig. 1, 347 A1-a); however, when trophozoites are incubated with the bisphosphonate calcium 348 metabolism regulator PAM, a complete alkalinization of endocytic vacuoles is produced 349 (Fig. 1, A1-b). Likewise, the distribution of acidic alveolar sacs coincides with the 350 distribution of cilia (Fig. 1, A2). 351 In the trophozoites, Ca2+ was primarily located in the alveolar sacs and some 352 internal vacuoles (Fig. 1B). The staining patterns obtained with acid dyes and the Ca2+ 353 probe were both consistent with the pattern of immunostaining obtained using a 354 recombinant anti-H +-PPase, with labelling of both internal vacuoles and alveolar sacs 355 located under the kinetia (Fig. 1C). 356 Examination by electron microscopy revealed spherical electro-dense structures 357 inside the alveolar sacs and also spread through the cytoplasm in the interior of the 358 cytoplasmic vacuoles (Fig. 2A). At the ultrastructural level, a close association between 359 the inner membrane of alveolar sacs and the immediately underlying mitochondria was 360 observed (Fig. 2B). 361 16 362 3.2. Regulatory effect of ATP and Ca2+ on the H+-translocating activity 363 An assay of proton pumping activity was performed to investigate the effect of 364 PPi and ATP on H+-translocation activity and acidification of intracellular 365 compartments, including the alveolar sacs, in P. dicentrarchi. Addition of PPi to DIG-366 permeabilized trophozoites induced translocation of H+ and the acidification of 367 intracellular compartments (Fig. 3A); however, the presence of ATP (0.01, 0.25, 0.5 and 368 1 mM) did not affect intracellular acidification, indicating that ATP does not stimulate 369 H+-translocation (Fig. 3B). In samples in which PPi-driven H+ translocation was 370 observed, this activity was inhibited by addition of 0.5 or 1 mM ATP (Fig. 3 C), 371 producing intracellular alkalinization. Addition (via a stock solution of 0.8 mM CaCl2) 372 of Ca2+(a known H+-PPase inhibitor), or the bisphosphonate analogous PAM at 0.1 mM, 373 inhibited the H+-translocating activity (Fig. 3D). 374 375 3.3. Role of ATP and Ca2+ in H+-PPase protein and gene expression 376 Western blots of the vesicle-enriched fractions treated with Ca2+ (0.8mM CaCl2) 377 and 1mM ATP revealed a significant decrease in the band intensity, measured by 378 densitometry software program, indicating a decrease in H+-PPase expression relative to 379 the untreated controls (Fig. 4 A, B). 380 The relative H+-PPase gene expression in P. dicentrarchi was quantified by RT-381 qPCR of cDNA from ciliates cultivated with the different treatments for 2 hours. Both 382 treatments (CaCl2 and ATP) induced a significant decrease in H+-PPase expression (Fig. 383 4C). 384 385 3.4. Effects of salinity on H+ translocating activity and intracellular Ca2+ levels 386 17 NaCl and KCl were used as sources of Na+ and K+, both of which are present in 387 seawater, to study the effects of salinity on the H+ translocation activity in P. 388 dicentrarchi. H+ pumping activity was induced by adding PPi in different buffers during 389 sample preparation and, in one of these, NaCl replaced the KCl in the standard 390 translocation assay buffer (100 mM KCl, 0.4 M glycerol, 1 mM Tris-EGTA, and 5mM 391 Tris-HCl, 1 mM PMSF and 1g/ml leupeptin, pH 8), at the same concentration (100 392 mM). Acidification only occurred when buffer containing KCl was used, and NaCl had 393 an inhibitory effect on the H+ translocation activity (Fig. 5A). With the aim of reversing 394 the effect of NaCl, the same concentration of KCl was added, in an attempt to induce H+ 395 translocation. However, the activity was not recovered. When NaCl was added to the 396 sample displaying H+ pumping activity that was prepared in buffer containing KCl, 397 addition of NaCl again resulted in alkalinization. 398 To analyze the effect of salinity and PPi on intracellular Ca2+ levels, ciliates 399 were cultured for 24 hours in saline solutions of 4 and 8 ‰, conditions considered 400 hypo-osmotic for these marine ciliates whose natural osmolarity is the 37 ‰. After the 401 DIG-permeabilization and the addition of 1 mM PPi, the ciliates were incubated for 30 402 min. The Ca2+ probe (Fluo-4 NW) was then added and the fluorescence (Ex: 494nm, 403 Em: 516nm) was measured after 1 hour. Addition of PPi caused a significant decrease 404 in internal Ca2+ levels in both cases (Fig. 5B). The Ca2+ levels were lower under hypo-405 osmotic conditions (Fig. 5B) 406 407 4. Discussion 408 Ciliates are among the most abundantly distributed protozoa in the marine 409 environment, and around 60 scuticociliate species have been described (Wang et al., 410 2008). The capacity of ciliates to adapt to changes in salinity and pH is essential to 411 18 enable littoral colonization (Nisbet, 1984). Vacuoles and other acidic organelles play an 412 essential role in this capacity, including storage, sequestration of toxic compounds and 413 maintenance of turgor (Pittman et al., 2011). Studies of plant vacuoles have shown that 414 regulation of vacuole acidification is crucial for secretory and endocytic routes 415 (Baltscheffsky et al., 1999; Gaxiola et al., 2007). Moreover, pH homeostasis in 416 intracellular compartments is essential under pathological conditions, and the enzymes 417 involved may therefore be good chemotherapeutic targets (Martínez et al., 2002; López 418 and Segura Latorre, 2008). As cells must be compartmentalized in unicellular 419 organisms, in the present study we mainly focused on Ca2+ regulation and acidic stores 420 to identify potential targets involved in bioenergetics, which can be modulated in 421 infected fish. The P. dicentrarchi H+-PPase is located in the membranes of endocytic 422 vacuoles and alveolar sacs (both acidic compartments), as demonstrated by acridine 423 orange and Lysotracker Red staining and which is consistent with our previous 424 observations (Mallo et al., 2015; 2016a). H+-PPase promotes acidification of these 425 compartments and could therefore interfere in the adaptive response of the ciliate to 426 maintaining osmoregulation and pH homeostasis, by performing similar functions as in 427 acidocalcisomes (Docampo et al, 2005; Pan et al., 2011; Mallo et al., 2015). In the 428 present study, we also observed a wide variation in the pH of alveolar sacs and 429 endocytic vacuoles, which probably reflects the role that these cellular compartments 430 play in regulating intracellular pH, as occurs with acidocalcisomes in other protozoan 431 parasites (Docampo et al., 2013). 432 Some electron-dense deposits were observed in the cytosolic region and alveolar 433 sacs, where the H+-PPase enzyme is located (Mallo et al., 2015). The same distribution 434 pattern was observed for Ca2+, with the Fluo-4 NW probe. These electron-dense 435 deposits are also characteristic of acidocalcisomes (Miranda et al., 2000, 2004, 2008; 436 19 Soares-Medeiros et al., 2005), which share some of their features with alveolar sacs of 437 P.dicentrarchi, such as their acidic origin, their function as Ca2+ stores and possession 438 of some of the enzymes involved in acidification (e.g. H+-PPase). Acridine orange 439 staining and enzyme immunolocalization showed that some alveolar sacs are located 440 under the cilia, indicating that they are involved in ciliary movement and/or 441 bioenergetics (Plattner and Klauke, 2001). 442 The presence of enzymes and exchangers that cooperate in pH maintenance in 443 acidocalcisomes, such as H+-PPase, H+- ATPase, Na+/H+ and Ca2+/H+ exchangers, may 444 interfere in release of Ca2+ from internal stores via modification of pH due to H+ 445 translocation and associated activity (Marchesini et al., 2000; Vercesi et al., 2000; 446 Saliba et al., 2003; Moriyama et al., 2003). 447 Movement of H+ between intracellular membranes can be divided into two 448 different categories: those coupled to ATP production and those with a purpose other 449 than energy production, such as acidification (Rudnick, 1987). Sequencing of the 450 complete genome of some ciliate species has led to identification of H+-ATPase in 451 organelle membranes of Paramecium (Plattner, 2010), and the main function of this 452 ATPase seems to be osmoregulation and pH homeostasis (Van der Heyden and 453 Docampo, 2002; Wassmer et al., 2005), i.e. similar functions to those of H+-PPase. H+-454 ATPase activity has been measured in different organisms by use of acridine orange as 455 an indicator of acidification. Unlike in other organisms, addition of ATP inhibits H+ 456 translocation in permeabilized specimens of P. dicentrarchi, resulting in alkalinization 457 of vacuoles, as occurs with addition of NH4Cl, used as a control for alkalinization at the 458 end of all experiments as it eliminates H+ from acidic compartments (Docampo et al., 459 1995; Rodrigues et al., 1999a; Ruíz et al., 2001). H+-ATPase has been described as a 460 Ca2+ insensitive enzyme, unlike H+-PPase (Rea et al., 1992), and total blockage of H+ 461 20 pumping activity by Ca2+ may indicate that this activity does not take place through the 462 H+-ATPase route, as occurs in P. dicentrarchi. This phenomenon has also been 463 observed in the trypanosomatid Herpetomonas (Soares Medeiros et al., 2005), in which 464 ATP did not promote H+ uptake. In Streptococcus faecalis, PPase is inhibited by ATP, 465 which competes with PPi for chelation of Mg2+ ions (Lahti and Lonnberg, 1985), in 466 which Mg2PPi is the main substrate. It is also possible that H+-PPase activity is inhibited 467 by excess Pi formed as a product of ATP metabolism (ADP + Pi), induced by excess 468 ATP. Another possible explanation for the inability of ATP 469 to induce H+ translocation may be related directly to the DIG used in permeabilization 470 of cells that could generate an alteration in cholesterol levels of intracellular 471 compartments, causing incapability to measure ATP-driven H+ transport in DIG-472 permeabilized cells (Rodrigues et al., 2001). Inhibition of H+-PPase by Ca2+ has been 473 described through formation of the CaPPi complex, which competes with the enzyme 474 substrate or the Ca2+ ion, mimicking Mg2+ and inhibiting enzymatic activity (Maeshima, 475 1991; Rea et al., 1992). Moreover, H+ release as a consequence of the addition of Ca2+ 476 in P. dicentrarchi suggests that an exchanger of Ca2+/H+ takes place in the membranes 477 of acidic compartments as occurs in the trypanosomatid Herpetomonas (Soares 478 Medeiros et al., 2005). 479 In P. dicentrarchi, NaCl enhances H+ release, thus leading to the alkalinization 480 of acidic compartments. Because H+ translocation in the ciliate mainly occurs via H+-481 PPase activity, NaCl may act as an inhibitor of this enzyme. Although this is not 482 common in H+-PPases (Fukuda et al., 2004), a subtype of H+-PPase that is inhibited by 483 Na+ has recently been characterized; however, this phenomenon has been only observed 484 in prokaryotes and is believed to be an evolutionary remnant (Luoto et al., 2013; 2015). 485 Similar observations have been made in the trypanosomatid parasite Herpetomonas, in 486 21 which NaCl-mediated inhibition of PPi-dependent H+ uptake has been detected (Soares 487 Medeiros et al., 2005), and also in Leishmania donovani, in which NaCl does not 488 stimulate H+ translocation (Sen et al., 2009). The NaCl probably reverses acidification, 489 as occurs in the parasite T. gondii, because of the presence of a Na+/H+ exchanger. The 490 effect of Na+ was observed independently of the addition of PPi, indicating that the 491 effect on acidification is not related to that promoted by PPi. NaCl was added during 492 preparation of the sample in KCl buffer, indicating that the observed effect was not due 493 to changes in osmolarity (Rohloff et al., 2011). 494 As ATP and Ca2+ induced alkalinization of compartments in P. dicentrarchi, 495 their effect on H+-PPase expression was evaluated, and it was found that they inhibited 496 both protein and gene expression. ATP and Ca2+ probably inhibit expression and 497 activity of the H+-PPase in the ciliate. If the Ca2+-ATPase pump is present in the ciliate, 498 any excess ATP and Ca2+ could interact with Ca2+ and Na+ exchangers, so that H+-PPase 499 activity would not be required for H+ translocation. 500 Regulation of Ca2+ has been investigated in depth in Paramecium and has been 501 implicated in multiple functions and cell survival. Maintenance of acidification is 502 responsible for Ca2+ retention in acidic compartments such as acidocalcisomes. The 503 contractile vacuole of Paramecium and Dictyostelium discoideum also appears to be 504 important in regulating the internal Ca2+ concentration (Stock et al., 2002, Pittman et 505 al., 2011; Martínez-Higuera et al., 2013). In Trypanosoma, the Ca2+-ATPase, H+-506 ATPase and H+-PPase enzymes are suggested as being responsible for Ca2+ 507 accumulation in acidocalcisomes (Docampo and Moreno, 2001; De Souza et al., 2002). 508 As salinity may modulate Ca2+ channels in P. dicentrarchi and the H+-PPase enzyme 509 interferes with acidification of the Ca2+ compartment, different concentrations of NaCl 510 and PPi were added to study how these compounds interfere in intracellular Ca2+ 511 28 Maroulis SL, Schofield PJ, Edwards MR., 2003. Osmoregulation in the parasitic 698 protozoan Tritrichomonas foetus. 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Pflugers Arch –Eur J Physiol 820 457, 599-607. 821 Wassmer T, Froissard M, Plattner H, Kissmehl R, Cohen J., 2005. The vacuolar proton-822 ATPase plays a major role in several membrane-bounded organelles in 823 Paramecium. J Cell Sci 118, 2813-2825. 824 Zhen RG, Kim EJ, Rea P., 1997. The molecular and biochemical basis of 825 pyrophosphatase energized proton translocation at the vacuolar membrane. Adv 826 Bot Res 25, 297-337. 827 34 Figures 828 Figure 1: Distribution of calcium and acidic compartments in P. dicentrarchi. A1 and 829 A2) Intracellular acidic compartments stained with acridine orange and 830 Lysotracker Red 99-DND. In A1, intense fluorescent staining of the alveolar 831 sacs with the dyes Lysotracker and acridine orange is observed (arrowheads). 832 The frame a) shows a P. dicentrarchi trophozoite dyed only with acridine orange 833 showing differences in intensity of fluorescence and colour 834 (red/orange/yellow/green) in the endocytic vacuoles (arrows). The frame b) 835 shows a trophozoite treated with the pyrophosphate analogous and regulator of 836 the Ca2+ metabolism, pamidronate (PAM) at 0.1 mM where a green fluorescent 837 labeling of the vesicles stained with acridine orange is observed indicating its 838 alkali state (arrows). In A2, staining of alveolar sacs with Lysotracker/acridine 839 orange is consistent with the ciliary pattern in kinetia (arrowheads). B) The Fluo 840 4-NW Calcium probe revealed the calcium stores distributed in alveolar sacs 841 (arrowheads) and endocytic internal vacuoles (arrows). C) Immunolocalization 842 of H+-PPase with recombinant anti-H+-PPase labelling of alveolar sacs 843 (arrowheads) and some internal vacuole membranes (arrows) of the ciliate. Bar 844 scale = 10 m. 845 Figure 2: Transmission electron microscope (TEM) photomicrograph of a P. 846 dicentrarchi trophozoite. A) Panoramic view of an ultrathin section showing 847 alveolar sacs beneath the plasma membrane and containing a spherical electron-848 dense body (arrows). The presence of electron-dense bodies was also observed 849 in the interior of the cytoplasmic vacuoles (arrowheads). B) Enlargement of the 850 surface of a trophozoite with an electron dense body (arrow) in the interior of 851 35 alveolar sacs (as). Mitochondria (m) are also observed in close contact with the 852 inner membrane of the alveolar sacs. N = nucleus. 853 Figure 3: Effect of ATP and Ca2+ on H+ translocating activity. H+ translocation activity 854 in the presence and absence of PPi (A), ATP (B), PPi and ATP (C) and Ca2+ or 855 pamidronate –PAM-(D). The concentrations used (indicated) were achieved by 856 addition of stock solutions prepared in assay buffer. The results show the mean 857 values ± standard error (n=5) of the variation (Δ) in fluorescence. Asterisks 858 indicate statistically significant differences (* P < 0.05; **P < 0.01). 859 Figure 4: Effect of ATP and Ca2+ on H+-PPase protein and gene expression. A) 860 Western blot showing the recognition pattern of an H+-PPase (rH+-Ppase) 861 recombinant polyclonal antibody against ciliate vesicle enriched samples in non-862 reducing conditions (without DTT). Ciliates were incubated with 1mM ATP 863 (lane 2) or with 0.8 mM CaCl2 (lane 3) for 6h. Lane 1 corresponds to the control. 864 Mw: molecular weight marker proteins. B) The band intensity of Western blot 865 assay (in arbitrary units) was measured with Total Lab, and statistical 866 comparisons were made relative to the control. C) H+-PPase gene expression 867 levels in ciliates treated for 24 hours with 1 mM ATP and 0.8 mM CaCl2. 868 Expression was analyzed by RT-qPCR. The P. dicentrarchi β-tubulin was used 869 as reference gene. As shown in both cases, calcium and ATP induced a decrease 870 in gene expression. The bars in the graphs show the results as mean ± standard 871 error (n= 5). Asterisks indicate the statistically significance * P < 0.05, ** P < 872 0.01 873 Figure 5: A) Effect of NaCl and KCl (100 mM) on the H+ translocating activity induced 874 by PPi. The results show the mean values ± standard error (n=5) of the variation 875 (Δ) in fluorescence. B) Effect of salinity and PPi on calcium levels. Ciliates were 876 36 incubated for 24 hours in saline solutions of 4 and 8 ‰. 1 m PPi was then 877 added and the ciliates were incubated for a further1 h. The calcium probe (Fluo-878 4 NW) was then added and after 1 hour, fluorescence (Ex: 494 nm, Em: 516 nm) 879 was measured, in arbitrary units (AU). Bars represent the mean (± standard 880 error; n= 5) and letters indicate statistically significance between groups (*P< 881 0.05, **P< 0.01). 882 Figure Click here to download high resolution image