Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite

This is the accepted manuscript of the following article: Folgueira I, Lamas J, De Felipe AP, Sueiro RA, Leiro JM. (2019). Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite. Fish Shellfish Immunol. 2019 Jul 5;92:802-812. doi: 10.1016/j.fsi.2019.07.008 © 2019 Elsevier Ltd. This manuscript version is made available under the CC-BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Highlights: Turbot immune serum induces mucoid encapsulation in P. dicentrarchi Ciliates agglutinated by the immune serum secrete mucin-like proteins Extrusomes are activated by calcium-dependent mechanisms The agglutination response activates the transcription of trichocyst matrix genes *Highlights Evidence for the role of extrusomes in evading attack by the 1 host immune system in a scuticociliate parasite 2 Iria Folgueiraa, Jesús Lamasb, Ana Paula De Felipea, Rosa Ana Sueiroa, José 3 Manuel Leiroa,* 4 aDepartamento de Microbiología y Parasitología, Instituto de Investigación y Análisis 5 Alimentarios, Campus Vida, Universidad de Santiago de Compostela, Spain 6 bDepartamento de Biología Funcional, Instituto de Acuicultura, Campus Vida, Universidad de 7 Santiago de Compostela, Spain 8 9 10 11 12 13 SHORT TITLE: Defensive role of extrusomes in scuticociliate parasites 14 15 16 17 18 19 20 21 22 23 24 25 *Author for correspondence:26 Laboratorio de Parasitología, 27 Instituto de Investigación y Análisis Alimentarios, 28 Universidad de Santiago de Compostela, 29 C/ Constantino Candeira s/n, Campus Vida, 30 15875, Santiago de Compostela, La Coruña, Spain. 31 2 Abstract 32 Like other ciliates, Philasterides dicentrarchi, the scuticociliate parasite of 33 turbot, produces a feeding-only or growing stage called a trophont during its life cycle. 34 Exposure of the trophonts to heat-inactivated serum extracted from the turbot host 35 and containing specific antibodies that induce agglutination / immobilization leads to 36 the production of a mucoid capsule from which the trophonts later emerge. We 37 investigated how these capsules are generated, observing that the mechanism was 38 associated with the process of exocytosis involved in the release of a matrix material 39 from the extrusomes. The extruded material contains mucin-like glycoproteins that 40 were deposited on the surface of the cell and whose expression increased with time of 41 exposure to the heat-inactivated immune serum, at both protein expression and gene 42 expression levels. Stimulation of the trophonts with the immune serum also caused an 43 increase in discharge of the intracellular storage compartments of calcium necessary 44 for the exocytosis processes in the extrusomes. The results obtained suggest that P. 45 dicentrarchi uses the extrusion mechanism to generate a physical barrier protecting 46 the ciliate from attack by soluble factors of the host immune system. Data on the 47 proteins involved and the potential development of molecules that interfere with this 48 exocytic process could contribute to improving the prevention and control of 49 scuticociliatosis in turbot. 50 51 Key words: Philasterides dicentrarchi; turbot; exocytosis; extrusomes; trichocyst 52 matrix proteins; mucin-like glycoproteins. 53 54 55 56 57 58 59 60 61 62 3 1. Introduction 63 Exocytosis may be an important mechanism of communication between 64 microbes. Indeed, some microorganisms can develop highly specialized exocytotic 65 organelles by extruding different materials with important roles in mechanisms that 66 enable adaptation to different environmental conditions [1]. Several groups of 67 protozoa possess different types of exocytotic extrusive organelles, known as 68 extrusomes. These organelles are associated with the cell membrane and have 69 different structures containing a material that is usually expelled or extruded from the 70 cell and that participates in different functions [2]. In ciliates, most extrusomes belong 71 to the trichocyst type, which are characteristically spindle shaped and can quickly 72 download their protein content in the form of a projectile in response to mechanical or 73 physical stimuli, and with a probable function in defence against predators [3]. Other 74 common extrusomes in some groups of ciliates include toxicysts and haptocysts, which 75 contain toxic material or can extrude material capable of penetrating the prey; both 76 have a possible predatory function in prey capture and food uptake [4,5]. The function 77 of trichite-type extrusomes, i.e. rod-shaped organelles circumferentially arranged in 78 plasma pockets [6], is not yet completely known. However, it is believed that they can 79 act as defensive or offensive elements [7]. Mucocysts and cortical granules, a special 80 type of mucocysts, secrete an amorphous mucilaginous protective material on the cell 81 surface. In some species, this material may be involved in the formation of cysts or 82 temporary capsules with a protective role and constituting a first line of defence 83 against predators in the ciliate, regulating cell ionic concentration and anchoring cells 84 to substrates [3, 8-10]. 85 Philasterides dicentrarchi is an amphizoic scuticociliate, originally free-living, 86 but which under certain conditions can be transformed into an opportunistic 87 histiophagous parasite in cultivated flat fish, causing a serious disease called 88 scuticociliatosis and causing high mortality rates [11,12]. In order to produce the 89 parasitic phase, the ciliate must develop various strategies of biochemical adaptation 90 to its new habitat [13,14]. In addition, it must evade attack by the fish immune system, 91 especially by lysis induced by soluble factors in the serum, such as complement. 92 Activation of complement via the classical pathway (in conjunction with antibodies), 93 together with activation of the coagulation system, causes destruction of the parasite 94 4 [15-17]. Two types of extrusomes have been characterized in P. dicentrarchi: one 95 fusiform, compatible with trichocysts, and the other spherical, compatible with 96 mucocysts, and which release a thin layer of mucus on the cell surface [18,19]. In 97 previous studies, we have observed that incubation (for 2h) of P. dicentrarchi 98 trophonts with serum from turbot that had survived a natural outbreak of 99 scuticociliatosis caused agglutination and immobilization of the ciliates and the 100 appearance of numerous capsules from which the trophonts later emerged. We 101 interpreted this phenomenon as a possible antigenic change and a mechanism of 102 evasion of the humoral immune response [20]. 103 In the present study, we aimed i) to elucidate the role of the extrusomes in 104 capsule production induced by incubation of the trophonts of P. dicentrarchi with 105 serum extracted from vaccinated turbot and that produces agglutinating and 106 immobilizing antibodies, ii) to characterize the proteins of the trichocysts and 107 mucocysts after extrusion, and iii) to demonstrate the role of the process of exocytosis 108 as a ciliate defence mechanism against attack by the soluble factors of the host 109 humoral immune system. 110 111 2. Materials and Methods 112 113 2.1. Parasites 114 Specimens of P. dicentrarchi (isolate I1) were collected under aseptic conditions 115 from peritoneal fluid obtained from experimentally infected turbot (Scophthalmus 116 maximus), as previously described [21]. The ciliates were cultured at 21 °C in complete 117 sterile L-15 medium, as previously described [20]. In order to maintain the virulence of 118 the ciliates, fish were experimentally infected every 6 months by intraperitoneal (ip) 119 injection of 200 μL of sterile physiological saline containing 5x105 trophonts, and the 120 ciliates were recovered from ascitic fluid and maintained in culture as described above. 121 122 2.2. Experimental animals 123 Turbot of approximately 50 g body weight were obtained from a local fish farm. 124 The fish were held in 250-L tanks with aerated recirculating sea water maintained at 14 125 5 °C. They were subjected to a photoperiod of 12L:12D and fed daily with commercial 126 pellets (Skretting, Burgos, Spain). The fish were acclimatized to laboratory conditions 127 for 2 weeks before the start of the experiments. 128 Swiss ICR (CD-1) mice (eight to ten weeks old), supplied by Charles River 129 Laboratories (USA), were bred and maintained in the Central Animal Facility of the 130 University of Santiago de Compostela (Spain). The mice were reared following the 131 criteria for the protection, control, care and welfare of animals and the legislative 132 requirements relating to the use of animals for experimentation (EU Directive 133 86/609/EEC), the Declaration of Helsinki, and/or the Guide for the Care and Use of 134 Laboratory Animals as adopted and promulgated by the US National Institutes of 135 Health (NIH Publication No. 85–23, revised 1996). The Institutional Animal Care and 136 Use Committee of the University of Santiago de Compostela approved all experimental 137 protocols. 138 139 2.3. Microscopic analysis 140 141 2.3.1. Scanning electron microscopy (SEM) 142 Ciliates treated with immune serum from turbot (see Immunization and serum 143 collection), were collected by centrifugation at 1000 x g and fixed with 2.5% (v/w) 144 glutaraldehyde in a cold solution of 4% paraformaldehyde in 0.1 M potassium 145 phosphate buffer (PB), pH 7.2 for 30 min. The samples were post-fixed for 30 minutes 146 with 1% (wt/v) osmium tetroxide in PB. The samples were then washed three times 147 with distilled water and dehydrated in a series of ethanol (50, 70, 90, 95, 100, 100% for 148 10 min each) and hexamethyldisilazane (HMDS, Sigma-Aldrich) (50 and 100% for 10 149 min each). Finally, the samples were mounted on aluminium stubs, sputter coated 150 with a layer of iridium, by using a Q150T-S sputter coater (Quorum Technologies, UK), 151 and viewed under a Zeiss Fesem ultra plus microscope (Zeiss, Germany) at 10 kV. 152 153 2.3.2. Transmission electron microscopy (TEM) 154 For TEM, we followed the technique described by [19]. Briefly, the cultured 155 ciliates were collected by centrifugation at 1000 x g for 5 min. Cells were fixed in 2.5% 156 (v/v) glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2. They were then washed 157 6 several times with 0.1 M cacodylate buffer and post-fixed in 1% (wt/v) OsO4, pre-158 stained in saturated aqueous uranyl acetate, dehydrated through a graded acetone 159 series and embedded in Spurr’s resin. Semi-thin sections were then cut using an 160 ultratome (Leica Ultracut UCT, Leica microsystems, Germany) and stained with 1% 161 toluidine blue for examination under a light microscope. Ultrathin sections were 162 stained in alcoholic uranyl acetate and lead citrate and viewed in a Jeol JEM-1011 163 transmission electron microscope (Jeol, Japan) at an accelerating voltage of 100 kV. 164 165 2.3.3. Histochemistry: Safranin-O Staining 166 167 For detection of mucin-type proteins, the cells were stained with Safranin-O. 168 Ciliates were incubated without turbot immune serum or with the serum for different 169 times. The ciliates were fixed in 10% buffered formalin (PBS, 0.01 M Na2HPO4, 0.0018 170 M KH2PO4, 0.0027 M KCl, 0.137 M NaCl, pH 7.0). The samples were then washed 2 171 times with distilled water and incubated for 5 min with an aqueous solution of 0.1% of 172 Safranin-O. After exhaustive washing with water to eliminate excess dye, the 173 preparation was air-dried and mounted using a permanent mounting medium 174 (Entellan, Merck). 175 176 2.4. Trichocyst associated proteins 177 The sequences of several mRNAs that encode proteins potentially related to 178 the trichocysts of Philasterides dicentrarchi were obtained in a previous RNAseq study 179 (unpublished results) carried out to compare the transcriptome of several P. 180 dicentrarchi strains, in collaboration with ZF-Screen (Holland). The assembled 181 sequences were analyzed using Blastgo software 5.0 (Biobam, Spain), to identify 182 homologous sequences, before functional annotation. Annotated sequences that 183 encode proteins potentially related to the trichocysts of ciliates were selected using 184 the BLASTx tool of the TGD Wiki (http://www.ciliate.org/blast/blast_link.cgi) where the 185 Tetrahymena thermophila gene and protein sequences database is located. To confirm 186 the nucleotide sequences that encode the proteins associated with the extrusomes 187 obtained by RNAseq, their cDNAs were amplified by RT-PCR and sequenced by Sanger 188 Sequencing (Eurofins Genomics, Germany). The selected proteins associated with the 189 7 extrusomes were the P. dicentrarchi trichocyst matrix protein T2A (TMPT2A) (GenBank 190 accession number MH412657.1) and the P. dicentrarchi trichocyst matrix protein T4-B 191 (TMPT4B) (GenBank accession number MH412658.1). 192 193 2.5. Production of recombinant proteins in yeast cells 194 In order to determine the complete nucleotide sequence that encoded 195 TMPT2A, codon usage was optimized to produce the recombinant protein in the yeast 196 Klyuveromyces lactis, by using the bioinformatics tool developed by Integrated DNA 197 Technology (IDT) (https://eu.idtdna.com/CodonOpt). The gene was then synthethized 198 by Invitro GeneArt Gene Synthesis (ThermoFisher Scientific). For expression of 199 recombinant protein in yeast, the K. Lactis Protein Expression kit (New England 200 Biolabs, UK) was used with the pKLAC2 vector, following the instructions provided by 201 the manufacturer. The synthesized nucleotide sequence was initially cloned in the 202 pSpark® II vector (Canvax, Spain), and the recombinant plasmid was subsequently 203 amplified in competent Escherichia coli strain DH-5. After extraction and purification 204 of the plasmid from the bacteria, PCR was carried out using the following primers: 205 FT2AKl 5’ CGCCTCGAGAAAAGAatgcgtgtctgaccgcacta-3’ / RT2AKl 5-’ 206 ATAAGAATGCGGCCGCTTAATGATGATGGTGATGGTGATGATGGTGATGatcggcacgctttacgtc207 ga-3’. The reverse primer includes 10 codons encoding histidine at the C-terminal end 208 of the protein. The yeasts were then transformed with the cloned pKLAC2 plasmid and 209 seeded in yeast carbon base agar medium plates containing 5 mM acetamide at 30°C 210 for 3–4 days until colony formation. Several of the colonies were collected and 211 inoculated in the YPGal medium at 30 ºC for 3-4 days with shaking at 250 rpm. When a 212 suitable cell density was reached, the medium was centrifuged at 6000 xg for 10 min, 213 and the supernatant was held at 4 ºC until use. The protein was extracted from the 214 supernatant by immobilized metal affinity chromatography (IMAC), by using columns 215 prepacked with Ni-Sepharose (HisTrapTM, GE Healthcare), in an ÄKTA Star protein 216 purification system (GE Healthcare) and following the manufacturer's instructions. 217 Once eluted, the protein was fully dialysed against distilled water using dialysis tubing 218 of pore size 3 kDa. Finally, the protein was lyophilized and stored at 4 º C until use. 219 220 14 We used RNA sequencing technology to identify any proteins contained in the 408 extrusomes. This enabled us to sequence the entire transcriptome of the ciliates and 409 to locate the protein sequences that may be related to the extrusomes. After 410 annotation of the genes that encode proteins of the parasite, using the BLASTx tool, 411 we were able to detect proteins associated with extrusomes in other ciliates. Thus, 412 homology analysis enabled us to detect two types of proteins related to extrusomes: 413 1) In P. dicentrarchi extrusomes are activated by calcium-dependent mechanisms T2-A 414 (TMPT2A) (accession MH412657.1) encoded by an 1134 bp mRNA that generates a 415 protein of 377 amino acids long (Fig. 5A), of estimated molecular weight 43502.79 416 daltons (Da) and a theoretical pI of 4.96 (Fig. 5C). According to the Phobius program, 417 this protein has a signal peptide between amino acid positions 1 and 18 (Fig. 5A), with 418 a cleavage site between positions 18 and 19, with the signal peptide C-region between 419 positions 15 and 18, the signal peptide H-region is located between positions 3 and 14 420 and the signal peptide N-region between positions 1 and 2. The TMPT2A protein 421 possesses 12 potential O-glycosylation sites at positions82, 189, 195-196, 202, 210, 422 222-224, 317, 348 and 366 (Fig. 5A), and, according to the prediction by 423 METALDECTETOR v2.0 (predictor of cysteine and histidine metal binding sites) binds to 424 metals in the cysteine at position 10 (which may be a Ca2+binding site). 2) P. 425 dicentrarchi trichocyst matrix protein T4-B (TMPT4B) (accession MH412658.1) encoded 426 by an 1113 bp mRNA, comprising 370 amino acids (Fig. 5B), of molecular weight 427 41996.11 Da and with a theoretical pI of 4.90 (Fig. 5D). The protein has a signal peptide 428 located between amino acid positions 1 and 16, according to the prediction by the 429 Phobius program (Fig. 5B); however, the Signal-3L program predicts a signal peptide 430 between positions 1 and 23 with the signal peptide C-region located between positions 431 13 and 16, the signal peptide H-region between positions 4 and 12, and the signal 432 peptide N-region between positions 1 and 3. This protein has 13 potential O-433 glycosylation sites at positions 21, 28, 54, 104, 111, 147, 218, 286, 291-293, 301 and 434 324 (Fig. 5B). BLAST analysis of the database including the Tetrahymena thermophila 435 genome (TGD) indicated that this protein is related to a similar protein encoded by the 436 GRL3 gene (Granule Lattice), which encodes the granule lattice protein and 437 corresponds to an acidic, calcium-binding structural protein of dense core granules, 438 contains coiled-coil region. This protein seems to possess a Cys at position 16, which 439 15 may be a Ca2+ binding site. As the predicted Ca2 + binding site corresponds to Cys 440 located in the SP, this site may not be functionally important. Modelling of the protein 441 structure (Swiss-model) indicates that the oligomeric state of the two P. dicentrarchi 442 trichocyst matrix proteins is monomeric (Fig. 5C-D). 443 The TMPT2A and TMPT4B proteins displayed very low sequence identity (23%). 444 The sequence identity was also very low in comparison with other ciliated proteins 445 (e.g. Paramecium, Ichtthyophthirius and Tetrahymena) with maximum sequence 446 identity scarcely exceeding 30%, for TMPT2A and TMPT4B (Figs. 6A, B; 7A, B). 447 Phylogenetically, the TMPT2A protein is closer to Paramecium (Fig. 6C), while the 448 TMPT4B protein is phylogenetically closer to the other ciliates analyzed (Fig. 7C). 449 When the trophonts were incubated with heat-inactivated preimmune serum 450 and stained with Safranin-O, only slight intracellular staining, which remained constant 451 over time in all ciliates, was observed (Fig. 8A). However, when the ciliates incubated 452 with turbot heat-inactivated immune serum were stained with safranin-O dye, a 453 progressive and time-dependent increase in the intensity of staining both in the 454 cytoplasm and in the external material surrounding the ciliate was observed (Fig. 8B-455 D). 456 457 3.3. Expression and location of extrusome proteins after stimulation with 458 immune serum from the host 459 In order to determine whether the proteins presumably associated with the 460 trichocysts are involved in the formation of the capsules observed during the 461 agglutination of the ciliates by the host immune serum, the recombinant protein was 462 generated in the yeast Kluyveromyces lactis. For this purpose, we expressed the 463 TMPT2A protein in the yeast (Fig. 9A), which was used to generate antisera in mice to 464 enable us to perform experiments to study expression of this protein after incubation 465 with the antiserum (Fig. 9B) and to determine the cytolocation (Fig. 9C). 466 First, the recombinant protein expressed by yeast has the biochemical 467 characteristics (e.g. molecular size) predicted for the original sequence obtained from 468 the ciliate, which indicates that this protein expression system is optimal for the 469 heterologous expression of this type of eukaryotic proteins (Fig. 9A). On the other 470 16 hand, the antibodies generated in mice against the rTMPT2A protein demonstrated 471 that the material produced after incubation of ciliates with the turbot immune serum 472 is related to this protein, as demonstrated by the FELISA, in which the absorbance 473 levels of these antibodies increase during the period of incubation with the immune 474 serum from turbot (Fig. 9B). An increase in fluorescence was observed in both the 475 cytoplasm of the agglutinated ciliates and in the material associated with the outer 476 surface throughout the incubation period (Fig. 9C). 477 478 3.4. Expression of the genes associated with extrusome proteins and 479 their association with the discharge of intracellular Ca2+ after stimulation 480 of the ciliates with host immune serum 481 We investigated expression of the genes encoding the trichocysts proteins 482 TMPT2A, TMPT4B after incubation with the turbot immune serum for different times. 483 Incubation of the ciliates with the turbot immune serum produced a significant 484 increase in the mRNA levels of the genes encoding these proteins throughout the 485 incubation period (Fig. 10A). Dibucaine, included as a positive control for the induction 486 of extrusion, also had a stimulatory effect on the expression of both mRNA levels 487 relative to the all trichocyst genes; however, the absolute mean values of the increase 488 were higher for the TMPT2A gene than for the TMPT4B gene (Fig. 10A). 489 Finally, we analyzed the effect of the addition of turbot immune serum on the 490 intracellular Ca2+ discharge by using the Fluo-4NW probe. Incubation of the trophonts 491 with the turbot immune serum induced discharge of intracellular Ca2+, as indicated by 492 the increase in fluorescence levels throughout the incubation time, while the 493 fluorescence increased only slightly over time in the ciliates not exposed to the serum 494 (Fig. 10B). 495 496 4. Discussion 497 In protists, extrusomes are specialized exocytotic and ejectable organelles 498 which can discharge their contents outside of the cell in response to external 499 mechanical or chemical stimuli and which may have offensive or defensive functions 500 during predation or in the acquisition of food [38]. In P. dicentrarchi, two types of 501 17 extrusomes have been described: a fusiform type (fibrous trichocysts) located in the 502 cortex, perpendicular to the plasma membrane, and a spherical type (mucocysts) with 503 an irregular distribution [18,19]. The mucocysts, which have an amorphous content, 504 merge with the plasma membrane and release their contents to the exterior giving rise 505 to a thin mucilaginous layer over the cell surface [19]. Although in free-living ciliates 506 the extrusomes can have a protective or defensive response to environmental 507 changes, in ciliated parasites such as P. dicentrarchi, the extrusomes may play a role in 508 providing protection from attack by the host immune system. The existence of the 509 production of capsules by the trophonts of P. dicentrarchi was initially obtained in 510 studies of ciliate agglutination caused by different immune sera from turbot and rabbit 511 [20]. In those studies, it was observed that when ciliates were incubated with the 512 immune sera (for 2h), abundant transparent capsule-like structures appeared. The 513 precise surface topography of the ciliate, including the somatic cilia, was observed, and 514 ciliates were also observed moving within the capsules [20]. At that time, it was 515 interpreted that their capsules probably made up of immunocomplexes between these 516 antigens and the agglutinating antibodies [20]. In the present study, we sequentially 517 monitored the agglutination of the trophonts by inactivated immune turbot serum in 518 order to investigate the capsule formation. The phenomenon of capsule formation has 519 already been described in the ciliates; e.g. Tetrahymena forms capsules when 520 exocytosis of mature mucocysts is induced by the secretagogue Alcian Blue 8GS [39-521 41]. In the environment, the ciliate mucocysts secrete an amorphous material to 522 protect the cell from osmotic shock or from predator attacks [43]. 523 The appearance of capsules during agglutination of the P. dicentrarchi 524 trophonts with immune serum suggests that the host antibodies induce the mucocysts 525 to extrude their mucilaginous content. This material is deposited on the surface of the 526 ciliate forming a protective layer, which eventually became a rigid capsule with an 527 external topology identical to that of the ciliate and which protects it from 528 agglutination. This process was clearly observed in this study by both optical 529 microscopy and SEM. 530 In ciliates such as Paramecium, trichocysts are characterized by a highly 531 constrained shape that reflects the crystalline organization of the proteins that they 532 contain and that are derived from the process of a broad family of precursor proteins 533 18 (coded by a family of some 100 co-expressed genes) that allow correct processing of 534 the crystalline core assembly necessary for functioning of the trichocyst [44,45]. The 535 trichocyst matrix proteins in Paramecium are of sizes ranging between 15-20 kDa, and 536 some are glycosylated; the isoelectric points are between 4.7 and 5.5 and the proteins 537 seem to be derived from the proteolytic processing of precursor proteins of size 538 between 40-45 kDa [46,47]. In our study, the TMPT2A and TMPT4B proteins were 539 about 43 kDa in size and the isoelectric points were close to 5.0, i.e. they are 540 compatible with the precursor proteins described in Paramecium. In addition, the 541 proteins from P. dicentrarchi possess sequences with a very low similarity to each 542 other, although with very similar isoelectric points and sizes. This may indicate that the 543 trichocyst matrix is composed of complex interrelated proteins, or of the proteolytic 544 processing during the maturation of secretory proteins [46], or of post-translational 545 modifications [48]. It has also been observed in Paramecium tetraurelia that proteins 546 released by exocytosis of trichocysts are glycoproteins [49]. 547 As previously mentioned, apart from the encysting stages of the ciliates, 548 capsule production is rare, but has been induced in vitro in several species [42]. The 549 capsule has been shown to consist of mucopolysaccharide material from mucocysts 550 [50,51]. Tetrahymena has mucocyst-type extrusomes characterized by containing 551 mucin-like acidic proteins of sizes between 40 and 80 kDa and that can bind to Ca2+ [8]. 552 O-glycosylation (or “mucin-type O-glycosylation”) indicates that these proteins carry 553 this type of glycan to the side-arm hydroxyl groups of serine and threonine residues 554 [52]. Safranin O staining has been used to detect glycosaminoglycans [53] and mucins 555 [54]. All mucins are highly O-glycosylated, and the biosynthesis and degradation are 556 perfectly integrated for protection of the cell against external aggressions [55]. The 557 present findings clearly show that the turbot immune serum acts as a stimulus that 558 leads to the production of mucin-like proteins, as shown by Safranin staining. The 559 stimulation also causes a significant increase in the expression of both the matrix 560 proteins and the expression of the genes that encode them. The immunological assays 561 revealed that the components of the capsule share epitopes with the matrix 562 glycoproteins of the extrusomes. 563 In ciliate secretion systems, Ca+2 is necessary for stimulus-secretion coupling 564 [56]. In Paramecium it has been shown that the exocytic release of the paracrystalline 565 19 secretor product derived from the trichocyte matrix depends on Ca2+, and the 566 secretory signal probably involves an influx of calcium [57,58]. The role of calcium in 567 exocytosis has been demonstrated in Paramecium following the application of Ca+2 568 ionophores, and direct microinjection of Ca+2 in the cells induces exocytosis of the 569 trichocysts [59]. On the other hand, in Tetrahymena, the addition of the anaesthetic 570 dibucaine induces the synchronous secretion of mature mucocysts [60] via an increase 571 in intracellular Ca+2 [61] and the release of flocculent mucin [8]. In this study, we 572 demonstrated that stimulation of P. dicentrarchi trophonts with turbot serum 573 containing agglutinating antibodies induces discharge of intracellular Ca+2 and 574 extrusion of mucoid material; this suggests that these processes are induced by IgM in 575 the ciliate. However, whether capsule formation is triggered by a mechanical effect, by 576 the interaction of IgM with specific membrane receptors or by both is an interesting 577 question that needs to be investigated. 578 In conclusion, our findings indicate that P. dicentrarchi can overcome the 579 agglutination generated by the specific antibodies produced by the host by generating 580 capsules through the extrusome-mediated secretion of O-glycosylated matrix proteins 581 that possess mucin-like characteristics, and whose release is regulated through Ca+2-582 mediated signalling. The findings show that the ciliate uses exocytosis as a defence 583 mechanism that probably allows evasion of the host immune response. 584 585 Acknowledgements 586 This study was financially supported by grants from the Ministerio de Economía 587 y Competitividad (Spain) and Fondo Europeo de Desarrollo Regional -FEDER588 (European Union) (AGL2017-83577-R) and from the Xunta de Galicia (Spain) 589 (ED431C2017/31) and also by the PARAFISHCONTROL project, which received funding 590 from the European Union's Horizon 2020 research and innovation programme under 591 grant agreement No. 634429. This publication reflects the views of the authors, and 592 the European Commission cannot be held responsible for any use which may be made 593 of the information contained herein 594 595 596 20 References 597 [1] G. Rosati, L. Mondeo, Extrusomes in ciliates: diversification, distribution, and 598 phylogenetic implications. J. Eukaryiot. Microbiol. 50 (2003), 383-402. 599 [2] W.D. Taylor, R.W. Sanders, Protozoa. In: Ecology and Classification of North 600 American Freshwater Invertebrates. Third Edition, (Thorp, JH & Covich AP, 601 Eds.). Academic Press (2010) pp: 49-90. 602 [3] B. Haacke-Bell, R. Hohenberger-Bregger, H. Plattner, Trichocysts of Paramecium: 603 secretory organelles in search of their function. Eur. J. Protistol. 25 (1990), 604 289-305. 605 [4] G. Benwitz, Die Entladung der Haptocysten von Ephelota gemmipara (Suctoria, 606 Ciliata). Z. Naturforsch. C 39 (1984), 812-817. 607 [5] N. Ricci, A. Morelli, F. Verni, The predation of Litonotus on Euplotes: a two-step 608 cell-cell recognition process. Acta Protozool. 35 (1996), 201-208. 609 [6] K.H. Krainer, Contribution to the morphology, infraciliature and ecology of the 610 planktonic ciliates Strombidium pelagicum n. sp., Pelagostromhidium mirabile 611 (Penard, 1916) n. g. n. comb., and Pelagostrombidium fullax (Zacharias, 1876) 612 n. g., n. comb. (Ciliophora, Oligotrichida). Eur. J. Protistol. 27 (1991), 60-70. 613 [7] L. Modeo, G. Pegroni, M. Bonaldi, G. Rosati, Trichites of Strombidium (Ciliophora, 614 Oligotrichida) are extrusomes. J. Eukaryot. Microbiol. 48 (2001), 95-101. 615 [8] M.K. Sauer, R.B. Kelly, Conjugation rescue of exocytosis mutants in Tetrahymena 616 thermophila indicates the presence of functional intermediates in the 617 regulated secretory pathway. J. Eukaryot. Microbiol. 42 (1995), 173-183. 618 [9] A. Miyake, F. Buonanno, P. Saltalamachia, M.E. Masaki, H. Lio, Chemical defence 619 by means of extrusive cortical granules in the heterotrich ciliate 620 Climacostomun virens. Eur. J. Protistol. 39 (2003), 25-36. 621 21 [10] J. Fyde, G. Kennaway, K. Adams, A. Warren, Ultrastructural events in the 622 predator-induced defence response of Colpidium kleini (Ciliophora: 623 Humenostomatia). Acta Protozool. 45 (2006), 461-464. 624 [11] R. Iglesias, A. Paramá, M.F. Álvarez, J. Leiro, J. Fernández, M.L. Sanmartín, 625 Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent 626 of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW 627 Spain). Dis. Aquat. Organ. 46 (2001), 47-55. 628 [12] A.P. De Felipe, J. Lamas, R.A. Sueiro, I. Folgueira, J.M. Leiro, New data on flatfish 629 scuticociliatosis reveal that Miamiensis avidus and Philasterides dicentrarchi 630 are different species. Parasitology 29 (2017), 1-18. 631 [13] N. Mallo, J. Lamas, J.M. Leiro, Evidence of an alternative oxidase pathway for 632 mitochondrial respiration in the scuticociliate Philasterides dicentrarchi. 633 Protist 164 (2013), 824-836. 634 [14] N. Mallo, J. Lamas, A.P. De Felipe, R.A Sueiro, F. Fontenla, Mallo N, Lamas J, de 635 Felipe AP, Sueiro RA, Fontenla F, J.M. Leiro, Role of H(+)-pyrophosphatase 636 activity in the regulation of intracellular pH in a scuticociliate parasite of 637 turbot: Physiological effects. Exp. Parasitol. 169 (2016) 59-68. 638 [15] M.C. Piazzon, G.F. Wiegertjes, J. Leiro, J. Lamas, Turbot resistance 639 to Philasterides dicentrarchi is more dependent on humoral than on cellular 640 immune responses. Fish Shellfish Immunol. 30 (2011), 1339-1347. 641 [16] M.C. Piazzon, J. Leiro, J. Lamas, Reprint of "fish immunity to scuticociliate 642 parasites". Dev. Comp. Immunol. 43 (2014), 280-289. 643 [17] V. Blanco-Abad, M. Noia, A. Valle, F. Fontenla, I. Folgueira, A.P. De Felipe, P. 644 Pereiro, J. Leiro, J. Lamas, The coagulation system helps control infection 645 caused by the ciliate parasite Philasterides dicentrarchi in the turbot 646 Scophthalmus maximus (L.). Dev. Comp. Immunol. 87 (2018), 147-156. 647 22 [18] A. Dragesco, J. Dragesco, F. Coste, C. Gasc, B. Romestand, J.C. Raymond, G. Bouix, 648 Philasterides dicentrarchi, n. sp. (Ciliophora, Scuticociliatida), a histophagous 649 opportunistic parasite of Dicentrarchus labrax (Linnaeus, 1758) a reared 650 marine fish. Eur. J. Protistol. 31 (1995), 327-340. 651 [19] A. Paramá, J.A. Arranz, M.F. Álvarez, M.L. Sanmartín, J. Leiro, Ultrastructure and 652 phylogeny of Philasterides dicentrarchi (Ciliophora, Scuticociliatia) from 653 farmed turbot in NW Spain. Parasitology 132 (2006), 555-564. 654 [20] R. Iglesias, A. Paramá, M.F. Alvarez, J. Leiro , F.M. Ubeira, M.L. Sanmartín, 655 Philasterides dicentrarchi (Ciliophora:Scuticociliatida) expresses surface 656 immobilization antigens that probably induce protective immune responses in 657 turbot. Parasitology 126 (2003), 125-134. 658 [21] A. Paramá, R. Iglesias, M.F. Álvarez, J. Leiro, C. Aja, M.L. Sanmartín, Philasterides 659 dicentrarchi (Ciliophora, Scuticociliatida): experimental infection and possible 660 routes of entry in farmed turbot (Scophthalmus maximus). Aquaculture 217 661 (2003), 73–80. 662 [22] R. Iglesias, J. Leiro, F.M. Ubeira, M.T. Santamarina, M.L. Sanmartín, 663 Anisakis simplex: antigen recognition and antibody production in 664 experimentally infected mice. Parasite Immunol. 15 (1993), 243-250. 665 [23] J. Lamas, M.L. Sanmartín, A. Paramá, R. Castro, S. Cabaleiro, M.V. Ruiz de 666 Ocenda, J.L. Barja, J. Leiro, Optimization of an inactivated vaccine against a 667 scuticociliate parasite of turbot: Effect of antigen, formalin and adjuvant 668 concentration on antibody response and protection against the pathogen. 669 Aquaculture 278 (2008), 22-26. 670 [24] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-671 time quantitative PCR and the 2-(delta delta C(T)) method. Methods 25 (2001), 672 402-408. 673 23 [25] S.A. Bustin, V. Benes, J.A. Garson, J. Hellemans, J. Huggett, M. Kubista, R. 674 Mueller, T. Nolan, M.W. Pfaffl, G.L. Shipley, J. Vandesompele, C.T. Wittwer, 675 The MIQE guidelines: minimum information for publication of quantitative 676 real-time PCR experiments. Clin. Chem. 55 (2009), 611-622. 677 [26] N. Mallo, J. Lamas, C. Piazzon, J.M. Leiro, Presence of a plant-like proton 678 translocating pyrophosphatase in a scuticociliate parasite and its role as a 679 possible drug target. Parasitology 142 (2015), 449–462. 680 [27] A.L. Mitchell, T.K. Attwood, P.C. Babbitt, M. Blum, P. Bork, A. Bridge, S.D. Brown, 681 H.Y. Chang, S. El-Gebali, M.I. Fraser, J. Gough, D.R. Haft, H. Huang, I. Letunic, R. 682 López, A. Luciani, F. Madeira, A. Marchler-Bauer, H. Mi, D.A. Natale, M. Necci, 683 G. Nuka, C. Orengo, A.P. Pandurangan, T. Paysan-Lafosse, S. Pesseat, S.C. 684 Potter, M.A. Qureshi, N.D. Rawlings, N. Redaschi, L.J. Richardson, C. Rivoire, 685 G.A. Salazar, A. Sangrador-Vegas, C.J.A. Sigrist, I. Sillitoe, G.G. Sutton, N. 686 Thanki, P.D. Thomas, S.C.E. Tosatto, S.Y. Yong, R.D. Finn, InterPro in 2019: 687 improving coverage, classification and access to protein sequence 688 annotations. Nucleic Acids Res. gky 689 28. L. Käll, A. Krogh, E.L.L. Sonnhammer, A Combined Transmembrane Topology and 690 Signal Peptide Prediction Method. J. Mol. Biol. 338 (2004), 1027-1036. 691 [29] H. Nielsen, Predicting Secretory Proteins with SignalP. In Kihara, D (ed): Protein 692 Function Prediction (Methods in Molecular Biology vol. 1611) (2017) pp. 59-693 73, Springer. 694 [30] Y.-Z. Zhang, H.-B. Shen, Signal-3L 2.0: A hierarchical mixture model for enhancing 695 protein signal peptide prediction by incorporating residue-domain cross level 696 features. J. Chem. Inf. Model 57 (2017), 988-999. 697 [31] C. Steentoft, S.Y. Vakhrushev, H.J. Joshi, Y. Kong, M.B. Vester-Christensen, K.T. 698 Schjoldager, K. Lavrsen, S. Dabelsteen, N.B. Pedersen, L. Marcos-Silva, R. 699 Gupta, E.P. Bennett, U. Mandel, S. Brunak, H.H. Wandall, S.B. Levery, H. 700 30 recombinant mouse antibody anti-P. dicentrarchi TMPT2A and revealed with an anti-872 mouse rabbit antibody conjugated with FITC. 873 874 Figure 10.- (A) Levels of mRNA expression of the genes that encode the P. dicentrarchi 875 trichocyst matrix protein T2-A (TMPT2A) and P. dicentrarchi trichocyst matrix protein 876 T4-B (TMPT4B) in ciliates incubated for different lengths of time with turbot immune 877 serum and dibucaine (D). The results are expressed as the relative gene expression 878 versus the P. dicentrarchi elongation factor 1-alpha (EF1). (B) Calcium response of 879 trophonts stimulated with turbot immune serum and Hanks’ balanced salt solution 880 (HBSS without Ca2+, Mg2+, and phenol red) quantified using the Fluo-4 NW calcium 881 assay kit. The graph represents the time course of the increase per min in fluorescence 882 (F/min) of the cell-permeable fluorescent dye. Each data point represents the mean  883 standard error (SE) for five replicates. Asterisks indicate a statistically significant 884 difference (P<0.01) relative to the control (time 0). 885 FIGURE 1 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image Figure 8 Click here to download high resolution image Figure 9 Click here to download high resolution image