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

  • Folgueira López, Iria
  • Lamas Fernández, Jesús
  • Felipe González, Ana Paula de
  • Sueiro Benavides, Rosa Ana
  • Leiro Vidal, José Manuel
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, Like other ciliates, Philasterides dicentrarchi, the scuticociliate parasite of turbot, produces a feeding-only or growing stage called a trophont during its life cycle. Exposure of the trophonts to heat-inactivated serum extracted from the turbot host and containing specific antibodies that induce agglutination/immobilization leads to the production of a mucoid capsule from which the trophonts later emerge. We investigated how these capsules are generated, observing that the mechanism was associated with the process of exocytosis involved in the release of a matrix material from the extrusomes. The extruded material contains mucin-like glycoproteins that were deposited on the surface of the cell and whose expression increased with time of exposure to the heat-inactivated immune serum, at both protein expression and gene expression levels. Stimulation of the trophonts with the immune serum also caused an increase in discharge of the intracellular storage compartments of calcium necessary for the exocytosis processes in the extrusomes. The results obtained suggest that P. dicentrarchi uses the extrusion mechanism to generate a physical barrier protecting the ciliate from attack by soluble factors of the host immune system. Data on the proteins involved and the potential development of molecules that interfere with this exocytic process could contribute to improving the prevention and control of scuticociliatosis in turbot, This study was financially supported by grants from the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional -FEDER- (European Union) (AGL2017-83577-R) and from the Xunta de Galicia (ED431C2017/31) and also by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429, SI



Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia)

  • Folgueira López, Iria
  • Felipe González, Ana Paula de
  • Sueiro Benavides, Rosa Ana
  • Lamas Fernández, Jesús
  • Leiro Vidal, José Manuel
This is the accepted manuscript of the following article: Folgueira, I., de Felipe, A.P., Sueiro, R.A., Lamas, J. & Leiro, J. (2018). Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia). Cryobiology, 80, 77-83. doi:
10.1016/j.cryobiol.2017.11.010, Philasterides dicentrarchi is a free-living marine ciliate that can become an endoparasite that causes a severe disease called scuticociliatosis in cultured fish. Long-term maintenance of this scuticociliate in the laboratory is currently only possible by subculture, with periodic passage in fish to maintain the virulence of the isolates. In this study, we developed and optimized a cryopreservation protocol similar to that used for the long-term storage of scuticociliates of the genus Miamiensis. The cryogenic medium comprised ATCC medium 1651 and a combination of 11% dimethylsulfoxide and 5% glycerol. We have verified that the most important factor ensuring the efficiency of the cryopreservation procedure is the growth phase of the culture, and that ciliates should be cryopreserved at the stationary phase (around the sixth day of culture). The cryopreservation protocol described here can be used for all strains of P. dicentrarchi as well as commercial strains of Miamiensis and enables the virulence of the strains to be maintained. Finally, this cryopreservation protocol has been shown to be more effective than others routinely applied to scuticociliates, yielding a higher survival rate with a lower initial concentration of ciliates. The results obtained indicate that the cropreservation protocol enables the long-term storage of scuticociliate parasites while maintaining the virulence of the isolates. The protocol is therefore suitable for use in vaccine production and related studies, This work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL),
by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain), SI



Enzymes involved in pyrophosphate and calcium metabolism as targets for anti-scuticociliate chemotherapy

  • Mallo Seijas, Natalia
  • Lamas Fernández, Jesús
  • Felipe González, Ana Paula de
  • Sueiro Benavides, Rosa Ana
  • Fontenla Iglesias, Francisco
  • Leiro Vidal, José Manuel
This is the peer reviewed version of the following article: Mallo, N., Lamas,
J., DeFelipe, A.P., Sueiro, R.A., Fontenla, F. and Leiro JM. (2016), Enzymes
Involved in Pyrophosphate and Calcium Metabolism as Targets for Antiscuticociliate Chemotherapy. J Eukaryot Microbiol 63(4): 505-15. doi:
10.1111/jeu.12294, which has been published in final form
at https://doi.org/10.1111/jeu.12294. This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for
Use of Self-Archived Versions, Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 μM and 80 ± 8 μM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 μM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis, This study was financially supported by grant AGL2014-57125-R from the Ministerio de Economía y Competitividad (Spain), by the European Commission, under the Horizon 2020 programme (Grant Agreement 634429, PARAFISHCONTROL), and 430 by grant GPC2014/069 from the Xunta de Galicia (Spain), SI



Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences

  • Mallo Seijas, Natalia
  • Lamas Fernández, Jesús
  • Felipe González, Ana Paula de
  • Castro de Antonio, María Eugenia de
  • Sueiro Benavides, Rosa Ana
  • Leiro Vidal, José Manuel
This article has been published in a revised form in Parasitology
[http://doi.org/10.1017/ S0031182015001997]. This version is free to view
and download for private research and study only. Not for re-distribution,
re-sale or use in derivative works. © 2017 Cambridge University Press, H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi, This work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL),
by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain), SI



Identification and Molecular Characterization of Superoxide Dismutases Isolated From A Scuticociliate Parasite: Physiological Role in Oxidative Stress

  • Folgueira López, Iria
  • Lamas Fernández, Jesús
  • Felipe González, Ana Paula de
  • Sueiro Benavides, Rosa Ana
  • Leiro Vidal, José Manuel
Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fsh. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity apable of inactivating the superoxide anion (•O2−) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise
three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34–44kDa and with very diferent AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2 − is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stress, This study was financially supported by grant AGL2017-83577-R awarded by the Ministerio de Economía
y Competitividad (Spain) and Fondo Europeo de Desarrollo Regional -FEDER- (European Union), by grant
ED431C2017/31 from the Xunta de Galicia (Spain), and by PARAFISHCONTROL project, which received
funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement
No. 634429, SI



Assembled transcripts obtained after RNA-seq analysis of Philasterides dicentrarchi B1, C1 and I1 isolates

  • Lamas Fernández, Jesús
  • Leiro Vidal, José Manuel
Funded European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429) (PARAFISHCONTROL project), Assembled transcripts obtained from raw sequence reads after RNA-seq analysis of cultured P. dicentrarchi B1, C1 and I1 strains, obtained after RNAseq of cultured ciliates. Ciliates were isolated from experimentally infected turbot and cultured for three weeks in L-15 medium with 10% FCS at 18 oC. Then, ciliates were subjected to transcriptome analysis by using RNAseq



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

  • Mallo Seijas, Natalia
  • Lamas Fernández, Jesús
  • Felipe González, Ana Paula de
  • Sueiro Benavides, Rosa Ana
  • Fontenla Iglesias, Francisco
  • Leiro Vidal, José Manuel
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, The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) 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 Ca(2+) storage). We evaluated the effects of Ca(2+) 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 Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) 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, This work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL),
by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R and by grant GPC2014/069 from the Xunta de Galicia (Spain), SI



Annotation genes Philasterides dicentrarchi strains B1, I1, C1

  • Lamas Fernández, Jesús
  • Leiro Vidal, José Manuel
Funded by PARAFISHCONTROL project, European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429, These files contain the annotated genes of P. dicentrarchi B1, I1 and C1 strains, obtained after RNAseq of cultured ciliates. Ciliates were isolated from experimentally infected turbot and cultured for three weeks in L-15 medium with 10% FCS at 18 oC. Then, ciliates were subjected to transcriptome analysis by using RNAseq



Characterization of the turbot Scophthalmus maximus (L.) myeloperoxidase. An insight into the evolution of vertebrate peroxidases

  • Noia Guldrís, Manuel
  • Fontenla Iglesias, Francisco
  • Valle Cao, Alejandra
  • Blanco Abad, Verónica
  • Leiro Vidal, José Manuel
  • Lamas Fernández, Jesús
We have completed the characterization of the turbot (Scophthalmus maximus) myeloperoxidase (mpx) gene and protein, which we partially described in a previous study. The turbot mpx gene has 15 exons that encode a protein of 767 aa, with a signal peptide, propeptide and light and heavy chains, and also with haem cavities, a Ca+2-binding motif and several N- and O-glycosylation sites. The mature protein forms homodimers of about 150 kDa and is very abundant in turbot neutrophils. In addition to the mpx (epx2a) gene, another three peroxidase genes, named epx1, epx2b1 and epx2b2, were identified in the turbot genome. Epx1, Epx2b1 and Epx2b2 proteins also have signal peptides and many structural characteristics of mammalian MPO and eosinophil peroxidase (EPX). Mpx was strongly expressed in head kidney, while epx2b1 and epx2b2 were strongly expressed in the gills, and epx1 was not expressed in any of the tissues or organs analysed. In vitro stimulation of head kidney leucocytes with the parasite Philasterides dicentrarchi caused a decrease in mpx expression and an increase in epx2b1 expression over time. In turbot infected experimentally with P. dicentrarchi a significant increase in mpx expression in the head kidney was observed on day 7 postinfection, while the other genes were not regulated. However, mpx, epx2b1 and epx2b2 were downregulated in the gills of infected fish, and epx1 expression was not affected. These results suggest that the four genes responded differently to the same stimuli. Interestingly, BLAST analysis revealed that Epx1 and Mpx showed greater similarity to mammalian EPX than to MPO. Considering the phylogenetic and synteny data obtained, we concluded that the epx/mpx genes of Gnathostomes can be divided into three main clades: EPX1, which contains turbot epx1, EPX2, which contains turbot mpx (epx2a) and epx2b1 and epx2b2 genes, and a clade containing mammalian EPX and MPO (EPX/MPO). EPX/MPO and EPX2 clades share a common ancestor with the chondrichthyan elephant shark (Callorhinchus milii) and the coelacanth (Latimeria chalumnae) peroxidases. EPX2 was only found in fish and includes two sister groups. One of the groups includes turbot mpx and was only found in teleosts. Finally, the other group contains epx2b1 and epx2b2 genes, and epx2b1-2b2 loci share orthologous genes with other teleosts and also with holosteans, suggesting that these genes appeared earlier on than the mpx gene., This study was financially supported by grant AGL2017-83577-R awarded by the Ministerio de Economía y Competitividad (Spain) and the European Regional Development Fund (ERDF, European Union), by grant ED431C2017/31 from the Xunta de Galicia (Spain) and by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 634429. FF-I was contracted by a grant from the Xunta de Galicia (Plan I2C), SI



Combined antiparasitic and anti‐inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis

  • Mallo Seijas, Natalia
  • Felipe González, Ana Paula de
  • Folgueira López, Iria
  • Sueiro Benavides, Rosa Ana
  • Lamas Fernández, Jesús
  • Leiro Vidal, José Manuel
This is the peer reviewed version of the following article: Mallo, N. , DeFelipe, A. P., Folgueira, I. , Sueiro, R. A., Lamas, J. and Leiro, J. M. (2017), Combined antiparasitic and anti‐inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis. J Fish Dis, 40: 205217. doi:10.1111/jfd.12503, which has been published in final form at https://doi.org/10.1111/jfd.12503. This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions, The histiophagous scuticociliate Philasterides dicentrarchi is the aetiological agent of scuticociliatosis, a parasitic disease of farmed turbot. Curcumin, a polyphenol from Curcuma longa (turmeric), is known to have antioxidant and anti-inflammatory properties. We investigated the in vitro effects of curcumin on the growth of P. dicentrarchi and on the production of pro-inflammatory cytokines in turbot leucocytes activated by parasite cysteine proteases. At 100 μm, curcumin had a cytotoxic effect and completely inhibited the growth of the parasite. At 50 μm, curcumin inhibited the protease activity of the parasite and expression of genes encoding two virulence-associated proteases: leishmanolysin-like peptidase and cathepsin L-like. At concentrations between 25 and 50 μm, curcumin inhibited the expression of S-adenosyl-L-homocysteine hydrolase, an enzyme involved in the biosynthesis of the amino acids methionine and cysteine. At 100 μm, curcumin inhibited the expression of the cytokines tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) produced in turbot leucocytes activated by parasite proteases. Results show that curcumin has a dual effect on scuticociliatosis: an antiparasitic effect on the catabolism and anabolism of ciliate proteins, and an anti-inflammatory effect that inhibits the production of proinflammatory cytokines in the host. The present findings suggest the potential usefulness of this polyphenol in treating scuticociliatosis, This work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429 (PARAFISHCONTROL) and by the Ministerio de Economía y Competitividad (Spain) under grant agreement AGL2014-57125-R, SI