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

Folgueira, I.; Lamas, Jesús; Sueiro, Rosa Ana; Leiro, J.

doi:10.1038/s41598-019-49750-5

Cited by 12 publications

(9 citation statements)

References 89 publications

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“…For example, it possesses mitochondria with two respiratory pathways: the cytochrome pathway, which exists in all aerobic organisms, and an alternative, cyanide-insensitive oxidase pathway, which enables the ciliate to survive and proliferate under normoxic and hypoxic conditions [ 15 ] and thus to become adapted to differences in oxygen levels in the host and seawater. In addition, P. dicentrarchi has potent antioxidant defence mechanisms that may be important during infection, including several superoxide dismutases that help the ciliate resist the reactive oxygen species released by the host [ 16 ]. Finally, it has been suggested that P. dicentrarchi can release its extrusome contents to create a protective barrier against soluble factors of the host immune system [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Interactions between the Parasite Philasterides dicentrarchi and the Immune System of the Turbot Scophthalmus maximus. A Transcriptomic Analysis

Valle

Leiro

Pereiro

et al. 2020

Biology

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The present study analyses the interactions between Philasterides dicentrarchi (a ciliate parasite that causes high mortalities in cultured flatfish) and the peritoneal cells of the turbot Scophthalmus maximus during an experimental infection. The transcriptomic response was evaluated in the parasites and in the fish peritoneal cells, at 1, 2 and 4 h post-infection (hpi) in turbot injected intraperitoneally (ip) with 107 ciliates and at 12 and 48 hpi in turbot injected ip with 105 ciliates. Numerous genes were differentially expressed (DE) in P. dicentrarchi, relative to their expression in control ciliates (0 hpi): 407 (369 were up-regulated) at 1 hpi, 769 (415 were up-regulated) at 2 hpi and 507 (119 were up-regulated) at 4 hpi. Gene ontology (GO) analysis of the DE genes showed that the most representative categories of biological processes affected at 1, 2 and 4 hpi were biosynthetic processes, catabolic processes, biogenesis, proteolysis and transmembrane transport. Twelve genes of the ABC transporter family and eight genes of the leishmanolysin family were DE at 1, 2 and 4 hpi. Most of these genes were strongly up-regulated (UR), suggesting that they are involved in P. dicentrarchi infection. A third group of UR genes included several genes related to ribosome biogenesis, DNA transcription and RNA translation. However, expression of tubulins and tubulin associated proteins, such as kinesins or dyneins, which play key roles in ciliate division and movement, was down-regulated (DR). Similarly, genes that coded for lysosomal proteins or that participate in the cell cycle mitotic control, glycolysis, the Krebs cycle and/or in the electron transport chain were also DR. The transcriptomic analysis also revealed that in contrast to many parasites, which passively evade the host immune system, P. dicentrarchi strongly stimulated turbot peritoneal cells. Many genes related to inflammation were DE in peritoneal cells at 1, 2 and 4 hpi. However, the response was much lower at 12 hpi and almost disappeared completely at 48 hpi in fish that were able to kill P. dicentrarchi during the first few hpi. The genes that were DE at 1, 2 and 4 hpi were mainly related to the apoptotic process, the immune response, the Fc-epsilon receptor signalling pathway, the innate immune response, cell adhesion, cell surface receptors, the NF-kappaB signalling pathway and the MAPK cascade. Expression of toll-like receptors 2, 5 and 13 and of several components of NF-κB, MAPK and JAK/STAT signalling pathways was UR in the turbot peritoneal cells. Genes expressing chemokines and chemokine receptors, genes involved in prostaglandin and leukotriene synthesis, prostaglandins, leukotriene receptors, proinflammatory cytokines and genes involved in apoptosis were strongly UR during the first four hours of infection. However, expression of anti-inflammatory cytokines such as Il-10 and lipoxygenases with anti-inflammatory activity (i.e., arachidonate 15-lipoxygenase) were only UR at 12 and/or 48 hpi, indicating an anti-inflammatory state in these groups of fish. In conclusion, the present study shows the regulation of several genes in P. dicentrarchi during the early stages of infection, some of which probably play important roles in this process. The infection induced a potent acute inflammatory response, and many inflammatory genes were regulated in peritoneal cells, showing that the turbot uses all the protective mechanisms it has available to prevent the entry of the parasite.

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Section: Introductionmentioning

confidence: 99%

Interactions between the Parasite Philasterides dicentrarchi and the Immune System of the Turbot Scophthalmus maximus. A Transcriptomic Analysis

Valle

Leiro

Pereiro

et al. 2020

Biology

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“…For de novo assembly of the parasite genome, the data sets were combined using TULIP software, v0.4 50 . For sequencing of the P. dicentrarchi transcriptome, we followed the RNAseq procedure as previously described 51 , 52 .…”

Section: Methodsmentioning

confidence: 99%

“…The membranes were immunoblotted with a 1: 100 dilution of the anti-rPdVP1 (α-rPdVP1) and anti-KLH-HK (α-KLH-HK) antibodies and then with a polyclonal peroxidase-conjugated rabbit anti-mouse antibody (Dakopatts, Denmark) at 1: 800 dilution. The blots were stained by adding a chromogenic enzyme substrate solution consisting of 0.003% H 2 O 2 and 0.06% 3,30-diaminobenzidine tetrahydrochloride with 0.03% NiCl 2 (DAB/NiCl 2 , Sigma, USA) 51 .…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization and transcriptional regulation of two types of H+-pyrophosphatases in the scuticociliate parasite Philasterides dicentrarchi

Folgueira

Lamas

Sueiro

et al. 2021

Sci Rep

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Proton-translocating inorganic pyrophosphatases (H+-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H+ translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg2+ binding sites and an additional K+ binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H+-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.

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“…The AOX gene nucleotide sequence was synthesized by Invitro GeneArt Gene Synthesis (ThermoFisher Scientific), amplified by PCR with the primers FaoxKl / RaoxKl: 5′-CGC CTC GAG AAA AGA ATG CAG TCC TTC GCC AGG AAA-3′/5′-ATA AGA ATG CGG CCG C TTA GTG GTG GTG ATG ATG GTG ATG ATG ATG ATG CTG CTC GGG GGA GTA AGG GTT 3′ and cloned in the integrative expression vector pKLAC2. The K. lactis Protein Expression kit (New England Biolabs, UK) was used to express the recombinant protein in yeast according to the manufacturer's instructions and the protein secretion strategy, as previously described 42 .…”

Section: Methodsmentioning

confidence: 99%

“…with 200 µg of rAOX. The antiserum was purified by immobilized metal affinity chromatography (IMAC) with Ni-Sepharose 42 and adsorbed for 30 min at room temperature in 200 µL of a 1% chitosan hydrogel adjuvant 42 (CH) (w/v) in PBS buffer, pH 7.0. At intervals of 15 and 30 days after the first immunization, mice were reimmunized with the same dose of rAOX and adjuvant.…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors

Folgueira

Lamas

Sueiro

et al. 2020

Sci Rep

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Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). in addition to a cytochrome pathway (cp), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AoX) in the mitochondrion. Although AoX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AoX in the ciliate has not previously been reported. in this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene (size 1,106 bp) contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AoX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with eXXH motifs associated with the diiron active centers. the aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AoX in respiration under conditions of low levels of o 2 and in protecting against oxidative stress generated during infection in the host. Alternative oxidase (AOX), also known as ubiquinol-oxidoreductase, is included within the diiron carboxylate superfamily. The enzyme is characterized by cyanide resistance and is associated with maintenance of metabolic homeostasis via mitochondrial electron transport in plants subjected to various stress conditions 1,2. In plants, AOX is considered a ubiquitous enzyme; however, in algae 3 , fungi 4-9 , protozoa 10-13 and several invertebrate species 14 it is quite sporadic. AOX is involved in the mitochondrial respiratory chain and provides an alternative pathway for the direct transfer of electrons from ubiquinone to oxygen, thus forming water 15. It facilitates mitochondrial respiratory activity and therefore the functioning of the Krebs cycle after inhibition of the cytochrome route due to the presence of nitric oxide, cyanide (both inhibitors of the complex IV) or antimycin A (complex III inhibitor) 1,12,16. However, AOX reduces the energy efficiency of respiration as only 1/3 of the ATP is produced

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

Cited by 12 publications

References 89 publications

Interactions between the Parasite Philasterides dicentrarchi and the Immune System of the Turbot Scophthalmus maximus. A Transcriptomic Analysis

Interactions between the Parasite Philasterides dicentrarchi and the Immune System of the Turbot Scophthalmus maximus. A Transcriptomic Analysis

Molecular characterization and transcriptional regulation of two types of H+-pyrophosphatases in the scuticociliate parasite Philasterides dicentrarchi

Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors

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