RESUMO
Trichomoniasis, is the most prevalent non-viral sexually transmitted disease worldwide. Although metronidazole (MDZ) is the recommended treatment, several strains of the parasite are resistant to MDZ, and new treatments are required. Curcumin (CUR) is a polyphenol with anti-inflammatory, antioxidant and antiparasitic properties. In this study, we evaluated the effects of CUR on two biochemical targets: on proteolytic activity and hydrogenosomal metabolism in Trichomonas vaginalis. We also investigated the role of CUR on pro-inflammatory responses induced in RAW 264.7 phagocytic cells by parasite proteinases on pro-inflammatory mediators such as the nitric oxide (NO), tumor necrosis factor α (TNFα), interleukin-1beta (IL-1ß), chaperone heat shock protein 70 (Hsp70) and glucocorticoid receptor (mGR). CUR inhibited the growth of T. vaginalis trophozoites, with an IC50 value between 117 ± 7 µM and 173 ± 15 µM, depending on the culture phase. CUR increased pyruvate:ferredoxin oxidoreductase (PfoD), hydrogenosomal enzyme expression and inhibited the proteolytic activity of parasite proteinases. CUR also inhibited NO production and decreased the expression of pro-inflammatory mediators in macrophages. The findings demonstrate the potential usefulness of CUR as an antiparasitic and anti-inflammatory treatment for trichomoniasis. It could be used to control the disease and mitigate the associated immunopathogenic effects.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antiparasitários/uso terapêutico , Curcumina/uso terapêutico , Terapia de Alvo Molecular , Compostos Fitoquímicos/uso terapêutico , Tricomoníase/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Antiparasitários/farmacologia , Curcumina/farmacologia , Citocinas/genética , Citocinas/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Parasitos/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Proteólise/efeitos dos fármacos , Piruvato Sintase/genética , Piruvato Sintase/metabolismo , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tricomoníase/parasitologia , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/genéticaRESUMO
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.
Assuntos
Infecções por Cilióforos/veterinária , Doenças dos Peixes/imunologia , Linguados , Imunidade Humoral/imunologia , Oligoimenóforos/fisiologia , Animais , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/parasitologia , Doenças dos Peixes/parasitologiaRESUMO
Scuticociliatosis is a severe disease in farmed flatfish. However, the causative agent is not always accurately identified. In this study, we identified two isolates of scuticociliates from an outbreak in cultured fine flounder Paralichthys adspersus. Scuticociliate identification was based on morphological data, examination of life stages and the use of molecular approaches. The isolates were compared with a strain of Philasterides dicentrachi from turbot Scophthalmus maximus and with a strain deposited in the American Type Culture Collection as Miamiensis avidus ATCC® 50180™. The use of morphological, biological and molecular methods enabled us to identify the isolates from the fine flouder as P. dicentrarchi. Comparison of P. dicentrachi isolates and M. avidus revealed some differences in the buccal apparatus. Unlike P. dicentrarchi, M. avidus has a life cycle with three forms: macrostomes (capable of feeding on P. dicentrarchi), microstomes and tomites. Additionally, we found differences in the 18S rRNA and α- and ß-tubulin gene sequences, indicating that P. dicentrarchi and M. avidus are different species. We therefore reject the synonymy/conspecificity of the two taxa previously suggested. Finally, we suggest that a combination of morphological, biological, molecular (by multigene analysis) and serological techniques could improve the identification of scuticociliates parasites in fish.
RESUMO
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.
Assuntos
Cálcio/metabolismo , Cilióforos/efeitos dos fármacos , Cilióforos/enzimologia , Difosfatos/metabolismo , Linguados/parasitologia , Pirofosfatase Inorgânica/metabolismo , Animais , Artemisininas/farmacologia , Cloroquina/farmacologia , Cilióforos/crescimento & desenvolvimento , Infecções por Cilióforos/parasitologia , Infecções por Cilióforos/veterinária , Doenças dos Peixes/parasitologia , Homeostase/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/genéticaRESUMO
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.
Assuntos
Infecções por Cilióforos/veterinária , Doenças dos Peixes/parasitologia , Linguados/parasitologia , Pirofosfatase Inorgânica/análise , Oligoimenóforos/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Cilióforos/parasitologia , DNA de Protozoário/análise , Imunofluorescência/veterinária , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/imunologia , Isoenzimas/análise , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal/veterinária , Microscopia Imunoeletrônica/veterinária , Dados de Sequência Molecular , Oligoimenóforos/genética , Oligoimenóforos/ultraestrutura , Filogenia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , RNA de Protozoário/isolamento & purificaçãoRESUMO
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.
Assuntos
Infecções por Cilióforos/veterinária , Doenças dos Peixes/parasitologia , Linguados/parasitologia , Pirofosfatase Inorgânica/metabolismo , Oligoimenóforos/enzimologia , Trifosfato de Adenosina/fisiologia , Animais , Western Blotting , Cálcio/fisiologia , Infecções por Cilióforos/metabolismo , Infecções por Cilióforos/parasitologia , Doenças dos Peixes/enzimologia , Doenças dos Peixes/metabolismo , Imunofluorescência , Concentração de Íons de Hidrogênio , Pirofosfatase Inorgânica/genética , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Oligoimenóforos/fisiologia , Oligoimenóforos/ultraestrutura , Concentração Osmolar , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , SalinidadeRESUMO
The proton-translocating inorganic pyrophosphatases (H(+)-PPases) are primary electrogenic H(+) pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H(+)-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61.7 kDa and an isoelectric point of 5.0. Several motifs characteristic of plant vacuolar H(+)-PPases (V-H(+)-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V-H(+)-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K(+)-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V-H(+)-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.
Assuntos
Pirofosfatase Inorgânica/genética , Oligoimenóforos/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Arabidopsis/enzimologia , Sequência de Bases , Infecções por Cilióforos/tratamento farmacológico , Infecções por Cilióforos/parasitologia , Infecções por Cilióforos/veterinária , DNA Complementar/química , Difosfatos/metabolismo , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/parasitologia , Linguados/parasitologia , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/imunologia , Pirofosfatase Inorgânica/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oligoimenóforos/classificação , Oligoimenóforos/efeitos dos fármacos , Filogenia , Inibidores da Bomba de Prótons/farmacologia , Proteínas Recombinantes/imunologia , Alinhamento de SequênciaRESUMO
Philasterides dicentrarchi causes a severe disease in turbot, and at present there are no drugs available to treat infected fish. We have previously demonstrated that, in addition to the classical respiratory pathway, P. dicentrarchi possesses an alternative mitochondrial respiratory pathway that is cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive. In this study, we found that during the initial phase of growth in normoxia, ciliate respiration is sensitive to the natural polyphenol resveratrol (RESV) and to Antimycin A (AMA). However, under hypoxic conditions, the parasite utilizes AMA-insensitive respiration, which is completely inhibited by RESV and by the antioxidant propyl gallate (PG), an alternative oxidase (AOX) inhibitor. PG caused significantly dose-dependent inhibition of the in vitro growth of the parasite under normoxia and hypoxia and an over-expression of heat shock proteins of the Hsp70 subfamily. RESV and PG may affect the protective role of the AOX against mitochondrial oxidative stress, leading to an impaired mitochondrial membrane potential and mitochondrial dysfunction, which the parasite attempts to neutralize by increasing the expression of Hsp70. In view of the antiparasitic effects induced by AOX inhibitors and the absence of AOX in their host, this enzyme constitutes a potential target for the development of new drugs against scuticociliatosis.
Assuntos
Antioxidantes/farmacologia , Antiparasitários/farmacologia , Linguados/parasitologia , Proteínas Mitocondriais/antagonistas & inibidores , Oligoimenóforos/enzimologia , Oxirredutases/antagonistas & inibidores , Proteínas de Plantas/antagonistas & inibidores , Galato de Propila/farmacologia , Animais , Antimicina A/farmacologia , Respiração Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oligoimenóforos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Resveratrol , Estilbenos/farmacologiaRESUMO
Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 µM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 µM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 µM). At doses of 50 to 100 µM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.
Assuntos
Antitricômonas/farmacologia , Hidrogenase/antagonistas & inibidores , Proteínas Ferro-Enxofre/antagonistas & inibidores , Organelas/efeitos dos fármacos , Piruvato Sintase/efeitos dos fármacos , Estilbenos/farmacologia , Trichomonas vaginalis/efeitos dos fármacos , Animais , Feminino , Humanos , Hidrogênio/metabolismo , Organelas/enzimologia , Testes de Sensibilidade Parasitária , Piruvato Sintase/metabolismo , Resveratrol , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/crescimento & desenvolvimento , Trichomonas vaginalis/isolamento & purificação , Trichomonas vaginalis/ultraestrutura , Regulação para CimaRESUMO
Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igµ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.
Assuntos
Linfócitos B , Centro Germinativo , Animais , Imunoglobulina M , Antígenos , Vertebrados , MamíferosRESUMO
Philasterides dicentrarchi is a scuticociliate that causes high mortalities in farmed fish. Although vaccination is an effective method to prevent scuticociliatosis caused by the homologous serotype, a universal vaccine has not been developed yet. Many compounds have been shown to be toxic to this ciliate species; moreover, most of them are toxic to aquatic life and cannot be used to prevent the disease. We have evaluated the toxicity to P. dicentrarchi of several compounds of natural origin to be used to reduce parasite levels in the seawater. Ciliates were exposed to several compound concentrations, and the mortality was determined at several incubation times. Tomatine, plumbagin and 2',4'-dihydroxychalcone displayed the highest anticiliate activity, with a dose-dependent response. The effects of these compounds on the EPC cell line were also evaluated, finding that 2',4'-dihydroxychalcone displayed the lowest toxicity to fish cells. At 7.54 µM, 2',4'-dihydroxychalcone inhibited 50% parasite growth but only killed about 10% of EPC cells after 24 h incubation. Finally, we evaluated the toxicity of Pseudomonas H6 surfactant (PS) to P. dicentrarchi, finding that PS was toxic to the ciliate but showed lower toxicity to EPC cells. At a concentration of 7.8 µg/mL (LC50 for the ciliate after 3 h incubation), PS killed 14.9% of EPC cells. We conclude that 2',4'-dihydroxychalcone, and PS could be used to reduce parasite levels in seawater, thus decreasing the risk of scuticociliatosis infection in cultured fish.
RESUMO
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.
Assuntos
Evolução Molecular , Proteínas de Peixes/genética , Linguados/genética , Neutrófilos/metabolismo , Peroxidase/genética , Animais , Proteínas de Peixes/metabolismo , Linguados/sangue , Linguados/imunologia , Linguados/metabolismo , Loci Gênicos , Neutrófilos/imunologia , Peroxidase/metabolismo , Multimerização Proteica/imunologia , Especificidade da EspécieRESUMO
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 O2 and in protecting against oxidative stress generated during infection in the host.
Assuntos
Respiração Celular/efeitos dos fármacos , Hipóxia/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Oligoimenóforos/enzimologia , Oligoimenóforos/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Expressão Gênica , Genoma de Protozoário , Genômica/métodos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/química , Modelos Moleculares , Oligoimenóforos/classificação , Oxirredutases/química , Filogenia , Proteínas de Plantas/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
Myeloperoxidase (MPO) is a conspicuous enzyme in neutrophils of many fish species. Although the MPO gene has been identified in some fish species, the structure and functions of the protein remain to be determined in these vertebrates. In the present study, we isolated turbot neutrophil MPO from kidney cells by affinity chromatography, with Ulva rigida acidic sulphated polysaccharides (ASP), some of which resemble glycosaminoglycans, and Sepharose. The product obtained, of approximately 150kDa molecular weight and with peroxidase activity, was examined by SDS-page electrophoresis under reduced conditions and immunoblotting, and a single band of about 75kDa was observed. The results obtained suggest that turbot MPO is a dimer and that the band of 75kDa probably corresponds to a monomer generated by treatment of the samples with the reducing agent. The band was analysed by electromatrix-assisted laser desorption ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization-ion trap mass spectrometry, dynamic exclusion mode (LC-ESI-IT DE), to determine the amino acid composition of some peptides. The peptides obtained were very similar to myeloperoxidases of other organisms, including other fish and mammals, and were used to design the primers for cDNA amplification. A 567bp product was amplified and the deduced amino acid sequence, which contains several putative N-glycosylation and O-glycosylation sites, was compared with other myeloperoxidases. As expected, turbot MPO was more similar to MPO from other fish species (67-86% identity), where the phylogenetic tree obtained agrees with the taxonomic hierarchy, than to MPO from mammals (55-57% identity) and other groups. The results obtained in the present study will also allow functional studies to be carried out with turbot neutrophil MPO enzyme, as well as analysis of MPO gene expression under different stimuli.
Assuntos
Linguados/genética , Peroxidase/genética , Peroxidase/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neutrófilos/enzimologia , Peptídeos/química , Peptídeos/genética , Peroxidase/química , Peroxidases/metabolismo , Filogenia , Polissacarídeos/metabolismo , Alga Marinha/química , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Reporter constructs of three interferon (IFN)-gamma-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included gammaIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The gammaIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-gamma stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-gamma and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-gamma, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-gamma reporter stable cell line.
Assuntos
Interferon gama/farmacologia , Oncorhynchus mykiss/genética , Regiões Promotoras Genéticas/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Quimiocina CXCL10/genética , Cisteína Endopeptidases/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Luciferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Elementos de Resposta/genética , Deleção de Sequência , Fatores de TempoRESUMO
Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fish. 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 capable of inactivating the superoxide anion (â¢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) 34-44 kDa and with very different 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 50 kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60 kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when â¢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.
Assuntos
Mitocôndrias/metabolismo , Oligoimenóforos/enzimologia , Oligoimenóforos/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/química , Sequência de Aminoácidos/genética , Animais , Doenças dos Peixes/parasitologia , Linguados/parasitologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Camundongos , Camundongos Endogâmicos ICR , Oligoimenóforos/genética , Estresse Oxidativo/fisiologia , Resveratrol/toxicidade , Azida Sódica/toxicidade , Superóxido Dismutase/genética , Superóxido Dismutase/isolamento & purificação , Raios UltravioletaRESUMO
Water-soluble acidic polysaccharides from the cell walls of Ulva rigida are mainly composed of disaccharides that contain glucuronic acid and sulphated rhamnose. The structure of disaccharides resembles that of glycosaminoglycans (GAGs) as they both contain glucuronic acid and sulphated sugars. Glycosaminoglycans occur in the extracellular matrix of animal connective tissues but can also be produced by leucocytes at inflammatory sites. Certain types of GAGs can even activate macrophages and therefore the acidic polysaccharides from U. rigida probably modulate macrophage activity. In the present study, we evaluated the effects of U. rigida polysaccharides on several RAW264.7 murine macrophage activities, including expression of inflammatory cytokines and receptors, nitric oxide and prostaglandin E2 (PGE(2)) production, and nitric oxide synthase 2 (NOS-2) and cyclooxygenase-2 (COX-2) gene expression. U. rigida acidic polysaccharides induced a more than two-fold increase in the expression of several chemokines (chemokine (C motif) ligand 1, chemokine (C-X-C motif) ligand 12, chemokine (C-C motif) ligand 22 and chemokine (C-X-C motif) ligand 14 (Cxcl14)) and in the expression of IL6 signal transducer and IL12 receptor beta 1. Incubation of macrophages with U. rigida polysaccharides also induced an increase in nitrite production, although this effect decreased considerably after desulphation of polysaccharides, suggesting that the sulphate group is important for the stimulatory capacity of these molecules. U. rigida polysaccharides also stimulated macrophage secretion of PGE(2) and induced an increase in COX-2 and NOS-2 expression. The results indicate that U. rigida acid polysaccharide can be used as an experimental immunostimulant for analysing inflammatory responses related to macrophage functions. In addition, these polysaccharides may also be of clinical interest for modifying certain macrophage activities in diseases where macrophage function is impaired or needs to be boosted.
Assuntos
Regulação da Expressão Gênica/imunologia , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Ulva/química , Animais , Linhagem Celular , Quimiocinas/biossíntese , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Fatores Imunológicos/química , Inflamação/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Polissacarídeos/química , Ácidos Sulfúricos/químicaRESUMO
The scuticociliatosis produced by the endoparasite Philasterides dicentarchi is a severe parasitic infection of farmed turbot (Scophthalmus maximus) characterized by several histopathological effects including extensive inflammation. Indomethacin is a nonsteroidal anti-inflammatory drug that specifically inhibits synthesis of the proinflammatory mediator prostaglandins. The effect of indomethacin on the in vitro growth of P. dicentrarchi was investigated. In vitro growth of the scuticociliate was significantly inhibited by treatment with 100 microM indomethacin for 48 h. Higher concentrations of indomethacin (mM levels) did not affect the gelatinolytic activity of the cysteine proteinases of P. dicentrarchi. In vitro treatment with 25, 50 or 100 microM indomethacin for 3 days did not significantly affect the enzymatic activity of cysteine proteinases, as assayed with p-nitroanilide as substrate. Immunoblot analysis with anti-cysteine proteinase antibodies revealed an increase in proteinase expression (molecular weights of 80, 32 and 40-45 kDa) in parasite lysates originating from in vitro cultures incubated with 25 microM indomethacin for 72 h. Degradation of genomic DNA of the ciliates was observed in cultures incubated with 100 microM indomethacin for 1, 3 and 7 days. The results suggest that indomethacin is capable of inhibiting in vitro growth of the scuticociliate P. dicentrarchi by a mechanism related to the induction of programmed cell death, without affecting the enzymatic activation of parasite proteinases, which demonstrates the potential therapeutic use of this drug in the control of turbot scuticociliatosis.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Infecções por Cilióforos/veterinária , Doenças dos Peixes/parasitologia , Linguados/parasitologia , Indometacina/farmacologia , Oligoimenóforos/efeitos dos fármacos , Animais , Anticorpos Antiprotozoários/metabolismo , Infecções por Cilióforos/parasitologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA/efeitos dos fármacos , DNA de Protozoário/análise , Pesqueiros , Hidrólise/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Oligoimenóforos/enzimologia , Oligoimenóforos/crescimento & desenvolvimentoRESUMO
We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, FreundÌs complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination.