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

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.

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

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 (•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 •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.

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

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.

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

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.

Presence of a plant-like proton-translocating pyrophosphatase in a scuticociliate parasite and its role as a possible drug target.

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.

Alternative oxidase inhibitors as antiparasitic agents against scuticociliatosis.

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.

Evidence of an alternative oxidase pathway for mitochondrial respiration in the scuticociliate Philasterides dicentrarchi.

The presence of an alternative oxidase (AOX) in the mitochondria of the scuticociliate P. dicentrarchi was investigated. The mitochondrial oxygen consumption was measured in the presence of KCN, an inhibitor of cytochrome pathway (CP) respiration and salicylhydroxamic acid (SHAM), a specific inhibitor of alternative pathway (AP) respiration. AOX expression was monitored by western blotting with an AOX polyclonal antibody. The results showed that P. dicentrarchi possesses a branched mitochondrial electron transport chain with both cyanide-sensitive and -insensitive oxygen consumption. Mitochondrial respiration was partially inhibited by cyanide and completely inhibited by the combination of cyanide and SHAM, which is direct evidence for the existence of an AP in this ciliate. SHAM significantly inhibited in vitro growth of trophozoites both under normoxic and hypoxic conditions. AOX is a 42kD monomeric protein inducible by hypoxic conditions in experimental infections and by CP inhibitors such as cyanide and antimycin A, or by AP inhibitors such as SHAM. CP respiration was greatly stimulated during the exponential growth phase, while AP respiration increased during the stationary phase, in which AOX expression is induced. As the host does not possess AOX, and because during infection P. dicentrarchi respires via AP, it may be possible to develop inhibitors targeting the AP as a novel anti-scuticociliate therapy.

Role of scuticociliate proteinases in infection success in turbot, Psetta maxima (L.).

Scuticociliatosis caused by Philasterides dicentrarchi is one of the most severe diseases of farmed turbot, Psetta maxima (L.). Immunized fish showed elevated levels of specific antibodies (Ab), which caused the destruction of parasites through the activation of complement by the alternative and classical pathways. By using affinity chromatography on bacitracin-sepharose columns, we demonstrated the existence of high levels of parasite proteinases in the serum and, to a lesser extent, in the ascitic fluid of experimentally infected fish, and the absence of such proteinases in the serum of uninfected fish. Serum from uninfected fish displayed haemolytic activity against sheep red blood cells. However, incubation of this serum with parasite proteinases led to a decrease in serum haemolytic activity, suggesting that proteinases are able to destroy fish complement. Proteinases isolated from serum or ascitic fluid of infected fish were also able to degrade turbot Ab. Preincubation of turbot serum containing specific Ab for P. dicentrarchi with the proteinases led to a significant decrease in the killing activity of the serum. The results confirm that P. dicentrarchi proteinases in serum from infected fish may provide a mechanism for circumventing normal host immunity by inactivating the Ab and complement factors required for complement activation.

Characterization of acid phosphatases from marine scuticociliate parasites and their activation by host's factors.

Scuticociliates are histophagous marine parasites that cause mortality in fish. Acid phosphatases (AcPs) are considered virulence factors and they are used by different parasites to dephosphorylate host molecules. The aim of this work was to characterize the AcPs from 3 scuticociliate species, Uronema marinum, Miamiensis avidus and Parauronema virginianum, which parasitize marine finfish species. We identified AcP activity (pH 5.2) with differential cellular distribution in the 3 parasite species. Native gel electrophoresis of ciliate lysates revealed the presence of 1 high molecular weight AcP activity band in M. avidus (tartrate-sensitive), several low molecular weight AcPs in U. marinum and 1 low molecular weight band only in P. virginianum (tartrate-resistant). Scuticociliate AcP was inhibited by specific inhibitors of tyrosine protein phosphatases. AcP decreased upon starvation but rapid reactivation occurred following exposure to skin mucus. Groper (Polyprion oxygeneios) peripheral blood leucocytes (PBLs) and, to a lesser extent, red blood cells, also increased AcP activity. Protein tyrosine phosphatase PTP1b was primarily detected in the plasma membrane of M. avidus and ingestion of groper PBLs upregulated its expression. M. avidus recovered from experimentally infected groper had greater levels of PTP1b expression than the injected suspension. The present results highlight the importance of PTPs in histophagous parasites and their interaction with fish host's factors.

Cloning, site-directed mutagenesis and expression of cathepsin L-like cysteine protease from Uronema marinum (Ciliophora: Scuticociliatida).

A cysteine protease gene (ScCtL) homologous to the cathepsin L genes was isolated from a cDNA library of the scuticociliate parasite (Uronema marinum). To express the ScCtL recombinant protein in heterologous system, 17 codons were redesigned to conform to the standard eukaryotic genetic code using PCR-based site-directed mutagenesis. The synthetic U. marinum procathepsin L (proScCtL) was expressed at high levels in E. coli BL21 (DE3) with pGEX-4T-1 vector, and successfully refolded and purified into a functional and enzymatically active form. The optimal pH for protease activity was found to be 4.5. Like any typical cysteine protease, the enzyme was inhibited by E-64 and leupeptin. A dot-blot immunoassay was conducted in an attempt to determine the reaction abilities and sensitivity of the anti-proScCtL polyclonal antibody to the cytosol and to the membrane fraction from the scuticociliate. Our results suggest that the biochemical characteristics of the recombinant ciliate proScCtL protein are similar to that of the cathepsin L-like cysteine protease, and that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form.

Scuticociliate cysteine proteinases modulate turbot leucocyte functions.

The effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes (PL) were investigated. The enzymes were tested at concentrations of 125, 250 and 500 microg ml(-1), and it was found that the viability of the leucocytes was not affected after treatment for 24h. Leucocyte migration was inhibited by the cysteine proteinases in a dose-dependent manner, whereas the ascitic fluid obtained from turbot experimentally infected with P. dicentrarchi induced high chemotactic activity in the turbot PL. The proteinases did not affect yeast cell phagocytosis but increased intracellular production of the superoxide anion (O2(-)). Stimulation with the proteinases did not alter the PGE2 levels in supernatants from 24-h cultures of PL, however, beta-glucans (100 microg ml(-1)) provoked a large increase in PGE2 levels, which were inhibited after addition of 10 microg ml(-1) of indomethacin, a non-selective inhibitor of COX2 enzymatic activity. The mean PGE2 level in ascitic fluid from turbot, experimentally infected with P. dicentrarchi, was 500 pg ml(-1), and the addition of low levels of PGE2 (62.5 pg ml(-1)) to PL cultures stimulated O2(-) production, although addition of PGE2 at concentrations higher than 250 pg ml(-1) blocked the increase in stimulation. Addition of cysteine proteinases to 24-h cultures of PL also increased mRNA levels in the pro-inflammatory cytokine interleukin-1beta. The results revealed the capacity of cysteine proteinases isolated from P. dicentrarchi to modulate the innate immune response of turbot, which together with the inflammation mediators produced during infection, may play an important role in pathogenesis of the disease and in the survival of the parasite.

In vitro activity of the nonsteroidal anti-inflammatory drug indomethacin on a scuticociliate parasite of farmed turbot.

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.

Uronema marinum: identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C.

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus.

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.