Extracellular Proteinases of Miamiensis Avidus Causing Scuticociliatosis Are Potential Virulence Factors

Research article Extracellular Proteinases of Miamiensis avidus Causing Scuticociliatosis are Potential Virulence Factors

Yukie Narasaki1,2†, Yumiko Obayashi2†, Sayami Ito2,3, Shoko Murakami2, Jun-Young Song4, Kei Nakayama1,2,3 and Shin-Ichi Kitamura1,2,3*

1Graduate School of Science and Engineering, Ehime University, Ehime 790-8577, Japan 2Center for Marine Environmental Studies (CMES), Ehime University, Ehime 790-8577, Japan 3Department of Biology, Faculty of Science, Ehime University, Ehime 790-8577, Japan 4Pathology Division, National Fisheries Research and Development Institute, Busan 619-902, Korea

(Received September 12, 2016)

ABSTRACT―Miamiensis avidus is the causative agent of scuticociliatosis in various marine fish species. The virulence factors of the parasite have not been identified, so far. In this study, we examined M. avidus extracellular proteinases (ECPs) as potential virulence factors, using culture supernatants as an ECPs source. We investigated the substrate specificity of ECPs using artificial peptides, and the cytotoxicity of the ECPs was examined using CHSE-214 cells. To elucidate the role of ECPs in ciliate growth, M. avidus was cultured on CHSE-214 cells in the presence of proteinase inhibitors. We detected proteinase activities from the supernatant of M. avidus. Viable CHSE-214 cells decreased significantly in number, when incubated in a medium supplemented with the culture supernatant of M. avidus. The growth of ciliates on CHSE-214 cells was delayed in the presence of PMSF (serine proteinase inhibitor) and E-64 (cysteine proteinase inhibitor). These results suggested that the culture supernatant contained ECPs showing cytotoxicity, and the proteinases facilitated nutrient uptake by the ciliates. Thus, ECPs may be responsible for virulence factors of M. avidus.

Key words: scuticociliatosis, Miamiensis avidus, extracellular proteinase, virulence factor

Scuticociliatosis is one of the most serious diseases Scuticociliatosis has also been observed in in cultured fish species, causing economic losses in Japanese flounder Paralichthys olivaceus, one of the marine aquaculture worldwide. A number of scuticocili- main cultured fish species in Japan and Korea. In addi- ate species have been detected and/or isolated from dis- tion to an unidentified scuticociliate, U. marinum, eased fishes, including Uronema marinum from aquar- Pseudocohnilembus persalinus and Miamiensis avidus ium fish, Philasterides dicentrarchi from sea bass (syn. Philasterides dicentrarchi) have been reported as Dicentrarchus labrax, turbot Scophthalmus maximus, causative agents of the disease in both countries the sea dragons Phycodurus eques and Phyllopteryx (Yoshinaga and Nakazoe, 1993; Jee et al., 2001; Kim taeniolatus (Cheung et al., 1980; Dragesco et al., 1995; et al., 2004a, 2004b; Jung et al., 2005). However, we Iglesias et al., 2001; Rossteuscher et al., 2008). Addi- demonstrated that M. avidus is the primary etiologic tionally, U. nigricans, Tetrahymena corlissi and agent of the disease based on the results of experimen- Anophyroides haemophilia were reported in southern tal infections with four scuticociliate species: U. marinum, bluefin tuna Thunnus maccoyii, guppy Poecilia reticulata P. persalinus, P. hargisi and M. avidus (Song et al., and American lobster Homarus americanus, respectively 2009a). Among M. avidus isolates, we identified three (Cawthorn et al., 1996; Munday et al., 1997; Imai et al., serotypes based on the results of immobilization assays 2000). The ciliates are endoparasites that generate a and western blotting: serotype I for the IyoI, JF05To, systemic infection, invading internal organs such as the RF05To and SK05Kyo isolates; serotype II for the brain, liver, intestine and other tissues (Jung et al., Nakajima isolate; and serotype III for the Mie0301 iso- 2007). The typical symptoms of scuticociliatosis are late (Song et al., 2009b). These serotypes corre- bleached spots, ulceration and hemorrhages on the skin. sponded to the cox1-genotypes of the strains but did not reflect their geographic origins, host species, or pathogenicity (Jung et al., 2011). Recently, it is revealed that † These authors are equally contributed in this study. * Corresponding author the serotype-specific polypeptides of three serotypes E-mail: [email protected] were ciliary membrane proteins (Motokawa et al., 2018). 2 Y. Narasaki, Y. Obayashi, S. Ito, S. Murakami, J.-Y. Song, K. Nakayama and S.-I. Kitamura

Regarding the outbreak of the disease, Takagishi et al. mentioned ciliate isolates. (2009) reported that lower-salinity conditions increased mortalities in challenged fish by experimental infection. MCA substrate assay As indicated above, the morphological, taxonomical, For MCA substrate assays, Escherichia coli JM109 and genetical characteristics of scuticociliates have been cells were employed as feed for the ciliates to avoid pro- extensively investigated, but the virulence factors of the teinase contamination from FBS. The bacterium was ciliates have yet to be conclusively identified. Gener- cultured for 12 h at 37°C in Luria-Bertani broth medium ally, proteinases are thought to play key roles in the supplemented with 1% glucose. The cultured bacte- infection process of protozoan parasites such as rium (6.6 × 108 colony forming units [CFU]/mL) was then Tritrichomonas foetus, Tetrahymena spp. and autoclaved, washed twice by centrifugation at 1,800 × g Leishmania spp. (Leibowitz et al., 2009; Olivier et al., for 10 min, and finally resuspended in MEM unsupple- 2012; Tolbert et al., 2014). In M. avidus isolated from mented with FBS (abbreviated as MEM-N hereafter). turbot in Spain, which was originally described as Ciliates cultured on CHSE-214 cells were centrifuged at Philasterides dicentrarchi and later synonymized to M. 1,000 × g for 5 min and then washed with MEM-N three avidus, intracellular proteinases and extracellular pro- times to remove the FBS. For each isolate, the ciliates teinases (ECPs) were also detected by Paramá et al. were resuspended in MEM-N to a density of 3.0 × 104 (2004). They reported that the intracellular proteinases ciliates/mL. A total of 3.0 × 105 ciliates and 1.0 × 1010 and ECPs of M. avidus were almost similar to each CFU of autoclaved E. coli were cultured with 15 mL of other in terms of their electrophoretic profiles in gelatin- MEM-N in 75-cm2 culture flasks at 20°C. After 6 days, SDS-PAGE. Based on the similarity between intracel- the culture medium was centrifuged at 430 × g for 5 min, lular and extracellular proteinases, they carried out the and the culture supernatant of M. avidus (abbreviated as study with the assumption that intracellular proteinases M. avidus-SUP hereafter) was collected. W e confirmed and ECPs were identical. For example, Paramá et al. that this centrifugation did not crush the ciliate cells. In (2007a, 2007b) demonstrated chemotaxis inhibition and order to completely remove the ciliates, this step was apoptosis induction by the proteinases in turbot pro- repeated. Finally, the M. avidus-SUP was centrifuged nephric leukocytes, using purified proteinases from at 2,330 × g for 5 min to remove E. coli cell debris. The homogenates of the ciliate (intracellular proteinases). M. avidus-SUPs were diluted with MEM-N in order to However, it seemed that a slight difference was present equalize the proteinase concentration based on the between the electrophoretical profiles in substrate-SDS- number of ciliates of each isolate and then used for the PAGE between the intracellular proteinases and ECPs MCA substrate assay. (Paramá et al., 2004), and the detailed substrate speci- The proteolytic activity of the M. avidus-SUP was ficity of ECPs has been unclear. Moreover, cytotoxicity assayed using 19 types of peptide analogue MCA sub- mediated by ECPs has not been directly demonstrated. strates (Peptide Institute) (Table 1). Each substrate In this study, therefore, we investigated the substrate was dissolved in dimethyl sulfoxide (DMSO) prior to use. specificity of ECPs derived from M. avidus using a pep- All substrates were prepared as 1 mM stock solutions in tide analogue 4-methylcoumaryl-7-amide (MCA) sub- DMSO. Incubation tubes were filled with 100-μL ali- strate assay and assessed the cytotoxicity of the ECPs quots of the substrate stock solution and stored at –20°C to Chinook salmon embryo cells (CHSE-214). More- until use. The assay was conducted as reported previ- over, in order to understand the role of the ECPs in cili- ously (Obayashi and Suzuki, 2005, 2008). For assays, ate growth, M. avidus was cultured on CHSE-214 cells 900 μL of the M. avidus-SUP was added to each tube to in a medium supplemented with proteinase inhibitors. yield a final substrate concentration of 100 μM; the concentration was previously confirmed as the saturation level for Z-Phe-Arg-MCA using a proteinase sample Materials and Methods from the Nakajima isolate (data not shown). Of the 19 Parasites substrates examined using the MCA assay, the superna- Three different serotypes of M. avidus (IyoI, tant of the Nakajima isolate exhibited the highest proteo- Nakajima and Mie0301) isolated from Japanese flounder lytic activity against this substrate. Sample and sub- during scuticociliatosis outbreaks at different farms in strate mixtures were incubated at 20°C for 2 h, before Japan (Song et al., 2009b) were used in this study. and after which the fluorescence of the hydrolytic product, 7-amino-4-methylcoumarin (AMC), was measured Cell culture using a spectrofluorometer (F-2500, Hitachi) at excita- CHSE-214 cells were maintained at 20°C in Eagle’s tion/emission wavelengths of 380/460 nm. After sub- minimum essential medium (MEM, Nissui Pharmaceuti- tracting the blank fluorescence intensity of each protein- cal) supplemented with 10% fetal bovine serum (abbre- ase sample (measured without substrate), the viated as MEM-FBS hereafter) in 25-cm2 culture flasks. concentration of AMC in the incubation tube was calcu- The CHSE-214 cell line was used to maintain the above- lated using a calibration curve obtained from several Extracellular proteinases of Miamiensis avidus 3

Table 1. MCA substrates used in this study Name Substrate for * Molecular weight Arg-MCA Aminopeptidase (Cathepsin H) 331.37 Leu-MCA Aminopeptidase 288.35 Ala-MCA Aminopeptidase 246.27 Lys-MCA Aminopeptidase 303.36 Phe-MCA Aminopeptidase 322.36 Bz-Arg-MCA Trypsin 435.48 Z-Phe-Arg-MCA Cathepsin B/L, Plasma kallikrein, Arg-Gingipain 612.69 Glt-Gly-Arg-MCA Urokinase 502.53 Boc-Leu-Gly-Arg-MCA Horseshoe Crab Clotting Enzyme 601.70 Boc-Leu-Thr-Arg-MCA Factor VIIa-Tf 645.76 Boc-Phe-Ser-Arg-MCA Tryptase, 73K Protease, Arg-Gingipain 665.75 Boc-Val-Pro-Arg-MCA α-Trombin 627.74 Boc-Leu-Ser-Thr-Arg-MCA Activated Protein C 732.83 Boc-Val-Leu-Lys-MCA Plasmin, Calpain 615.77 Boc-Glu-Lys-Lys-MCA Plasmin 660.77 Suc-Ala-Ala-Pro-Phe-MCA Chymotrypsin 661.71 Suc-Leu-Leu-Val-Tyr-MCA Ingensin/Proteasome, Calpain 763.89 Suc-Ala-Ala-Ala-MCA Elastase 488.50 Suc-Ala-Pro-Ala-MCA Elastase 514.54 * according to product information from manufacturer (Peptide Institute) concentrations of standard AMC. For the control, the cell densities of CHSE-214 cells (3.1 × 105, 4.9 × 105 supernatant of a culture without M. avidus was assayed and 1.6 × 106 cells). The data are expressed as rela- using the same method described above and used to tive values to initial (pre-treated) cell numbers. Results determine the level of non-enzymatically produced AMC. were analyzed using Two-way ANOVA followed by the The substrate hydrolysis rates were estimated from the Tukey-Kramer multiple comparison tests, and differ- change in the concentration of AMC in the incubation ences were considered significant at p < 0.05. tube after the concentration of non-enzymatically produced AMC was subtracted. Growth of M. avidus on CHSE-214 cells with proteinase inhibitor Cytotoxicity of the culture supernatant of M. avidus In order to examine the importance of the ECPs in Miamiensis avidus Iyo I isolate was cultured on breaking food down into nutrients utilizable for ciliate CHSE-214 cells. The M. avidus-SUP collected as growth, we cultured the ciliates on CHSE-214 cells in described above. An aliquot of the M. avidus-SUP was media with or without proteinase inhibitors. Miamiensis heated for 5 min to prepare heat-killed control of M. avidus Iyo I cells were cultured, washed, and resus- avidus-SUP. As a control, the cytoplasm solution of pended in MEM-FBS to a density of 1.0 × 104 cells/mL. CHSE-214 cells were also collected by sonication for 3 A proteinase inhibitor cocktail containing aprotinin, min in MEM-N on ice using a Sonifier 250 (Branson) bestatin, E-64, leupeptin and pepstatin A (Sigma-Aldrich) operated at 30% duty cycle and output 3. The samples was diluted 300- or 900-fold with DMSO as recom- were concentrated by 15 times using Amicon Ultra-10K mended by the manufacturer. Next, 50 μL of ciliate (Millipore). suspension (2.0 × 102 cells) and inhibitor was added to To test the cytotoxicity of M. avidus-SUP, CHSE- CHSE-214 cells cultured in MEM-FBS in a 25-cm2 flask. 214 cells were placed in a 24-well plate and cultured at As controls, the same volume of MEM-N or DMSO was 20°C in four different conditions: culture in MEM-FBS added to the cells. At 30 h after inoculation, the same (no treatment control) and cultures in MEM-FBS supple- volume of proteinase inhibitor was added again. The mented (100 μL/well) with cytoplasm solution of CHSE- ciliates were cultured until 114 h, with the number of cili- 214, with M. avidus-SUP and with heated-M. avidus- ates determined using a OneCell Counter at 54, 66, 78, SUP. After 12 h incubation, the culture supernatant 90 and 114 h. Experiments were carried out in tripli- was gently removed and the cells were treated with tryp- cate. Data are given as the mean ± S.D. sin for 1 min. The cells in each well were then sus- To reveal proteinase types which contribute to the pended with 1 mL of MEM-N and enumerated using a digestion of CHSE-214 cells, five individual proteinase OneCell Counter (Bio-Medical Science). The experi- inhibitors were employed in inhibitor assay: phenylmeth- ments were carried out in triplicate using different initial ane sulfonyl fluoride (PMSF; serine proteinases inhibitor, 4 Y. Narasaki, Y. Obayashi, S. Ito, S. Murakami, J.-Y. Song, K. Nakayama and S.-I. Kitamura

1 mM final conc.), leupeptin (cysteine and serine protein- Z-Phe-Arg-MCA (substrate for cathepsin B/L, plasma ases inhibitor, 20 μM final conc.), E-64 (cysteine protein- kallikrein and Arg-Gingipain). The substrates Boc-Glu- ases inhibitor, 50 μM final conc.), pepstatin A (aspartic Lys-Lys-MCA and Boc-Val-Leu-Lys-MCA for plasmin proteinases inhibitor, 10 μM final conc.) and EDTA were also well hydrolyzed by the M. avidus-SUPs. (metallo-proteinases inhibitor, 1 mM final conc.). These were purchased from Sigma-Aldrich. Each proteinase Cytotoxicity of the culture supernatant of M. avidus inhibitor was dissolved with distilled water or ethanol as At 12 h post-treatment, the cell numbers in the no recommended by the manufacturer. Experiments were treatment control, and treatments with cytoplasm solu- carried out by the same procedure as above. tion of CHSE-214, M. avidus-SUP and heated-M. Prior to analysis, the cell number was logarithmi- avidus-SUP groups were 78%, 78%, 62% and 78% cally transformed. Significant differences in the number compared with pretreatment cell numbers, respectively between each inhibitor treatment and respective solvent (Fig. 2). The number of viable cells in the M. avidus- control were tested by Mann-Whitney U test. SUP treatment group was significantly lower than those in the control and the other treatments.

Results Effect of proteinase inhibitor on M. avidus cultured on Substrate specificity of the culture supernatant of M. CHSE-214 cells avidus At 30 h post-infection (hpi), CHSE-214 cells were The results of MCA substrate assays are shown in lysed by histophagous M. avidus in the control and sol- Fig. 1. Several substrates were hydrolyzed by the M. vent-control groups (Fig. 3A and B), whereas cell lysis avidus-SUP, and the enzymatic hydrolysis pattern was was inhibited in the presence of proteinase inhibitor similar among the three different serotypes of M. avidus cocktail (Fig. 3C and D). Most cells were lysed by the described in our previous study (Song et al., 2009b). ciliates at 66 hpi in all groups (data not shown). Among the tested substrates, 5 substrates, namely Ciliate growth was monitored until 114 hpi. At 54 Z-Phe-Arg-MCA, Boc-Leu-Thr-Arg-MCA, Boc-Phe-Ser- hpi, the density of ciliates in the negative control, solvent Arg-MCA, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys- control and low-dose proteinase inhibitor cocktail groups MCA, were readily hydrolyzed by these three M. avidus- was 31.5 × 104, 23.8 ×104 and 19.9 ×104 cells/mL, SUPs, whereas low or no hydrolytic activity was detected respectively (Fig. 4). At this time point, the ciliate den- for substrates of aminopeptidase and elastase. The sity in the high-dose proteinase inhibitor cocktail group highest proteinase activity was detected against could not be determined because of the low number of

Enzymatic hydrolysis (nmol/L/h)

0 200 400 600 800 1000 1200 1400 1600 1800 Arg-MCA Leu-MCA Aminopeptidase Ala-MCA Lys-MCA Phe-MCA Bz-Arg-MCA Z-Phe-Arg-MCA Glt-Gly-Arg-MCA Boc-Leu-Gly-Arg-MCA Boc-Leu-Thr-Arg-MCA Boc-Phe-Ser-Arg-MCA Boc-Val-Pro-Arg-MCA Boc-Leu-Ser-Thr-Arg-MCA Boc-Val-Leu-Lys-MCA Boc-Glu-Lys-Lys-MCA Suc-Ala-Ala-Pro-Phe-MCA Suc-Leu-Leu-Val-Tyr-MCA Suc-Ala-Ala-Ala-MCA Elastase Suc-Ala-Pro-Ala-MCA

Fig. 1. Hydrolysis pattern of substrates by the culture supernatant of three serotypes of M. avidus. □; Iyo I isolate, ■; Nakajima isolate, ■Fig.; Mie 01.30 Hydrolysis1 isolate. Err opatternr bars sh oofw ssubstratestandard devi abytion the of tr icultureplicates. supernatant of three serotypes of M. avidus. □; Iyo I isolate, ; Nakajima isolate, ■; Mie0401 isolate. Error bars show standard deviation of triplicates.

17 Extracellular proteinases of Miamiensis avidus 5

100

80 *

20 Relative cell numbers compared with that of pretreatment with that of pretreatment (%) compared cell numbers Relative 0 No-treatment CHSE-214 M. avidus- Heated-M. avidus control cytoplasm SUP SUP Fig. 2. Percent change of ceFig.ll nu 2.m bPercenters at change12 h in of c ocellntr onumbersl (no tr eata t12m ehoursnt ), incy controltoplasm (no so treatmentlution of ),C cytoplasmHSE-214 cells, culture supernatant of M. avidus and heatsolutioned-cult uofre CHSE-214 supernat acells,nt o fculture M. avidus supernatant treatm ofen M.t. avidus The c andell n heated-cultureumbers were supernatant compared with that of pretreatment cells. Asterisk ofin dM.ic aavidustes a streatment.igniﬁcan tThe dif fcellere nnumbersce (p < were0.05 )compared in multip withle c othatmp aofr ipretreatson tesmentt. cells. Asterisk indicates a significant difference (p < 0.05) in multiple comparison test.

A B

C D

Fig. 3. CHSE-214 cells at 30 h post-infection with M. avidus Iyo I isolate. A: negative control, B: solvent (DMSO) control, C: low doseFig. of p r3.ot eCHSE-214inase inhibito rcells, D: h iatgh 30dos hourse of pro postteina sinfectione inhibitor. with The rM.e a reavidus many cIyoiliate Is isolate.on the ce A:lls. Lysed cell spots are shownegativen by dot c icontrol,rcle. Sc aB:le bsolventar = 100 (DMSO)μm. control, C: low dose of protease inhibitor, D: high dose of protease inhibitor. There are many ciliates on the cells. Lysed cell spot are ciliates. Thshowne max ibymu mdot d ecircle.nsity i nScale the n barega t=iv 100e co nμm.trol, proteinase inhibitor cocktail at 78, 90 and 114 hpi com- 19 solvent control and low- and high-dose inhibitor-treated pared to the solvent control. groups was 49.6 × 104 cells/mL at 114 hpi, 60.4 × 104 In assays using each individual inhibitor, the mean cells/mL at 66 hpi, 42.8 × 104 cells/mL at 66 hpi and cell numbers at 72 hpi in PMSF, leupeptin and E-64 35.1 × 104 cells/mL 114 hpi, respectively. The numbers were (4.2 ± 1.2) × 103, (6.7 ± 2.4) × 103 and (4.2 ± 1.2) × of ciliates were significantly lower with high dose of 103 cells/mL, respectively, whereas that in their solvent 6 Y. Narasaki, Y. Obayashi, S. Ito, S. Murakami, J.-Y. Song, K. Nakayama and S.-I. Kitamura

Fig. 4. Growth curve of M. avidus cultured on CHSE-214 cells with proteinase inhibitor cocktail. The number of ciliates could not be counted due to small number of the cells until 54 h (66 h for high-dose inhibitor ﬂask). Asterisk indicates signiﬁcant differences (p < 0.05) to solvent control.

12 cells/mL) cells/mL) 3

10 10 ×

8 * * 6

4 Number ciliates the Number of ( 2

Fig. 5. The numberFig. of M. 5. avidus The number cultured ofon M.CH SavidusE-214 culturedcells with oonr w CHSE-214ithout protei ncellsase inwithhibit oorrs .without The fin proteinaseal concentra tion of solvent ethanolinhibitors. in PMSF, l eTheupe pfinaltin, a nconcentrationd E-64 was 0.1 of% ,solvent wherea sethanol those w inere PMSF, 1% and leupeptin, 0% in pep sandtatin E-64 A an dwas ED TA, respectively. Asterisk indicates significant differences (p < 0.05) to the control. 0.1%, whereas those were 1% and 0% in pepstatin A and EDTA, respectively. Asterisk indicates significant differences (p < 0.05) to the control. control (0.1% ethanol) was (12 ± 2.4) × 103 cells/mL the cell numbers of M. avidus in PMSF (serine protein- (Fig. 5). The numbers in pepstatin A and its control ases inhibitor) and E-64 inhibitor (cysteine proteinases (1% ethanol) were (4.2 ± 1.2) × 103 and (3.3 ± 1.2) × 103 inhibitor) treatments were significantly lower (p < 0.05) at cells/mL, while those in EDTA and its control (0% etha- 72 hpi. The number of M. avidus in leupeptin (cysteine nol) were (4.0 ± 2.5) × 103 and (8.3 ± 2.4) × 103 cells/mL, and serine proteinases inhibitor) was slightly lower than respectively. Comparing to respective solvent controls, that in control, although the difference was not Extracellular proteinases of Miamiensis avidus 7 statistically significant (p = 0.10). Later than 72 hpi, the As M. avidus causes a systemic infection (Jung et al., growth inhibition effect was not observed in all treat- 2007; Moustafa et al., 2010), the cathepsin-like ments. proteinase(s) detected in this study might play roles in the degradation of host tissues and evasion of the host immune response. Discussion We also examined whether M. avidus-SUP was Until now, ECPs have been detected from free-liv- cytotoxic, using CHSE-214 cells as model host fish cells. ing ciliates such as Paranophrys marina, Tetrahymena Our results showed that the proportion of intact CHSE- pyriformis and Paramecium tetraurelia, and were 214 cells treated with M. avidus-SUP decreased signifi- thought to be involved in digestion of nutrients in aquatic cantly, in comparison with untreated control cells and environments (Völkel et al., 1996; Suzuki et al., 1998; cells treated with heated-M. avidus-SUP (Fig. 2). The Thao et al., 2005). ECPs have also been observed in cytotoxicity and proteolytic activities in the M. avidus- free-living ciliate M. avidus (Paramá et al., 2004). SUP suggested ECPs were responsible for cell lysis. Since the ciliate is also well known as an opportunistic To our knowledge, this is the first report demonstrating pathogen, ECPs in particular may be required for host direct cytotoxic effects of ciliate ECPs on live cells, invasion, evading host immunity and degrading host pro- although histolytic activity of intracellular proteinases iso- teins. However, the characters and function of ECPs in lated from M. avidus has been reported so far (Paramá M. avidus have yet to be fully elucidated. In the pres- et al., 2004). In order to investigate the role of the ent study, using an MCA assay, we confirmed that M. ECPs during natural infection, we then cultured ciliates avidus produced various ECPs. The profile of ECP on CHSE-214 cells with and without proteinase inhibi- activity was very similar among the three serotypes of tors. As a result, with the proteinase inhibitor cocktail, M. avidus (Fig. 1). PMSF (serine proteinase inhibitor) or E-64 (cysteine pro- The highest proteinase activity was detected against teinase inhibitor), lysis of CHSE-214 cells and growth of Z-Phe-Arg-MCA, which is a substrate for cathepsin B/L, ciliates were significantly inhibited (Figs. 4 and 5). With plasma kallikrein and Arg-Gingipain. Similar proteoly- leupeptin (cysteine and serine proteinases inhibitor), sis of substrates for cathepsin B was reported for the growth of ciliates seemed to be inhibited, however, the intracellular proteinases derived from Spanish isolates of difference was not statistically significant. The discrep- M. avidus, and the activity was inhibited by E-64 and Ac- ancy between the result with leupeptin and those with Leu-Val-Lys aldehyde, potent inhibitors of cysteine pro- PMSF and E-64 might be due to their mechanism of inhi- teinases of the cathepsin B family (Paramá et al., 2004). bition; PMSF and E-64 inhibit cysteine/serine protein- It was assumed that the proteinases were secreted, ases by binding directly to their active site, whereas leu- based on the similar proteinase activity profiles between peptin is a competitive transition state inhibitor and the intracellular and extracellular proteinases as determined inhibition is not effective with excess substrates. In by substrate analysis using SDS-PAGE (Paramá et al., these growth experiments with proteinase inhibitors, it 2004). In the present study, we also found the inhibi- remained a possibility that the inhibition of growth of M. tion effect of E-64 proteinase inhibitor on growth of M. avidus resulted from inhibition of intracellular protein- avidus fed CHSE-214 cells, which was consistent with ases which were required to their cell growth or cell divi- the report by Paramá et al. (2004). In addition, the sion. However, it was more likely that inhibition of gene encoding a cathepsin L-like proteinase in M. ECPs caused growth inhibition of M. avidus, considering avidus was recently identified and characterized (Shin et the consistent results of MCA assay (Fig. 1) and cytotox- al., 2014). Together with the results of MCA substrate icity (Fig. 2) of the M. avidus-SUP. All of these results assays and inhibition assay in the present study, these suggest that the cysteine and serine-proteinase in ECPs data suggest that M. avidus produces and secretes are necessary for lysis of host cells and nutrient uptake cathepsin B- and/or cathepsin L-like proteinases as by M. avidus, indicating that the ECPs are ciliate viru- ECPs. Cathepsin B/L are classified as cysteine pro- lence factors. teinases, and their function as virulence factors in Two of the highly hydrolyzed substrates by M. numerous parasites was reviewed by Sajid and avidus-SUP in our experiment (Fig. 1 and Table 1), Boc- McKerrow (2002). The proteinases degrade host pro- Glu-Lys-Lys-MCA and Boc-Val-Leu-Lys-MCA, are sub- teins such as hemoglobin, immunoglobulin and fibronec- strates for plasmin, a proteinase known to degrade tin (Sajid and McKerrow, 2002; Ruszczyk et al., 2008; thrombus for vascular patency (Castellino and Ploplis, Sako et al., 2011). The degradation of host proteins 2005). As a part of the general host immune response, such as immunoglobulins and complement components the blood clotting system is initiated to trap pathogens and the induction of apoptosis in leukocytes was also invading the blood vessels. As M. avidus causes a reported for proteinases derived from M. avidus homog- systemic infection via the circulation (Jung et al., 2005; enates, although the causative proteinase(s) was not Moustafa et al., 2010), blood clots could serve as obsta- identified (Paramá et al., 2007a; Piazzon et al., 2011). cles to the spread of infection. The proteinases 8 Y. Narasaki, Y. Obayashi, S. Ito, S. Murakami, J.-Y. Song, K. Nakayama and S.-I. Kitamura detected in the present study appear to play an impor- Jung, S. J., S. I. Kitamura, J. Y. Song, I. Y. Joung and M. J. Oh tant role in evading these obstacles via degradation of (2005): Complete small subunit rRNA gene sequence of the scuticociliate Miamiensis avidus pathogenic to olive thrombi. Although no reports have described plasmin flounder Paralichthys olivaceus. Dis. Aquat. Organ., 64, in scuticociliates, further investigation of these protein- 159–162. ases is worthwhile in order to clarify the details of the Jung, S. J., S. I. Kitamura, J. Y. Song and M. J. Oh (2007): infection process of M. avidus. Miamiensis avidus (Ciliophora: Scuticociliatida) causes In conclusion, we detected similar extracellular pro- systemic infection of olive flounder Paralichthys olivaceus and is a senior synonym of Philasterides dicentrarchi. teinase activities in culture supernatants of three differ- Dis. Aquat. Organ., 73, 227–234. ent serotypes of M. avidus. These M. avidus-SUPs Jung, S. J., E. Y. Im, M. C. Strüder-Kypke, S. I. Kitamura and P. readily hydrolyzed substrates for cathepsin B/L and plas- T. K. Woo (2011): Small subunit ribosomal RNA and mito- min in particular, suggesting that ECPs of M. avidus chondrial cytochrome c oxidase subunit 1 gene sequences might play important roles in the degradation of host pro- of 21 strains of the parasitic scuticociliate Miamiensis avidus (Ciliophora, Scuticociliatia). Parasitol. Res., 108, teins and the destruction of fibrin clots to facilitate inva- 1153–1161. sion via the blood vessels. The M. avidus-SUPs were Kim, S. M., J. B. Cho, E. H. Lee, S. R. Kwon, S. K. Kim, Y. K. also cytotoxic in vitro and ECPs contained in the super- Nam and K. H. Kim (2004a): Pseudocohnilembus persali- natant were likely involved in the cytotoxicity. The nus (Ciliophora: Scuticociitida) is an additional species growth of ciliates on CHSE-214 cells was delayed in the causing scuticociliatosis in olive flounder Paralichthys olivaceus. Dis. Aquat. Organ., 62, 239–244. presence of proteinase inhibitor cocktail, PMSF and Kim, S. M., J. B. Cho, S. K. Kim, Y. K. Nam and K. H. Kim E-64, suggesting that the serine and cysteine-protein- (2004b): Occurrence of scuticociliatosis in olive flounder ases facilitated nutrient uptake by the ciliates. Our find- Paralichthys olivaceus by Phiasterides dicentrarchi (Cili- ings thus suggest that the ECPs we described are viru- ophora: Scuticociliatida). Dis. Aquat. Organ., 62, 233– lence factors of M. avidus. 238. Leibowitz, M. P., R. Ofir, A. Golan-Goldhirsh and D. Zilberg (2009): Cysteine proteases and acid phosphatases con- Acknowledgements tribute to Tetrahymena spp. pathogenicity in guppies, Poe- cilia reticulata. Vet. Parasitol., 166, 21–26. This study was partially supported by a grant from Motokawa, S., Y. Narasaki, J. Y. Song, Y. Yokoyama, E. Matsuoka Research Institute for Science. Hirose, S. Murakami, S. J. Jung, M. J. Oh, K. Nakayama and S. I. Kitamura (2018): Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miam- References iensis avidus. Parasitol. Int., 67, 196–202. Moustafa, E. M. M., N. Tange, A. Shimada and T. Morita Castellino, F. J. and V. A. Ploplis (2005): Structure and function (2010): Experimental scuticociliatosis in Japanese floun- of the plasminogen/plasmin system. Thromb. Haemost., der (Paralichthys olivaceus) infected with Miamiensis avi- 93, 647–654. dus: pathological study on the possible neural routes of Cawthorn, R. J., D. H. Lynn, B. Despres, R. MacMillan, R. invasion and dissemination of the scuticociliate inside the Maloney, M. Loughlin and R. Bayer (1996): Description of fish body. J. Vet. Med. Sci., 72, 1557–1563. Anophyroides haemophila n. sp. (Scuticociliatida: Orchi- Munday, B. L., P. J. O’Donoghue, M. Watts, K. Rough and T. tophryidae), a pathogen of American lobsters Homarus Hawkesford (1997): Fatal encephalitis due to the scutico- americanus. Dis. Aquat. Organ., 24, 143–148. ciliate Uronema nigricans in sea-caged, southern bluefin Cheung, P. J., R. F. Nigrelli and G. D. Ruggieri (1980): Studies tuna Thunnus maccoyii. Dis. Aquat. Organ., 30, 17–25. on the morphology of Uronema marinum Dujardin (Cili- Obayashi, Y. and S. Suzuki (2005): Proteolytic enzymes in atea: Uronematidae) with a description of the histopathol- coastal surface seawater: Significant activity of endopepti- ogy of the infection in marine fishes. J. Fish Dis., 3, 295– dases and exopeptidases. Limnol. Oceanogr., 50, 722– 303. 726. Dragesco, A., J. Dragesco, F. Coste, C. Gasc, B. Romestand, Obayashi, Y. and S. Suzuki (2008): Occurrence of exo- and J. C. Raymond and G. Bouix (1995): Philasterides dicen- endopeptidases in dissolved and particulate fractions of trarchi, n. sp., (Ciliophora, Scuticociliatida), a histopha- coastal seawater. Aquat. Microb. Ecol., 50, 231–237. gous opportunistic parasite of Dicentrarchus labrax Olivier, M., V. D. Atayde, A. Isnard, K. Hassani and M. T. Shio (Linnaeus, 1758), a reared marine fish. Eur. J. Protistol., (2012): Leishmania virulence factors: focus on the metal- 31, 327–340. loprotease GP63. Microbes Infect., 14, 1377–1389. Iglesias, R., A. Paramá, M. F. Alvarez, J. Leiro, J. Fernández Paramá, A., R. Iglesias, M. F. Álvarez, J. Leiro, F. M. Ubeira and M. L. Sanmartín (2001): Philasterides dicentrarchi and M. L. Sanmartín (2004): Cysteine proteinase activities (Ciliophora, Scuticociliatida) as the causative agent of scu- in the fish pathogen Philasterides dicentrarchi (Ciliophora: ticociliatosis in farmed turbot Scophthalmus maximus in Scuticociliatida). Parasitology, 128, 541–548. Galicia (NW Spain). Dis. Aquat. Organ., 46, 47–55. Paramá, A., R. Castro, J. Lamas, M. L. Sanmartín, M. T. Imai, S., S. Tsurimaki, E. Goto, K. Wakita and K. Hatai (2000): Santamarina and J. Leiro (2007a): Scuticociliate protein- Tetrahymena infection in guppy Poecilia reticulata. Fish ases may modulate turbot immune response by inducing Pathol., 35, 67–72. apoptosis in pronephric leucocytes. Int. J. Parasitol., 37, Jee, B. Y., Y. C. Kim and M. S. Park (2001): Morphology and 87–95. biology of parasite responsible for scuticociliatosis of cul- Paramá, A., R. Castro, J. A. Arranz, M. L. Sanmartín, J. Lamas tured olive flounder Paralichthys olivaceus. Dis. Aquat. and J. Leiro (2007b): Scuticociliate cysteine proteinases Organ., 47, 49–55. modulate turbot leucocyte functions. Fish Shellfish Extracellular proteinases of Miamiensis avidus 9

Immunol., 23, 945–956. Song, J. Y., K. Sasaki, T. Okada, M. Sakashita, H. Kawakami, Piazzon, C., J. Lamas and J. M. Leiro (2011): Role of scutico- S. Matsuoka, H. S. Kang, K. Nakayama, S. J. Jung, M. J. ciliate proteinases in infection success in turbot, Psetta Oh and S. I. Kitamura (2009b): Antigenic differences of the maxima (L.). Parasite Immunol., 33, 535–544. scuticociliate Miamiensis avidus from Japan. J. Fish Rossteuscher, S., C. Wenker, T. Jermann, T. Wahli, E. Dis., 32, 1027–1034. Oldenberg and H. Schmidt-Posthaus (2008): Severe scuti- Suzuki, K. M., N. Hayashi, N. Hosoya, T. Takahashi, T. Kosaka cociliate (Philasterides dicentrarchi) infection in a popula- and H. Hosoya (1998): Secretion of tetrain, a Tetrahymena tion of sea dragons (Phycodurus eques and Phyllopteryx cysteine protease, as a mature enzyme and its identifica- taeniolatus). Vet. Pathol., 45, 546–550. tion as a member of the cathepsin L subfamily. Eur. J. Ruszczyk, A., M. Forlenza, M. Joerink, C. M. S. Ribeiro, P. Biochem., 254, 6–13. Jurecka and G. F. Wiegertjes (2008): Trypanoplasma bor- Takagishi, N., T. Yoshinaga and K. Ogawa (2009): Effect of reli cysteine proteinase activities support a conservation of hyposalinity on the infection and pathogenicity of Miamien- function with respect to digestion of host proteins in com- sis avidus causing scuticociliatosis in olive flounder Parali- mon carp. Dev. Comp. Immunol., 32, 1348–1361. chthys olivaceus. Dis. Aquat. Organ., 86, 175–179. Sajid, M. and J. H. McKerrow (2002): Cysteine proteases of Thao, N. V., A. Nozawa, Y. Obayashi, S. I. Kitamura, T. parasitic organisms. Mol. Biochem. Parasitol., 120, Yokokawa and S. Suzuki (2015): Extracellular proteases 1–21. are released by ciliates in defined seawater microcosms. Sako, Y., K. Nakaya and A. Ito (2011): Echinococcus multilocu- Mar. Environ. Res., 109, 95–102. laris: Identification and functional characterization of Tolbert, M. K., S. H. Stauffer, M. D. Brand and J. L. Gookin cathepsin B-like peptidases from metacestode. Exp. Par- (2014): Cysteine protease activity of feline Tritrichomonas asitol., 127, 693–701. foetus promotes adhesion-dependent cytotoxicity to intes- Shin, S. P., S. Y. Han, J. E. Han, J. W. Jun, J. H. Kim and S. C. tinal epithelial cells. Infect. Immun., 82, 2851–2859. Park (2014): Expression and characterization of cathepsin Völkel, H., U. Kurz, J. Linder, S. Klumpp, V. Gnau, G. Jung and L-like cysteine protease from Philasterides dicentrarchi. J. E. Schultz (1996): Cathepsin L is an intracellular and Parasitol. Int., 63, 359–365. extracellular protease in Paramecium tetraurelia. Purifica- Song, J. Y., S. I. Kitamura, M. J. Oh, H. S. Kang, J. Lee, S. tion, cloning, sequencing and specific inhibition by its Tanaka and S. J. Jung (2009a): Pathogenicity of Miamien- expressed propeptide. Eur. J. Biochem., 238, 198–206. sis avidus (syn. Philasterides dicentrarchi), Pseudocoh- Yoshinaga, T. and J. Nakazoe (1993): Isolation and in vitro cul- nilembus persalinus, Pseudocohnilembus hargisi and tivation of an unidentified ciliate causing scuticociliatosis in Uronema marinum (Ciliophora, Scuticociliatida). Dis. Japanese flounder (Paralichthys olivaceus). Fish Pathol., Aquat. Organ., 83, 133–143. 28, 131–134.