Characterisation of an Extracellular Vibriolysin of the Fish Pathogen
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Veterinary Microbiology 136 (2009) 326–334 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Characterisation of an extracellular vibriolysin of the fish pathogen Moritella viscosa Bryndis Bjornsdottir a, Olafur H. Fridjonsson b, Steinunn Magnusdottir b, Valgerdur Andresdottir a, Gudmundur O. Hreggvidsson b,c, Bjarnheidur K. Gudmundsdottir a,* a Institute for Experimental Pathology, University of Iceland, Keldur, Vesturlandsvegi, 112 Reykjavik, Iceland b Matis-Prokaria, Gylfaflot 5, 112 Reykjavik, Iceland c Department of Biology, University of Iceland, Sturlugotu 7, 101 Reykjavik, Iceland ARTICLE INFO ABSTRACT Article history: Moritella viscosa causes winter ulcer disease in salmonids. The aim of the present work was Received 29 September 2008 to isolate and partially characterise an extracellular peptidase from M. viscosa, and to study Received in revised form 25 November 2008 its role in virulence. The peptidase, termed MvP1, was a 38-kDa metallopeptidase Accepted 28 November 2008 produced in late exponential growth. The optimum temperature for MvP1 was 40 8C, but the enzyme was active over a broad range of temperatures. MvP1 was non-lethal to Keywords: salmon at concentrations up to 0.22 mg/g fish, but extracellular products were lethal to Moritella viscosa salmon. MvP1 degraded casein, gelatin and collagen from lumpfish skin. It caused MvP1 considerable tissue necrosis and hemorrhages at the site of injection, and affected cell–cell Extracellular products Vibriolysin adhesions in EPC and BF-2 cell lines, but was not highly cytotoxic. The peptidase partially Virulence factor degraded fish IgM heavy chain but was non-hemolytic. The mvp1 gene was sequenced and encoded a 734-aa polypeptide containing a signal sequence, an N-terminal propeptide, a mature peptidase domain and a C-terminal propeptide. The MvP1 propeptide undergoes both N-terminal and C-terminal processing and different C-terminal processing results in the formation of several active isoforms of the mature peptidase. The catalytic domain showed highest sequence similarity with several vibriolysins (EC 3.4.24.25) originating from Pseudoalteromonas strains, showing up to 80% aa identity. The results indicate that MvP1 is a previously unknown vibriolysin that might affect M. viscosa virulence by aiding in the invasion and dissemination of the bacterium in its host, by causing tissue destruction. ß 2008 Elsevier B.V. All rights reserved. 1. Introduction disease causes significant mortalities and financial losses in Atlantic salmon, Salmo salar (L.) and rainbow trout, Moritella viscosa causes winter ulcer disease in fish Oncorhynchus mykiss (Walbaum), and causes infections in reared at low temperatures in North Atlantic countries, Atlantic cod, Gadus morhua (L.). Furthermore, the bacter- mainly in Norway (reviewed in Gudmundsdottir and ium has been isolated from several other fish species Bjornsdottir, 2007). Large ulcers often appear on infected (reviewed in Gudmundsdottir and Bjornsdottir, 2007). fish, and hemorrhages and tissue necrosis are detectable Lipooligosaccharides and a 17–19 kDa outer membrane internally (Lunder et al., 1995; Bruno et al., 1998). The antigen have recently been shown to be protective antigens (Heidarsdottir et al., 2008). Pathogenic bacteria produce a variety of virulence * Corresponding author. Tel.: +354 5855100; fax: +354 5674714. factors that affect their hosts, allowing the bacteria to E-mail address: [email protected] (B.K. Gudmundsdottir). multiply and produce a disease. Several extracellular 0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.11.020 B. Bjornsdottir et al. / Veterinary Microbiology 136 (2009) 326–334 327 bacterial peptidases, mainly metallopeptidases, have been Remaining caseinolytic activity was determined using shown to have either direct or indirect roles in pathogen- azocasein assay. esis and virulence, although most are known for nutrient acquisition for bacterial growth (Miyoshi and Shinoda, 2.3. Peptidase purification 2000). The Pseudomonas aeruginosa elastase and Vibrio vulnificus and Vibrio cholerae vibriolysins have been ECP proteins were precipitated by adding ammonium implicated with bacterial virulence. They cause various sulphate to 85% saturation. The precipitate was recovered actions, such as tissue destruction and hemorrhages, and by centrifugation and dissolved in 20 mM Tris (pH 8). are believed to play roles in colonization, invasiveness, and Precipitated proteins were desalted using PD-10 columns dissemination within the host (Booth et al., 1984; (Amersham Biosciences), equilibrated with the same Finkelstein et al., 1992; Komori et al., 2001; Silva et al., buffer at 4 8C. Desalted samples were loaded onto a 2003; Miyoshi, 2006). MonoQ HR 5/5 column (Pharmacia), preequilibrated with This paper describes the isolation and characterisation 20 mM Tris (pH 8). The column was eluted at 15 8C with a of a previously unknown extracellular peptidase from the linear gradient of 0–0.8 M NaCl in the same buffer. Eluate fish pathogenic bacterium M. viscosa. fractions were collected and analyzed for caseinolytic activity. Pooled fractions containing the highest case- inolytic activities were concentrated by ultrafiltration 2. Materials and methods using Amicon Ultra-15 filter (Millipore, MWCO 10,000) at 2.1. Bacterial strain, growth conditions, and production of 4 8C. Concentrated fractions were further fractionated on a ECP Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech), equilibrated and eluted with 50 mM Na-phos- Moritella viscosa, strain K58, was isolated and identified phate buffer (pH 7), containing 15 mM NaCl (SD-200 in 1992 at the Institute for Experimental Pathology, buffer) at 15 8C. Fractions with major caseinolytic activities University of Iceland, from the head kidney of diseased were pooled and used as the purified enzyme, following Atlantic salmon from a farm in SW-Iceland (Benedikts- analysis with SDS-PAGE and Western blotting. dottir et al., 1998, 2000). The strain was passaged in salmon prior to the study, by reisolation from kidney of i.p. 2.4. Electrophoresis and isoelectric focusing infected fish. The bacterium was grown on 5% horse blood agar supplemented with 2% NaCl at 4 8C for 4 days. M. Standard SDS-PAGE was carried out according to the viscosa growth curve was determined by averaging three Laemmli method (Laemmli, 1970) in 12% polyacrylamide identical batch cultures cultivated in Brain-Heart infusion gels and protein bands visualised by silver staining (Silver broth (BHI, BD) containing 1.5% NaCl (BHI-NaCl) at 9 8C, Stain Plus Kit, BioRad). Zymography was performed as with agitation (200 rpm). Growth was estimated in log previously described (Gudmundsdottir, 1996), using 0.03% CFU/ml by serial dilution and plate counting. casein, 0.1% collagen (isolated from lumpfish skin), or 0.1% Extracellular products (ECP) were isolated from batch gelatin as substrates. Gels were incubated for 2 h at 30 8C. cultures grown at 4 or 9 8C for 5 days by centrifugation, and Isoelectric focusing was carried out in Phast system then filtered (Whatman, 22 mm). For isolation of ECP, used (Pharmacia), using PhastGel IEF 4–6.5 (GE healthcare), and for toxicity testing, M. viscosa was cultivated using the standards pH 3–10 (Pharmacia). Gels were fixed in 20% cellophane overlay method (Liu, 1957) on Tryptone soya trichloroacetic acid and visualised with silver staining. broth (Difco) agar with 1.5% NaCl at 15 8C for 3 days. 2.5. Immunoreactivity of MvP1 isoforms 2.2. Proteolytic activity, protein content, and inhibition studies Isolated MvP1 was electrophorised and visualised using Western blotting as described previously (Gudmundsdot- Caseinolytic activity was assayed using azocasein tir et al., 2003). Blotted membranes were incubated with (Sigma) as previously described (Gudmundsdottir, 1996), polyclonal mouse antibodies against the 38 kDa MvP1 and incubated at 30 8C for 60 min. Samples were assayed in isotype (I1, see definition in Section 3.2) that were duplicate and each duplicate measured twice. One unit (U) prepared in mouse ascitic fluid using a previously of caseinase activity was defined as an increase in described method (Gudmundsdottir et al., 2003), and with absorbance of 0.01 under the assay conditions. an estimated dose of 10 mg MvP1 injected each time. Protein content was measured with a protein assay kit Antibodies were diluted 1:2000. Washed membranes were (Coomassie Plus, Pierce) and bovine serum albumin then incubated with goat anti-mouse antibodies conju- (Sigma) used for plotting a standard curve. Absorbance gated with alkaline phosphatase (Dako), diluted 1:1000. was measured at 590 nm and all samples measured in duplicate. 2.6. Optimal temperature and thermostability The pure peptidase was incubated with inhibitors for 10 min at 22 8C. Stock solutions of the dissolved metallo- For estimating the optimal temperature for MvP1, the peptidase inhibitors EDTA (Merck) and OPA (1,10-phe- catalytic activity of the pure peptidase was measured at nanthroline) (Sigma–Aldrich) and the serine peptidase different temperatures between 5 and 80 8C for 1 h, by the inhibitor PMSF (Sigma) were prepared and added to the azocasein assay. For thermostability testing the purified MvP1 peptidase in final concentrations of 1 or 10 mM. protein was incubated at 30, 40, 50 and 60 8C for 30 min in 328 B. Bjornsdottir et al. / Veterinary Microbiology 136 (2009) 326–334 SD-200 buffer and then centrifuged before performing