Área de Microbiologia, Departamento de Biologia Funcional, Facultad de Medicina, Instituto Universitario de Biotecnologia de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain1
SERIDA, Laboratorio de Sanidad Animal de Jove, 33299 Gijon, Spain2
Author for correspondence: J. A. Guijarro. Tel: +34 985 104 218. Fax: +34 985 103 148. e-mail: jaga{at}sauron.quimica.uniovi.es
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ABSTRACT |
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Keywords: enteric redmouth disease, pathogenicity, type I secretion system
a The GenBank accession numbers for the sequences reported in this paper are AJ318052 (yrp1) and AJ421517 (yrpDEF and inh).
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INTRODUCTION |
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The early development of a vaccine in the prevention of this disease is probably one of the reasons why virulence determinants have not been studied in depth. Commercial vaccines for Y. ruckeri are composed of formalin-killed whole bacterial cells and they are administered by immersion, providing an effective reduction in mortality (Stevenson, 1997 ). However, in spite of vaccination, fish farm outbreaks occur from time to time. The micro-organism has been found in the intestinal lining of fish (Busch & Lingg, 1974
) and its presence in carrier fish could be the cause of the appearance of outbreaks under stress conditions (Everlyn, 1996
). Thus, it is important to know the pathogenic mechanisms of the bacterium for new approaches to vaccination development, new alternatives or for improving the vaccine.
Proteases have been suggested as one of the extracellular factors contributing to the virulence of micro-organisms. In particular, in fish-pathogenic bacteria, indirect experiments have indicated the participation of proteolytic enzymes in pathogenesis (Sakai, 1985 ; Griffin, 1987
; Bertolini et al., 1994
; Gunnlaugsdottir & Gudmundsdottir, 1997
; Denkin & Nelson, 1999
; Secades et al., 2001
). In addition, genetic approaches, such as the truncation of the encoding proteolytic genes, have shown that, although not as a general rule, proteolytic enzymes are involved in the virulence of fish-pathogenic bacteria (Leung & Stevenson, 1988
; Norqvist et al., 1990
; Milton et al., 1992
; Cascón et al., 2000b
). Although their role in pathogenesis is not clear, it seems that colonization and invasion could be one of their major mechanisms of action during hostpathogen interaction, at the same time providing nutrients for the micro-organisms. Thus, enzymes degrading connective and muscular tissues, such as collagenases, elastases, gelatinases, etc., may play a relevant role in pathogenesis.
In Y. ruckeri, extracellular proteolytic activity has been related to virulence (Romalde & Toranzo, 1993 ; Secades & Guijarro, 1999
). A 47 kDa extracellular protease from Y. ruckeri has been purified and characterized (Secades & Guijarro, 1999
). We became interested in further characterizing this proteolytic activity and we cloned the gene encoding the 47 kDa protease (Yrp1) of Y. ruckeri 150 and tested its role in virulence. The amino acid sequence of Yrp1 showed the absence of an N-terminal signal sequence and a high degree of homology with metalloproteases of several pathogenic bacteria, particularly with PrtA (Ghigo & Wandersman, 1992a
), PrtC (Dahler et al., 1990
) and PrtG (Ghigo & Wandersman, 1992b
) proteases from Erwinia chrysanthemi, the serralysin from Serratia marcescens (Nakahama et al., 1986
; Braunagel & Benedin, 1990
) and the metalloproteases from Pseudomonas aeruginosa (Doug et al., 1992
) and Pseudomonas fluorescens (Liao & McCallus, 1998
). The DNA sequence around the yrp1 gene was analysed and a gene cluster homologous to the one found in Erw. chrysanthemi, P. aeruginosa and S. marcescens, described by Binet et al. (1997)
and encoding a complete ABC exporter, was identified and sequenced. Out of the yersiniabactin ABC components from human-pathogenic Yersinia (Carniel, 1999
) and the Yfe and Yfu systems of cation transport found in Yersinia pestis (Bearden & Perry, 1999
; Gong et al., 2001
) and Yersinia enterocolitica (Saken et al., 2000
), the presence of a haemophore-dependent haem-acquisition system secreted by an ABC transporter system in Y. pestis has recently been reported (Rossi et al., 2001
). However, to our knowledge there is no description of an ABC export protease system in the genus Yersinia. To define the role of the ABC cassette in Yrp1 secretion as well as in the implied role of the protease in pathogenesis, two insertion mutants were generated. We showed, using SDS-PAGE and immunoblot analysis, that the mutants lacked the 47 kDa protein as well as the proteolytic activity using azocasein or gelatin as substrates. The mutants showed a similar growth curve to the parental strain, but LD50 experiments indicated that they were attenuated for rainbow trout. To our knowledge, this is the first time that a protein has been shown to play a role in the pathogenesis of Y. ruckeri.
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METHODS |
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Sequencing grade Taq polymerase (Perkin-Elmer) was employed. Plasmids used in this work were propagated in E. coli strains and isolated by the alkaline lysis method (Birnboim & Doly, 1979 ).
DNA from Y. ruckeri 150 was obtained from a 500 ml stationary-phase NB culture by the alkaline method (Sambrook et al., 1989 ). The DNA was partially digested with Sau3AI and size-fractioned by centrifugation at 215000 g for 16 h at 17 °C into a 1040% (v/v) sucrose gradient in TEN buffer [10 mM Tris/HCl (pH 8), 1 mM EDTA (pH 8), 100 mM NaCl]. Fragments from 5 to 10 kb were ligated to BamHI-digested and dephosphorylated pUC19 plasmid. The ligated DNA was then used for electroporation of E. coli DH10B strain in a Gene Pulser apparatus (Bio-Rad) set at 2·5 kV, 25 µF and 200
. Following electroporation, bacteria were incubated in 1 ml NB at 37 °C and 250 r.p.m. for 1 h without selection and then E. coli recombinants were selected by plating the transformed mixture onto NA supplemented with gelatin, ampicillin, X-Gal and IPTG, and incubated for 24 h at 37 °C. Further incubation for 24 h at 15 °C was needed for gelatin hydrolysis visualization.
Subcloning of the yrp1 gene and DNA sequence.
An E. coli DH10B clone with an approximately 8·4 kb BamHI insert in the pUC19 vector was analysed by fragment subcloning. Synthetic oligonucleotide primers were used to sequence the fragment by the dideoxy chain-termination method, using a DR Terminator Taq FS Sequencing kit (Applied Biosystems). DNA sequence was carried out according to the manufacturers specifications in an ABI-PRISM 310 A automated DNA sequencer from Perkin-Elmer. Sequencing data were analysed using the BLASTX computer program.
N-terminal amino acid sequencing of the protein.
Yrp1 was obtained as described previously (Secades & Guijarro, 1999 ). Approximately 25 µg of the 47 kDa protein were dialysed against 4 l 10 mM HEPES buffer, pH 6·8, concentrated by lyophilization and the N-terminal sequence was determined by automated Edman degradation at the Newcastle University protein sequencing facility. Multiple alignments of protein sequence were generated by using the PILEUP alignment program.
Construction of the derivative plasmids pLPY1 and pLPY2 carrying yrp1 and yrpE internal sequence.
The primers used were as follows: forward primers YRPP1 and YRPE1; 5'-GCCAAGATCTCATTACAATCTTGGGCA-3' (nt 314330 in bold type) and 5'-GCCAAGATCTGGGTTGACTGCGCAATA-3' (nt 313329 in bold type), respectively; reverse primers YRPP2 and YRPE2; 5'-CGTTAGATCTCGCACCATGTAATTCAT-3' (nt 10801064 in bold type) and 5'-CGTTAGATCTGTTTCACGGTGCCTTCT-3' (nt 10821098 in bold type), respectively. The primers were used to amplify the 787 and 820 bp internal regions of the yrp1 and the yrpE genes, respectively. Thermal cycling was conducted with a Perkin-Elmer 9700 GeneAmp thermocycler with an initial denaturation cycle at 94 °C for 5 min, followed by 25 cycles of amplification (denaturation at 94 °C for 30 s; annealing at 43 and 47 °C for yrp1 and yrpE, respectively, for 30 s and extension at 72 °C for 1 min) and a final 7 min elongation period at 72 °C. The primers contained a BglII site (underlined) and four additional bases at the 5' end. The generated amplicons were digested with BglII and the products were ligated with pIVET8 previously digested with the same enzyme. Aliquots of the ligation mixture were used to transform E. coli SM10 pir by electroporation under the conditions described above. Selection of transformants was performed on NA plates containing ampicillin and plasmid DNA was analysed with restriction endonucleases to identify plasmids carrying the correct inserts. The corresponding clones containing the pLPY1 (yrp1 insert) and the pLPY2 (yrpE insert) plasmids were then used for conjugation.
Bacterial conjugation.
Conjugation was performed as follows: 250 µl of a mid-exponential-growth culture of the donor E. coli SM10 pir strain containing the pLPY1 or the pLPY2 plasmids and 2 ml of the recipient Y. ruckeri 150R strain were mixed together in 10 ml distilled water and then filtered through a sterile 0·45 µm pore-size filter (Millipore). The filter was placed on an NA plate and incubated at 28 °C for 6 h, then washed by vortexing with 2 ml NB and the bacteria were plated onto NA containing gelatin (5 g l-1), ampicillin (100 µg ml-1) and rifampicin (50 µg ml-1) and incubated at 28 °C for 2 days. Following this, plates were transferred to 15 °C for 24 h to determine the proteolytic activity on gelatin.
Southern blot analysis.
Genomic DNA isolated from Y. ruckeri 150R and the mutant strains 150RI4 and 150RI6 was digested with BamHI and separated on a 0·6% (w/v) agarose gel. DNA was transferred to a nylon membrane (Amersham), fixed by UV irradiation and hybridized with the 787 or 820 bp PCR-generated internal fragments from the yrp1 and yrpE genes, respectively, previously purified using the Qiagen kit. Probe labelling, hybridization and developing were performed with the DIG DNA labelling and detection kit from Boehringer Mannheim following the manufacturers instructions. Prehybridization (1 h) and hybridization (16 h) were carried out at 68 °C in 5xSSC, 0·1% (w/v) N-lauroylsarcosine, 0·02% (w/v) SDS and 1·5% (w/v) kit blocking reagent. Membranes were washed for 1 h at room temperature and then at 68 °C for 30 min. Immunological detection was performed using anti-DIG/AP conjugate (1:5000). Membranes were developed until a suitable signal was obtained.
Complementation of mutants.
Complementation of mutants was carried out using plasmid pUK21 (Vieira & Messing, 1991 ). Three different fragments containing a 2·9 kb BamHI with the yrp1 and inh genes, a 5·6 kb ClaI with the inh, yrpD, yrpE and yrpF genes and a 8·4 kb EcoRIPstI with all genes, were ligated into the pUK21 plasmid, generating pUK21B, pUK21C and pUK21T, respectively. Y. ruckeri strains 150RI4 and 150RI6 were transformed by electroporation as described above for E. coli with pUK21B and pUK21C, respectively. Both strains were also transformed with pUK21T. Expression in E. coli DH10B of pUK21T with the 8·4 kb cloned fragment in both orientations in relation to the lac promoter was also determined. In all cases, the presence of proteolytic activity was analysed by halo production on NA plates containing kanamycin and ampicillin and supplemented with gelatin.
LD50 determination.
Rainbow trout (Oncorhynchus mykiss) weighing between 8 and 10 g were kept on 60 l tanks at 18±1 °C in continually flowing dechlorinated water. Groups of 10 fish were challenged by intraperitoneal injection of 0·1 ml serial tenfold dilutions of an exponential-phase culture of bacteria over a range of 102106 cells. The micro-organisms were previously washed and resuspended in PBS. Control fishes were injected with 0·1 ml PBS. The LD50 determinations were calculated according to the method of Reed & Muench (1938) .
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RESULTS |
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The inserted plasmid DNAs were maintained in a stable fashion inside the bacteria when grown in the presence or absence of ampicillin as a selection marker. The mutations were also stable after rainbow trout infection experiments, since colonies isolated from the infected fish retained the ampicillin resistance and were devoid of proteolytic activity (data not shown).
As can be observed in Fig. 4a, when parental and yrp1 mutant strains were grown in NB medium at 18 °C, although the mutant showed a slight growth delay, no significant differences were observed in the growth curve. However, when the proteolytic activity of supernatants from both strains was assayed using azocasein as a substrate, no activity at all was found in the Y. ruckeri 150RI4 mutant in contrast with the 412 UE ml-1 found in the parental strain (Fig. 4a
). On the other hand, when both strains were grown in NB medium and both supernatants were analysed by SDS-PAGE for the presence of the 47 kDa protein, this was only present in the parental Y. ruckeri 150R strain supernatant, but absent in the Y. ruckeri 150RI4 supernatant (Fig. 4b
). This result was confirmed by immunoblot analysis using antibodies raised against this protein (Fig. 4c
). Similar results to that obtained in the Y. ruckeri 150RI4 were found when Y. ruckeri 150RI6 was analysed (data not shown).
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Decreased virulence of yrp1 and yrpE mutants
To determine the effect of the absence of Yrp1 on the virulence of Y. ruckeri, the LD50 of three mutant strains was compared with that of the parental strain in a trout model. As little as approximately 102 c.f.u. of the wild-type strain resulted in 50% of the fish dying 7 days after inoculation (Table 3). By contrast, the 150RI4 and 150RI6 mutants presented an LD50 two orders of magnitude higher. Thus, these significant differences between the wild-type and the mutant strains indicate that Yrp1 activity appears to be an important virulence factor.
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DISCUSSION |
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The Yrp1 enzyme belongs to the serralysin subfamily of family M12, as defined by Rawlings & Barrett (1995) , having a primary structure very similar to proteases PrtC and PrtA from Erw. chrysanthemi (Dahler et al., 1990
; Ghigo & Wandersman, 1992a
), the SM protease from S. marcescens (Braunagel & Benedin, 1990
) and an alkaline metalloprotease from P. fluorescens (Liao & McCallus, 1998
). Thus, amino acid sequences share a percentage of identity between 50 and 67%. The sequence contains the Gly-rich sequences GGXGXD, where Ca2+ is bound, a consensus sequence HEXXH responsible for zinc binding, the zinc ion being an essential component, as well as the 4 aa reported at the end of the C-terminal region involved in some way in the secretion of the protein (Binet et al., 1997
). Moreover, the terminal 10 aa of the purified 47 kDa protein matched the deduced N-terminal A15 to V24 amino acid sequence of the yrp1 ORF. This, together with the lack of the 47 kDa protein in the mutant strain shows that the 47 kDa protease previously characterized (Secades & Guijarro, 1999
) is indeed encoded by the yrp1 gene. Enzymic properties of Yrp1 (Secades & Guijarro, 1999
) are also similar to the related serralysin proteases. On the other hand, when the yrp1 gene was insertionally truncated, 100% of the proteolytic activity was lost upon growth in NB medium when azocasein or gelatin was used as enzyme substrate. This might be the only extracellular metalloprotease produced by this bacterium. The yrp1 protease also has a propeptide of 14 aa at the N-terminal end, which should be excised to activate the protease. This suggests that the Yrp1 protein is synthesized and secreted as a zymogen without a signal peptide by an ABC export system, as occurs for the serralysin group of proteases (Binet et al., 1997
). Thus, a sequence downstream of the yrp1 gene indicates significant homology with the protease ABC export systems found in Erwinia, Serratia and Pseudomonas (Binet et al., 1997
). With the exception of the yersiniabactins, the Yfu and Yfe cation transporter system and the haemophore system, this type I export system is different from the type III system found in other species of the genus Yersinia (Huek, 1998
). The organization of the Y. ruckeri protease production and export system is similar to P. fluorescens, the yrp1 and yrpDEF transporter genes being situated downstream from the yrp1 gene, but different from the systems found in P. aeruginosa and Erw. chrysanthemi. This different gene organization among species does not have an obvious evolutionary significance. As expected, the cluster of genes required for the production of extracellular Yrp1 seems to form an operon as indicated by the polar mutation defined by complementation experiments in the 150RI4 mutant. Therefore, the cloned 8·4 kb DNA fragment contains the regulatory sequence necessary for the expression of the operon in Y. ruckeri according to the protease production obtained when the fragment was introduced in the 150RI4 strain through the pUK21T plasmid in both orientations in relation to the lac promoter.
Differential pathogenicity for Y. ruckeri strains after lethality studies in rainbow trout has been documented (Romalde & Toranzo, 1993 ). These differences have been attributed to factors such as adhesive properties (Romalde et al., 1990
), protease and haemolytic activities (Romalde & Toranzo, 1993
), iron acquisition systems (Davies, 1991
; Romalde & Toranzo, 1993
) and the presence of a heat-sensitive factor (Doug et al., 1992
; Everlyn, 1996
). However, until now, neither of these factors has been confirmed to be clearly involved in virulence. The present work defines the Yrp1 protease as a virulence factor for Y. ruckeri. Thus, when the isogenic 150RI4 and 150RI6 mutants were compared with the parental strain for virulence in intraperitoneally inoculated rainbow trout, the mutants showed a 100-fold higher LD50. Although a large difference in lethality between the parental and the mutant strains was not obtained, it was significant. As virulence is probably the result of the interaction of a variety of individual factors that, acting together, allow the progression of the infection, this LD50 difference between both strains should be sufficient to consider the involvement of the Yrp1 protease in the progress of infection caused by Y. ruckeri. It must be pointed out that the cells recovered from the infected fish with both mutant strains retained the gelatin-negative phenotype. Different results have been obtained in studies carried out with bacterial proteases from fish pathogens as virulence factors. Thus, in Aeromonas salmonicida, a chemically obtained protease-deficient mutant showed a significant loss in virulence (Sakai, 1985
), although Vipond et al. (1998)
reported that a strain of A. salmonicida carrying a marker replacement of an internal deletion in the serine protease aspA gene was as virulent as the parental strain. In this particular case, this conflicting result could be explained by the fact that in chemically mutated strains, multiple or specific mutations in genes involved in global pathogenesis regulation cannot be excluded. By contrast, in Aeromonas hydrophila, a clear difference has been established by insertion mutation experiments in the implication in virulence of two proteases. Thus, the AhpA serine protease was shown not to be essential for virulence (Cascón et al., 2000a
), whereas an elastase was needed for the pathogenesis of the micro-organism (Cascón et al., 2000b
). As occurred in Y. ruckeri, the differences in LD50 between the parental and mutant strains were in the order of 102. Therefore, according to these results, it seems that a general rule cannot be established about the involvement of exocellular proteolytic enzymes produced by bacteria in fish pathogenesis. Proteolytic enzymes can have a wide range of effects, such as tissue degeneration, invasion, protoxin activation, etc., as well as nutrient acquisition, and probably not all these protease functions are related to virulence, the potential functions of the proteases in vivo being difficult to establish. The present study shows that in Y. ruckeri a metalloprotease is involved in the virulence of the bacterium, although the knowledge of the importance and role of this enzyme in bacterial infection is still to be developed.
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ACKNOWLEDGEMENTS |
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Received 4 February 2002;
accepted 11 March 2002.