APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/97/$04.0010

Oct. 1997, p. 3770–3775 Vol. 63, No. 10

Copyright © 1997, American Society for Microbiology

Further Characterization of Renibacterium salmoninarumExtracellular Products

TROY A. BARTON,1 LAURA A. BANNISTER,1 STEVEN G. GRIFFITHS,2

AND WILLIAM H. LYNCH1*

Department of Biology, University of New Brunswick,1 and Research andProductivity Council of New Brunswick,2 Fredericton,

New Brunswick, Canada

Received 14 February 1997/Accepted 7 July 1997

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrationsof extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDaprotein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and itsderivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to producedegradation and aggregation products. One-dimensional electrophoretic separation of extracellular proteinrevealed a number of proteolytically active bands from >100 to approximately 18 kDa associated with various57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracel-lular protein showed that continued degradation and aggregation, similar both in location and behavior tosome of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after theirseparation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mech-anism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested thatthe 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically activepolypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellularproducts and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted withO-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity betweenthese polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa proteinderivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.

Renibacterium salmoninarum is a fastidious, slow-growinggram-positive bacterium which causes bacterial kidney disease(BKD) in salmonid fishes (for a review, see reference 7). R. sal-moninarum produces large amounts of extracellular polypep-tides detected both in situ (24) and in laboratory cultures (8).The extracellular polypeptides are derived almost entirelyfrom a 57-kDa protein, referred to as antigen F (8) and morerecently as p57 (25), which is associated with virulence of thebacterium (2, 3). Also, this p57 protein is loosely associatedwith the cell surface of R. salmoninarum (4). By immunoelec-tron microscopy, aggregates of p57 appear as short fimbria-likeprojections (6) in addition to a layer of capsular material whichcovers the cells (5).

The p57 protein is unstable within the extracellular products(ECP), its degradation producing many smaller-molecular-mass fragments (12, 22). Similar fragments of p57 are found intissues of infected fish, although their significance to the patho-genesis of BKD is unknown (21, 24). The degradation of p57within the ECP is inhibited by phenylmethylsulfonyl fluoride(PMSF), leading to suggestions that it is caused by a serineproteinase (6, 12, 22). Also, a proteolytic activity product witha molecular mass of .100 kDa was detected within the ECPafter autodigestion to remove p57, leading to the conclusionthat it was due to a proteinase of .100 kDa (22). However,instability was observed within p57 and several of its degrada-tion products, suggesting that proteolytic activity might be as-sociated with several p57 derivatives (12).

Due to the high concentrations of p57 and its degradationproducts in tissues of infected fish and the involvement of p57in the virulence of R. salmoninarum, further investigation tocharacterize the ECP was undertaken.

MATERIALS AND METHODS

Bacterial strains and cultivation. The Canadian east coast R. salmoninarumF91 strain was isolated from Atlantic salmon (Salmo salar L.) as reported pre-viously (17). The west coast R. salmoninarum WC strain was isolated fromchinook salmon (Oncorhynchus tshawytscha) and kindly provided by J. Brackett.Bacteria were cultivated on selective kidney disease medium agar (1) for 3 to 4weeks at 15°C, and the ECP were harvested in phosphate-buffered saline (PBS)at 4°C as reported previously (12) and stored at 220°C until processed.

Sources (infected Atlantic salmon) and cultivation of other bacterial strainsused, i.e., Aeromonas salmonicida Asa92, A. salmonicida Ast5, Pseudomonas flu-orescens F, and Vibrio ordallii B, were as reported elsewhere (11).

Fractionation of R. salmoninarum extracellular polypeptides. ECP prepara-tions were concentrated (5 to 6 mg of protein ml21) with Minicon B-15 mac-rosolute concentrators (Amicon, Inc., Beverly, Mass.), and aliquots (approximate-ly 1,800 mg of protein) were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) at room temperature as described elsewhere(27) with a Mini-Protean II gel electrophoresis system (Bio-Rad Laboratories,Ltd., Hercules, Calif.) in a single wide well. The polypeptides were transblottedto Immobilon membranes (Millipore Corp., Bedford, Mass.) as described bySzewczyk and Summers (23). The membrane was wetted with 95% (vol/vol)methanol prior to equilibration in transfer buffer. Electrophoretic transfer was at20 V for 1.25 h in a semidry electrophoretic transfer cell (Bio-Rad). Aftertransfer, the membrane was stained with 0.2% (wt/vol) amido black to locatepolypeptides and destained with deionized distilled H2O. Horizontal strips con-taining polypeptide bands were excised from the Immobilon membrane andeluted by washing with 1 ml of 1% (vol/vol) Triton X-100 and 2% (wt/vol) SDS.The eluates (fractions) were dialyzed overnight at 4°C against deionized distilledH2O. Western blotting (immunoblotting) of the isolated fractions (1 ml) wasperformed with 100-ml aliquots of fractions 1, 2, 3, and 6 and with 50-ml aliquotsof fractions 4 and 5 (see Fig. 1) as described previously (13), except that the firstbinding agent was a monoclonal anti-p57 protein antibody (4D3; Diagxotics, Inc.,Wilton, Conn.) solution of 30 ml added to 10 ml of 10 mM Tris-HCl–150 mMNaCl–0.5% (vol/vol) Tween 20 (pH 7.5; TTBS) and the second binding agent was

* Corresponding author. Mailing address: Department of Biology,University of New Brunswick, Bag Service #45111, Fredericton, NewBrunswick, Canada E3B 6E1. Phone: (506) 453-4733. Fax: (506) 453-3583.

3770

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

biotinylated goat anti-mouse immunoglobulin G (Amersham Corp., ArlingtonHeights, Ill.) solution of 20 ml added to 10 ml of TTBS.

G-PAGE. For one-dimensional gelatin-SDS-PAGE (G-PAGE), the gels wereprepared as described for SDS-PAGE except that 0.1% (wt/vol) gelatin wasincluded in the lower gel and aliquots of ECP were added directly to samplebuffer (16) prior to electrophoresis. Subsequent to electrophoresis, the gels weretreated as described elsewhere (22) to detect proteolytic activity.

For two-dimensional G-PAGE, aliquots of ECP in sample buffer were sepa-rated as described previously (12), except that gel strips containing the separatedpolypeptides from the one-dimensional SDS-PAGE gel were incubated for 1 h at20°C in 2% (vol/vol) Triton X-100 and then for 4 h in PBS and 10 min in samplebuffer. Subsequently, the gel strip was inserted on top of the two-dimensionalstacking gel (0.5 cm) without the aid of molten agarose, and the running gel usedcontained 0.1% gelatin. Proteolytic activity was detected as described above.

Two-dimensional Western blots of R. salmoninarum ECP. Two-dimensionalSDS-PAGE gels were run as described for two-dimensional G-PAGE gels exceptthat 0.1% (wt/vol) gelatin was omitted. Western blotting of the two-dimensionalgel was done as reported previously (12).

Effects of DTT and proteinase inhibitors on protein digestion by the R. sal-moninarum ECP. Dithiothreitol (DTT) and proteinase inhibitors were dissolvedin PBS and added to give the final concentrations indicated (see Fig. 4 andResults) to aliquots of the ECP in PBS (1-ml total volume). PMSF was firstdissolved in isopropanol and then diluted in PBS. The mixtures were left for 10min at room temperature prior to the addition of bovine serum albumin (BSA;10 mg in 1 ml of PBS) and incubation at 35°C. Aliquots of the incubation mixturewere removed at the times indicated and precipitated with trichloroacetic acid(10% [vol/vol] final concentration), and samples equivalent to 20 mg of BSA atthe start of incubation were processed by SDS-PAGE. Digestion of BSA wasvisualized by staining the gels with Coomassie blue (LKB application note 321).Also, DTT and inhibitors were added to aliquots of the ECP in PBS andincubated at 35°C. Samples were removed at the times indicated, precipitatedwith trichloroacetic acid, and subjected to one-dimensional Western blotting (12)to observe autodigestion of the p57 derivatives.

Polysaccharide detection in R. salmoninarum ECP. Aliquots of ECP and kid-ney tissue homogenates (stored at 220°C) from Atlantic salmon, either clinicallyinfected with R. salmoninarum or showing no external or internal signs of infec-tion (17), were incubated at the concentrations indicated (see Fig. 5) at 35°C inPBS containing pronase (final concentration, 2 mg ml21; Calbiochem-BehringCorp., La Jolla, Calif.). Samples were removed at the designated times, precip-itated with trichloroacetic acid, and examined by Western blotting. The Westernblots were developed with as the first binding agent either rabbit anti-whole ECP(from R. salmoninarum K2A2) antiserum (12) or rabbit antiserum to R. salmo-ninarum K2A2 cells boosted with pronase-digested cell lysate. The latter anti-serum was produced by initially injecting rabbits with a 0.5-ml volume containing108 CFU of R. salmoninarum emulsified in Freund’s incomplete adjuvant (FIA)at a ratio of 1 part bacterial suspension in sterile PBS to 3 parts FIA (12) toincrease the possibility of a strong immune response to polysaccharide (14). Theprocedure was repeated 4 weeks later. Subsequent boosters were prepared bysuspending R. salmoninarum cells from cellophane overlay selective kidney dis-ease medium agar cultures (12) in sample buffer, boiling for 3 min, and incu-bating overnight at 37°C in the presence of pronase (100 mg ml of cell lysate21)on a Nutator model 1105 rocking device (Clay Adams, Parsippany, N.J.). Re-sidual pronase activity was destroyed by boiling the protein-digested lysate for 5min. The digested lysates were concentrated with a p-200 ultrafiltration device(Amicon). The concentrated digested lysate was divided into 1-ml aliquots andlyophilized. The absence of protein material was observed by Western blottingthe digest followed by gold staining (Bio-Rad). The lyophilized digest was sus-pended in sterile PBS at a concentration of 1 mg (dry weight) ml21 and emul-sified in FIA at a ratio of 1 part suspended digest to 3 parts FIA. Aliquots of 0.5ml were injected into the left haunch of the rabbit at week 8 and again at week12. Antiserum was collected as described previously (12). To detect carbohydratein the pronase-digested ECP, Western blots were developed with the GlycoTrackcarbohydrate detection kit (Oxford GlycoSystems, Inc., Rosedale, N.Y.) as de-scribed in manufacturer’s instructions or the blots were subjected to periodate(0.5 mM) oxidation as described elsewhere (28), except that TTBS was used forthe final wash (three times) prior to development with the antiserum preparedagainst whole cells boosted with protein-free cell lysate as the first binding agent.To examine cross-reactivity of the antiserum prepared against whole cellsboosted with protein-free cell lysate, this antiserum was used as the first bindingagent to develop Western blots of the lipopolysaccharide, whole outer mem-brane, and extracellular fractions isolated from gram-negative bacteria as re-ported previously (10).

RESULTS

Instability of R. salmoninarum extracellular polypeptides.Western blots of the R. salmoninarum whole ECP (Fig. 1, lanee) showed a typical profile (25) of the p57 protein, its break-down products, and larger-molecular-mass aggregates. To ob-serve instability within various p57 derivatives, fractions of the

ECP, separated on the basis of molecular mass to contain alimited number of these polypeptides, were examined by West-ern blotting. Most of the fractions (Fig. 1, lanes 1 to 4) con-tained fragments of p57 smaller than the expected molecularmass for the material in the eluted fraction, suggesting contin-ued degradation within the separated polypeptides. Also, mostfractions (Fig. 1, lanes 2 to 6) contained immunoreactive pep-tides migrating above the range of expected molecular mass,suggesting that aggregates of p57 or its fragments are notcompletely dissociated during SDS-PAGE. This is supportedby the observation that fractions isolated from the larger-mo-lecular-mass zone above p57, when subjected to further SDS-PAGE (Fig. 1, lane 1), were found to contain immunoreactivepeptides migrating to the positions of p57 and its breakdownproducts. Western blots of the ECP fractions isolated fromPMSF-stabilized ECP contained the larger-molecular-mass ag-gregates but much less of the degradation products (e.g., Fig.1B), showing that PMSF inhibited degradation within the sep-arated fractions. Similar results were obtained with both R.salmoninarum isolates used in the study, although the extent ofECP degradation and the number of degradation productsdetected varied with different ECP preparations from eachstrain.

R. salmoninarum extracellular proteolytic activity. ECPpreparations were subjected to one-dimensional G-PAGE tolocate proteolytic activity. For both R. salmoninarum strains,usually a broad band of proteolytic activity was seen at .100kDa (Fig. 2A, lane 2) as reported previously (22). However, inseveral ECP preparations, a variable number of additionalproteolytic bands from .100 kDa to approximately 18 kDawere also detected (Fig. 2A, lane 1). The location of proteo-lytic bands (e.g., at approximately .57, 57, 37, 33, and 18 to 22kDa) was similar to that of some of the major p57 derivativesdetected on Western blots (e.g., Fig. 1, lanes 2 to 7). The ECPpolypeptides were subjected to SDS-PAGE, and gel strips con-taining the separated polypeptides were incubated (4 h at 20°)prior to two-dimensional electrophoretic separation. The two-dimensional G-PAGE profiles of the proteolytic bands (Fig.3A) resembled two-dimensional Western blot profiles of thep57 derivatives (Fig. 3B) in that some proteolytic activity wasobserved to migrate both below and above the expected mo-lecular mass positions (diagonal) of the proteolytic bands onthe two-dimensional gel.

FIG. 1. Western blot of separated fractions of R. salmoninarum F91 ECP.(A) The fractions were separated from whole ECP (lane e) from locationsbetween the arrowheads shown on the left of the figure. Fractions 1 to 6 areshown in lanes 1 to 6, respectively. Lane 7 contains prestained molecular massmarkers including phosphorylase b (140 kDa), BSA (87 kDa), ovalbumin (48kDa), carbonic anhydrase (34 kDa), soybean trypsin inhibitor (29 kDa), andlysozyme (21 kDa). The blot was developed with a monoclonal anti-57-kDaprotein antibody, and the location of the 57-kDa protein is indicated on the rightof the figure. (B) The Western blot containing fraction 2 separated from ECPstabilized by the addition of PMSF (1.4 mM) prior to separation is shown.

VOL. 63, 1997 R. SALMONINARUM EXTRACELLULAR PRODUCTS 3771

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

Effects of inhibitors and reducing agents on the R. salmoni-narum extracellular proteolytic activity. Digestion of the au-tologous p57 protein in the R. salmoninarum ECP was re-ported to be inhibited by 1 to 2 mM PMSF (6, 12, 22). Also,digestion of unrelated proteins such as BSA by the ECP pro-teolytic activity was reported to require or was stimulated bythe addition of SDS or DTT (22). The connection betweenthese observations and the multiple proteolytic bands detectedin the ECP was investigated.

For both R. salmoninarum strains, addition of PMSF to theECP inhibited the activity of all proteolytic bands digestinggelatin on G-PAGE gels, and higher concentrations of PMSF(6 mM) were required to inhibit the proteolytic activity againstgelatin in ECP preparations containing DTT (10 mM) (Fig.2B). Similarly, stimulation of BSA digestion by the ECP pro-teolytic activity in the presence of DTT (10 mM) (Fig. 4A, lane9) was partially prevented by PMSF at concentrations below 6mM (e.g., 1.4 mM) (Fig. 4A, lane 7). Similar effects on BSAdigestion were observed when other sulfhydryl group protein-ase inhibitors, including iodoacetate (10 mM), iodoacetamide(10 mM), N-ethylmaleimide (10 mM), and 2,2-dipyridyl disul-fide (0.25 mM), replaced PMSF (data not shown). Also, auto-digestion of the autologous p57 protein derivatives in the ECP

was stimulated by DTT (Fig. 4B) and inhibited by the sulfhy-dryl group proteinase inhibitors mentioned above in a similarmanner (data not shown).

R. salmoninarum extracellular polysaccharide-like material.To determine the nature of extra nonproteinaceous material inthe ECP from both strains, Western blots of pronase-digestedECP were developed with rabbit polyclonal antisera preparedagainst either the whole ECP or R. salmoninarum cells withprotein-free cell lysate boosters. The antiserum to ECP reactedstrongly with the p57-derived polypeptides, which were subse-quently removed by treatment with pronase (Fig. 5A). The useof antiserum to R. salmoninarum cells boosted with protein-free cell lysate and higher ECP concentrations better revealedthe presence of a pronase-resistant ladder-like series of bandsas a major constituent in ECP mainly at positions below 40kDa (Fig. 5B), typical of bacterial capsular polysaccharide (foran example, see reference 19). The polysaccharide-like mate-rial was detected also on Western blots of water-soluble ma-terial from the bacterial cell surface (data not shown). Westernblots of pronase-digested kidney tissue homogenates from in-fected Atlantic salmon (S. salar L.) revealed the presence ofsimilar polysaccharide-like material (Fig. 5E), which was notdetected in kidney tissue homogenates from fish without clin-ical signs of BKD. The presence of carbohydrate moietiesassociated with this polysaccharide-like material was detected

FIG. 2. G-PAGE of ECP from R. salmoninarum collected in PBS containing10 mM DTT. (A) ECP preparation from strains WC (lane 1) and F91 (lane 2);(B) ECP preparation from strain WC to which 0 (lane 2), 2 mM (lane 3), 4 mM(lane 4), and 6 mM (lane 5) PMSF had been added prior to electrophoresis. Eachlane contained 60 mg of ECP protein, and following electrophoresis, the gelswere washed and incubated overnight at 20°C before staining with Coomassieblue and destaining. Prestained molecular mass markers as described in thelegend to Fig. 1 are indicated (lane 1).

FIG. 3. Two-dimensional separation of the R. salmoninarum WC extracellu-lar proteolytic activity (A) and polypeptides (B). (A) ECP (125 mg of protein)from the ECP preparation described in the legend to Fig. 2 were separated in thefirst dimension by SDS-PAGE and in the second dimension by G-PAGE. Fol-lowing electrophoresis, the gel was treated as described in the legend to Fig. 2 todetect proteolytic activity. One-dimensional separation of prestained molecularmass markers (as described in the legend to Fig. 1) on the two-dimensional gelis shown to the right of the panel. (B) ECP (75 mg of protein) were separated inboth dimensions by SDS-PAGE prior to Western blotting to detect polypeptides.

FIG. 4. Effects of DTT and PMSF on the digestion of BSA (A) or autologousextracellular polypeptides (B) by the ECP proteolytic activity of R. salmoninarumF91. (A) Coomassie blue-stained SDS-PAGE profiles of BSA (5 mg ml21)before and after incubation for 12 h at 35°C with ECP (225 mg of protein ml21)(lanes 2 and 3), ECP plus 1.4 mM PMSF (lanes 4 and 5), ECP plus 1.4 mMPMSF, and 10 mM DTT (lanes 6 and 7), and ECP plus 10 mM DTT (lanes 8 and9). Molecular mass markers including BSA (67 kDa), ovalbumin (43 kDa), andcarbonic anhydrase (30 kDa) are shown in lane 1. (B) Western blot of ECP (135mg of protein ml21) incubated at 35°C in the presence of 10 mM DTT for 0 (lane2), 2 (lane 3), 4 (lane 4), and 6 (lane 5) h or in the absence of DTT for 0 (lane6), 2 (lane 7), 4 (lane 8), and 6 (lane 9) h. Samples were applied to give anequivalent of 4 mg of protein at 0 h of incubation. Biotinylated molecular massmarkers including BSA (69 kDa), ovalbumin (45 kDa), carbonic anhydrase (31kDa), and soybean trypsin inhibitor (21 kDa) are shown in lane 1.

3772 BARTON ET AL. APPL. ENVIRON. MICROBIOL.

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

on Western blots (Fig. 5C) by use of a carbohydrate detectionsystem (GlycoTrack; Oxford GlycoSystems). Furthermore, pe-riodate treatment (28) to disrupt these carbohydrate moietiesblocked the immunoreaction of the antiserum with the poly-saccharide material (Fig. 6). Interestingly, this antiserum pre-pared against R. salmoninarum cells boosted with protein-freecell lysate cross-reacted on Western blots with O polysac-chaides in outer membrane lipopolysaccharide fractions fromA. salmonicida but not with O polysaccharides from othergram-negative bacteria tested, i.e., V. ordallii or P. fluorescens(Fig. 7). No cross-reaction of this antiserum with the A. sal-monicida extracellular or outer membrane proteins was de-tected (data not shown).

DISCUSSION

Polypeptides in the R. salmoninarum ECP, consisting mainlyof p57, its breakdown products, and aggregates, are digested byan autologous proteolytic activity (22). In the current study, therelease of p57-derived fragments from separated ECP compo-nents suggested that proteolytic activity is present in or is acore component of many molecular mass fractions. The abil-ity of PMSF to inhibit the generation of fragments withinthe separate fractions supported this suggestion. Interesting-ly, fractions containing p57 or its breakdown products alsoappeared to form larger-molecular-mass aggregates.

The use of one-dimensional G-PAGE to locate ECP proteo-lytic activity against gelatin produced variable results with dif-ferent ECP preparations. Often, a broad band of proteolyticactivity was detected at .100 kDa as reported previously (22).

However, a variable number of additional proteolytic bands atpositions as low as approximately 18 kDa were also often seenat locations occupied by some of the major p57 derivatives.The observation that some proteolytic activity migrated belowand above the expected molecular mass positions for several ofthese proteolytic bands on two-dimensional G-PAGE gels im-

FIG. 5. Western blot detection of extracellular polysaccharide produced by R. salmoninarum. ECP (300 mg of protein ml21) from R. salmoninarum F91 (A, B, andC) or kidney tissue homogenate (50 ml ml21) from Atlantic salmon (S. salar L.) clinically infected with R. salmoninarum (D and E) were digested with pronase (2 mgml21) at 35°C. Samples equivalent to 8 (A) and 40 (B and C) mg of ECP protein or to 0.25 (D) and 0.5 (E) ml of kidney tissue at 0 h of incubation were applied togels after 1 (lanes 1), 3 (lanes 2), 6 (lanes 3), and 24 (lanes 4) h of incubation with pronase. The Western blots of panels A and D were developed with polyclonalanti-whole R. salmoninarum ECP antiserum, whereas blots of panels B and E were developed with polyclonal anti-pronase-digested cell lysate-boosted whole-cellantiserum. The blot of panel C was developed with a carbohydrate detection system (GlycoTrack; Oxford GlycoSystems). Prestained molecular mass markers asdescribed in the legend to Fig. 1 are shown (B and C, lanes 5), and the position of the 57-kDa protein is indicated to the left of the figure. Native molecular mass markersreacting with the GlycoTrack system, including BSA (67 kDa), ovalbumin (43 kDa), and a-lactalbumin (14.4 kDa), are also shown (C, lane 6).

FIG. 6. Western blot of the effect of periodate oxidation on the immunode-tection of the R. salmoninarum extracellular polysaccharide. ECP of R. salmo-ninarum F91 were digested with pronase for 24 h as described in the legend toFig. 5, and samples equivalent to 30 mg of ECP protein prior to digestion wereapplied to the gels (lanes 1). Samples of undigested ECP, equivalent to 10 mg ofECP protein, are also shown (lanes 2). (A) The blot was subjected to periodateoxidation by the method of Woodward et al. (28). (B) The blot was subjected tothe same procedure as that used for panel A except that periodate was omittedfrom the acetate buffer. The blots in both panels were developed with thepolyclonal anti-pronase-digested cell lysate-boosted whole-cell antiserum.

VOL. 63, 1997 R. SALMONINARUM EXTRACELLULAR PRODUCTS 3773

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

plied that degradation and aggregation occurred within theproteolytic bands after their separation in the first dimension.Variations in the proteolytic bands (and peptide bands) ob-served in different ECP preparations may be due, in part, tothe effects of processing the samples by SDS-PAGE as well asthe long incubation times at 15°C required to cultivate R. sal-moninarum (12, 22). However, the resemblance of essentiallyall of the observable proteolytic activity both in its locationsand behavior on two-dimensional G-PAGE gels to that ob-served for several of the p57-derived polypeptides on two-dimensional Western blots suggested that the proteolytic ac-tivity was associated with p57 and was retained by severalbreakdown products of its autolysis. Not all p57 breakdownproducts appeared to be associated with proteolytic activity.This might be expected because a number of the fragmentswould not contain the active portion of the molecule. Autolysisof p57 would explain the finding that fusion proteins expressedfrom plasmids containing most of the p57 gene inserted intoE. coli exhibit instability similar to that of the native p57 pro-tein in the R. salmoninarum ECP (9). Polypeptides, retainingproteolytic activity, were not observed to migrate below 18 to22 kDa. Cleavage of larger polypeptides with the release ofdegradation products, some retaining proteolytic activity, con-tinued after their electrophoretic separation. Therefore, theproteolytic activity observed at various molecular mass loca-tions on G-PAGE gels may be the result of cleavage of thelarger separated polypeptides (perhaps inactive precursors)with the release of proteolytically active fragments (18 to 22kDa) during their incubation in the gels. This is presentlyunder investigation.

The ability of PMSF to inhibit proteolytic activity againstgelatin in all of the p57 derivatives and of DTT to prevent thisinhibition indicated that a common mechanism existed amongthe active polypeptides. Also, DTT appeared to activate ECPproteolytic activity against both an unrelated protein, BSA,and the autologous p57 protein and to be able to prevent theinhibition caused by PMSF. These results could be interpretedas being characteristics of a thiol proteinase (20). Inhibition ofthe ECP proteolytic activity by other sulfhydryl group-reactive

inhibitors lends support to this interpretation. Accordingly,one reason for the variable detection of proteolytic bands indifferent ECP preparations by G-PAGE may be oxidation orinactivation of an enzyme relying on sulfhydryl groups for itsactivity. Also, this may explain the apparent stability of p57isolated by isoelectric focusing (22), a procedure reported toinactivate thiol proteinases under nonreducing conditions (15).

During the course of these investigations, polysaccharidewas detected as a major component of the R. salmoninarumECP and in kidney tissue from Atlantic salmon clinically in-fected with the pathogen. The pronase-resistant, carbohydrate-positive series of bands migrating mainly below 40 kDa onWestern blots of the ECP are characteristic of polysaccharide(19) and appeared to represent released capsular material re-ported to cover R. salmoninarum cells in addition to a surface-located fraction of p57 (5). Periodate oxidation (28) of thisECP material blocked its immunodetection on Western blots,which further confirmed its polysaccharide nature. Wood andKaattari (26) reported that formalin-killed cells of R. salmoni-narum, depleted of p57, produced enhanced antibody re-sponses to cell surface carbohydrate moieties in chinooksalmon (O. tshawytscha), suggesting the moieties were large-ly masked by p57. These immunogenic cell surface carbohy-drate moieties may be representative of the polysaccharidematerial detected in the ECP. Cross-reaction of antiserumagainst the R. salmoninarum ECP polysaccharide with the Opolysaccharides from A. salmonicida, an unrelated pathogen ofsalmonids, suggested some structural similarity in the polysac-charides which may be associated with common cellular recep-tors in host fish. Interestingly, it has been reported (18) thatformalin-killed R. salmoninarum cells produced a potentiatingeffect in protecting coho salmon (O. kisutch), vaccinated witha formalin-killed A. salmonicida bacterin, against subsequentchallenge with A. salmonicida.

The results presented here show that proteolytic activity isassociated with p57 and several of its derivatives. Becausethese polypeptides are found in large concentrations in tissuesof salmonids infected with R. salmoninarum (22) along with theextracellular polysaccharide, further studies on the potentialroles of the proteolytic activity and the polysaccharide will beimportant to the eventual understanding of the pathogenesis ofBKD infections and may lead to the development of potentialvaccines.

ACKNOWLEDGMENTS

The research was supported by a grant from the Natural Sciencesand Engineering Research Council of Canada.

We gratefully acknowledge the capable technical assistance ofJ. Roy and M. Adams (Research and Productivity Council of NewBrunswick) and B. Forward and K. Wong (University of New Bruns-wick).

REFERENCES

1. Austin, B., T. M. Embley, and M. Goodfellow. 1983. Selective isolation ofRenibacterium salmoninarum. FEMS Microbiol. Lett. 17:111–114.

2. Bruno, D. W. 1988. The relationship between autoagglutination, cell surfacehydrophobicity and virulence of the fish pathogen Renibacterium salmonina-rum. FEMS Microbiol. Lett. 58:135–140.

3. Bruno, D. W. 1990. Presence of a saline-extractable protein associated withvirulent strains of the fish pathogen Renibacterium salmoninarum. Bull. Eur.Assoc. Fish Pathol. 10:8–10.

4. Daly, J. G., and R. M. W. Stevenson. 1990. Characterization of the Renibac-terium salmoninarum haemagglutinin. J. Gen. Microbiol. 136:949–953.

5. Dubreuil, D., R. Lallier, and M. Jacques. 1990. Immunoelectron microscopicdemonstration that Renibacterium salmoninarum is encapsulated. FEMS Mi-crobiol. Lett. 66:313–316.

6. Dubreuil, D. J., M. Jacques, L. Graham, and R. Lallier. 1990. Purification,and biochemical and structural characterization of a fimbrial haemagglutininof Renibacterium salmoninarum. J. Gen. Microbiol. 136:2443–2448.

FIG. 7. Western blot showing cross-reactivity of the polyclonal antiserumprepared against pronase-digested R. salmoninarum cell lysate-boosted whole-cell antiserum with O polysaccharides from A. salmonicida. Lipopolysaccharidefractions equivalent to 8 mg of outer membrane protein from A. salmonicida Asa92 (lane 1), A. salmonicida Ast5 (lane 2), P. fluorescens F (lane 3), and V. ordalliiB (lane 4) or extracellular products equivalent to 100 mg of protein from R.salmoninarum F91 (lane 5) were subjected to Western blotting after digestionwith pronase. Prestained molecular mass markers as described in the legend toFig. 1 are shown in lane 6.

3774 BARTON ET AL. APPL. ENVIRON. MICROBIOL.

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

7. Evenden, A. J., T. H. Grayson, M. L. Gilpin, and C. B. Munn. 1993. Reni-bacterium salmoninarum and bacterial kidney disease—the unfinished jigsaw.Annu. Rev. Fish Dis. 3:87–104.

8. Getchell, R. G., J. S. Rohovec, and J. L. Fryer. 1985. Comparison of Reni-bacterium salmoninarum isolates by antigenic analysis. Fish Pathol. 20:149–159.

9. Grayson, T. H., A. J. Evenden, M. L. Gilpin, and C. B. Munn. 1995. Pro-duction of the major soluble antigen of Renibacterium salmoninarum inEscherichia coli K12. Dis. Aquat. Org. 22:227–231.

10. Griffiths, S. G., and W. H. Lynch. 1989. Characterization of Aeromonassalmonicida mutants with low-level resistance to multiple antibiotics. Anti-microb. Agents Chemother. 33:19–26.

11. Griffiths, S. G., and W. H. Lynch. 1990. Characterization of Aeromonassalmonicida variants with altered cell surfaces and their use in studyingsurface protein assembly. Arch. Microbiol. 154:308–312.

12. Griffiths, S. G., and W. H. Lynch. 1991. Instability of the major solubleantigen produced by Renibacterium salmoninarum. J. Fish Dis. 14:55–66.

13. Griffiths, S. G., G. Olivier, J. Fildes, and W. H. Lynch. 1991. Comparison ofWestern blot, direct fluorescent antibody and drop-plate culture methods forthe detection of Renibacterium salmoninarum in Atlantic salmon (Salmosalar L.). Aquaculture 97:117–129.

14. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual, p. 154–155.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

15. Jacobs, S. 1973. Effect of isoelectric focusing on the amino acid compositionof proteins. Analyst 98:25–33.

16. Laemmli, J. K. 1970. Cleavage of structural proteins during assembly of thehead of bacteriophage T4. Nature 227:680–685.

17. Lovely, J. E., C. Cabo, S. G. Griffiths, and W. H. Lynch. 1994. Detection ofRenibacterium salmoninarum infection in asymptomatic Atlantic salmon. J.Aquat. Anim. Health 6:126–132.

18. Nikl, L., L. J. Albright, and T. P. T. Evelyn. 1991. Influence of seven

immunostimulants on the immune response of coho salmon to Aeromonassalmonicida. Dis. Aquat. Org. 12:7–12.

19. Pelkonen, S., and J. Finne. 1989. Polyacrylamide gel electrophoresis ofcapsular polysaccharides of bacteria. Methods Enzymol. 179:104–110.

20. Rawlings, N. D., and A. J. Barrett. 1994. Families of cysteine peptidases.Methods Enzymol. 244:461–486.

21. Rockey, D. D., L. L. Gilkey, G. D. Wiens, and S. L. Kaattari. 1991. Mono-clonal antibody-based analysis of the Renibacterium salmoninarum antigen Fprotein in spawning chinook (Oncorhynchus tshawytscha) and coho salmon(O. kisutch). J. Aquat. Anim. Health 3:23–30.

22. Rockey, D. D., P. S. D. Turaga, G. D. Wiens, B. A. Cook, and S. L. Kaattari.1991. Serine proteinase of Renibacterium salmoninarum digests a majorautologous extracellular and cell-surface protein. Can. J. Microbiol. 37:758–763.

23. Szewczyk, B., and D. Summers. 1988. Preparative elution of proteins blottedto Immobilon membranes. Anal. Biochem. 108:48–53.

24. Turaga, P. S. D., G. D. Wiens, and S. L. Kaattari. 1987. Analysis of Reni-bacterium salmoninarum antigen production in situ. Fish Pathol. 22:209–214.

25. Wiens, G. D., and S. L. Kaattari. 1991. Monoclonal antibody characteriza-tion of a leukoagglutinin produced by Renibacterium salmoninarum. Infect.Immun. 59:631–637.

26. Wood, P. A., and S. L. Kaattari. 1996. Enhanced immunogenicity of Reni-bacterium salmoninarum in chinook salmon after removal of the bacterialcell surface-associated 57 kDa protein. Dis. Aquat. Org. 25:71–79.

27. Wood, S. C., R. N. McCashion, and W. H. Lynch. 1986. Multiple low-levelantibiotic resistance in Aeromonas salmonicida. Antimicrob. Agents Chemo-ther. 29:992–996.

28. Woodward, M. P., W. W. Young, Jr., and R. A. Bloodgood. 1985. Detectionof monoclonal antibodies specific for carbohydrate epitopes using periodateoxidation. J. Immunol. Methods 78:143–153.

VOL. 63, 1997 R. SALMONINARUM EXTRACELLULAR PRODUCTS 3775

on July 2, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from