Toxins produced by Aeromonas hydrophila strain A10 in the muscle of carp at the site of injection were studied. Carps injected with the crude toxins showed swelling and reddening of the body surface as those injected with the live bacteria. The toxic substance was partially purified from a extract of the necrotized muscles by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration. As a result, it was suggested that α-type hemolysin was implicated in the toxicity. The hemolysin was heat-labile, and inactivated by EDTA, trypsin and papain, but stable at pH value between 4.0 and 11.2

Kanai Kinya

Takagi Yasuki

Nagasaki University's Academic Output SITE: NAOSITE

j!!o.;\jijliJ!~ Fish Pathology 21 (4) 245-250, 1986. 12Alpha-hemolytic Toxin of Aeromonas hydrophila Produced in VivoKinya KANAI and Yasuki TAKAGIFaculty of Fisheries, Nagasaki University,1-14 Bunkyo-machi, Nagasaki 852, Japan(Received July 3, 1986)Toxins produced by Aeromonas hydrophila strain A10 in the muscle of carp at the site of injec-tion were studied. Carps injected with the crude toxins showed swelling and reddening of the bodysurface as those injected with the live bacteria. The toxic substance was partially purified from aextract of the necrotized muscles by ammonium sulfate precipitation, ion-exchange chromatographyand gel filtration. As a result, it was suggested that a-type hemolysin was implicated in the toxicity.The hemolysin was heat-labile, and inactivated by EDTA, trypsin and papain, but stable at pHvalue between 4.0 and 11.2.Aeromonas hydrophila is an important pathogenof freshwater fishes. The disease was charac-terized by hemorrhages of the body surface andformation of hemorrhagic and necrotic regions inthe internal organs (EGUSA, 1978). From bac-teriological and histopathological studies, the siteof bacterial multiplication was generally con-fined, e.g. the eyes in the infectious disease of ayu(MIYAZAKI and Jo, 1985) and the intestines in reddisease of eel (KANAI et ai., 1977; MIYAZAKI,1980). Therefore it seemed that these pathologicalchanges were due to the actions of some toxicsubstances produced by the bacterium. In fact,A. hydrophila is known to produce various toxicsubstances such as hemolysins (BERNHEIMER andAVIGAD, 1974; LJUNGH et al., 1981; BUCKLEYet al., 1981), proteases (THUNE et al., 1982; KANAIand WAKABAYASHI, 1984) and enterotoxin(JOHNSON and LIOR, 1981; BURKE et al., 1981) inartificial media. However, their productions havenot been demonstrated in vivo.As a result of several trials for extracting toxicsubstances from the artificially infected fish, A.hydrophila strain AI0 was found to produce suchan extractable toxin in vivo. An injection of thetoxin to carp caused swelling and reddening of itsbody surface. This report describes partial puri-fication and characterization of the toxin.Materials and MethodsBacterial strainA. hydrophila strain AIO, isolated from theintestine of a diseased eel in 1979, was used in thisstudy. The organism was suspended in nu-trient broth added with 10% glycerin and storedat -80°C to preserve its toxin productfvity. Thestrain is known to produce hemolysin and proteasein vitro (WAKABAYASHI et al., 1981).Production of in vivo toxinsTwenty-four to 48-hour-old culture of the or-ganism on nutrient agar was suspended in 0.01 Mphosphate buffered saline, pH 6.8, (PBS) at aconcentration of 0.01 mg/ml by wet weight.Carp weighing 30 to 80 g were injected intramus-cularly through several points of the dorsal areawith 0.1 ml of the cell suspension per point. Theinoculated fish were placed in an aquarium at23 t025°C. After 12 to 24 hr dying fish weredipped up out of water, and the muscle of thedorsal area was excised and homogenized with 3volumes (w/v) of ice-cooled PBS. After cen-trifugation at 10,000 g for 30 min. the supernatantfluid was sterilized by millipore filter and storedat 4°C.Partial purification of in vivo toxinsOne hundred to 120 ml of crude extracts ob-tained from five carps were used as a starting246 K. KANAI and Y. TAKAGImaterial.Solid ammonium sulfate was added to the crudeextracts to 30 % saturation. The precipitate wasremoved by centrifugation, and ammonium sulfatewas added again until the saturation reached60 %. The p,:ecipitate was collected and dissolvedin a small volume of 0.01 M phosphate buffer,pH 7.8. The solution was dialyzed against thephosphate buffer and applied to a DEAE-Toyo-pearl 650M (Toyo Soda) column, equilibratedwith the same buffer. Elution was performedwith a linear gradient of 0 to 0.5 M NaCI. Frac-tions which possessed toxic activities were pooledand cqncentrated by 30 % solution of polyethyl-eneglycol 20,000 and dialyzed against 0.1 Mphosphate buffer, pH 6.8. The dialyzed solutionwas applied to a Sephacryl S-300 column (2.5 x90 cm), equilibrated with the phosphate bufferand eluted with the same buffer. Fractions whichpossessed toxic activities were pooled and storedat 4°C.Seven reserves on the way to purification werecombined and concentrated with polyethyl-eneglycol solution. The concentrated and dialyzedsolution was subjected to re-gel filtration onSephacryl S-300 by the same way as above.Toxic fractions eluted from the column weredesignated as a partially purified toxin.Preparation 0/ in vitro toxinsBrain heart infusion broth (Nissui) was sup-plemented with filter sterillzed ammonium sulfateto a final concentration of 2 % for reducingprotease production (Lru and HSIEH, 1969).Fresh culture of strain AI0 was inoculated to themedium and shaken for 20 hr on a reciprocalshaker at 25°C. Cells harvested by centrifuga-tion were washed twice with 0.85 % NaCI solu-tion and suspended in 0.05 M phosphate buffer,pH 6.8, at a concentration of 0.5 glml by wetweight. The cell suspension was subjected tosonic disruption for 5 min. per 1 m!. The super-natant was dialyzed against the phosphate bufferand stored at 4°C with 0.02 % sodium azide as apreservative.The culture supernatant was added with am-monium sulfate to 66.7 % saturation. The pre-cipitate was dissolved in 0.05 M phosphate buffer,pH 6.8, and dialyzed against the buffer. Theclear solution was stored at 4°C with 0.02%sodium azide.Measurement 0/ enzyme activityHemolytic activity on rabbit erythrocytes wasassayed in PBS using a serial twofold dilutionmethod. Mixture of 0.5 ml of sample and 1%suspension of the erythrocytes was incubated for1 hr at 37°C followed by for 2 hr in an ice-cooledbath. Hemolytic unit (HU1m!) was expressed asthe reciprocal of the highest dilution which lysedat least 50 % of the erythrocytes.Proteolytic activity was determined accordingto KANAI and WAKABAYASHI (1984), using caseinas a substrate.Toxicity testToxic activities of preparations were studied byinjecting intramuscularly carp (10 to 30 g) witha dosage of 0.5 ml per 100 g body weight.Skin reaction was examined by injecting in-tracutaneously to the dorsal area of guinea-pigwith a dosage of 0.1 m!.Polyacrylamide gel electrophoresisDisc electrophoresis was carried out as de-scribed by DAVIS (1964), using 7.5% gels with aconstant current of 2 mAoResultsToxic activities were recognized in the hindpart of a large peak eluted from the secondchromatography on Sephacryl S-300 (Fig. 1).These fractions were accompanied with hemolyticactivies, and the fractions which contained morethan 512 HU/ml were toxic to carp (Table 1).The concentrated solution of a pool of the frac-tions between 43 and 49 was used as a partiallypurified toxin, which was separated to one majorand several minor bands by polyacrylamide gelelectrophoresis. The partially purified toxinpossessed an hemolytic activity of 1024 HUImland a proteolytic activity of 22 p,g/m!.Carp injected with live bacteria of strain AI0died, showing swelling and scale protrusion at theinjected site and characteristic reddening of bodysurface, especially the head and the abdomen(Fig. 2A). Upon injection of the partially purifiedtoxin with 256 HU per 100 g body weight, quitesimilar symptoms to those developed by liveAlpha-hemolytic Toxin of A. hydrophila 247A2805.0Afll'IIIIIIIIITIf[ffijlrnnlrljlllljlllllllll[IIIIIllfIjllllllllllllll11111111111111111111111111123. 56789---<>-·HU/ml1024512256128643216<1610 20 30 40 50 60 70 80 90Fraction numberO'---'----'---.L......:::-'----'-'=......--'---'--_.J.......Jo2.03.04.01.0Fig. 1. Re-gel filtration of the toxic fractions fromthe first chromatography on a Sephacryl S- B300 column. Column size, 2.5 x 90 cm;buffer, 0.1 M phosphate, pH 6.8; fraction,5 ml/tube.Table 1. Hemolytic activity and tOXICIty of thefractions from re-chromatography on Se-phacryl S-300Fraction*' Hemolyticnumber activityToxicity*2(HU/m/)39 <16 *341 <1643 3245 512 +47 1024 +49 512 +51 <1653 <16Fig. 2. A. Carp with reddish head and abdomen,20 hI' after an inoculation of the live bacteriaof A. hrdrophila strain AIO. B. Carp withreddish head and abdomen, 6 hI' after aninjection of the intramuscularly producedtoxin of A. hydrophila.*1 See Fig. ].+ Swelling at the injected site and reddening ofthe body surface were occurred upon intramu-scular injection to carp.- No symptoms were observed.*3 In this case toxicity was tested about the con-centrated solution of the pooled fractions be-tween 39 and 42.bacteria were observed within 8 hI' (Fig. 2B).Severe hemolysis of the peripheral blood was alsoobserved as well as in the case of the live bacteria.However, these fish merely died and recoveredwithin a week. In some cases experimental fishlaid themselves down to the bottom of anaquarium immediately after injection and some-times showed twitching motion as if the motornerves injured. But most fish did not showreddening of the body surface nor die.Injection site' of guinea-pig skin became togreenish yellow in color upon Injections of thepartially purified toxin. Minimum reaction dosewas 6.4 HU per injection. Hemorrhages were notobserved upon injections of the toxin up to 102.4HU.Hemolytic activities of a sonic extract of thebacterial cells and a concentrate of the culturesupernatant were estimated to be 512 HU/m!.and 8192 HU/m/, respectively. Swelling and scaleprotrusion at the injection sites were observedupon intramuscular injections of both the cell-extract and the culture supernatant to carp witha dosage of 256 HUI and 4096 HU per 100 g bodyweight, respectively. However, these fish did notshow reddening of the body surface.Susceptibilities of the erythrocytes of variousanimal species to the partially purified toxin wereshown in Table 2. Among six species rabbiterythrocytes were most sensitive. To the cell-248 K. KANA! and Y. TAKAGITable 2. Sensitivity of erythrocytes from differentspecies to the intramuscularly producedtoxin (1M), culture supernatant (CS), andsonic extract of cells (CE) of A. hydrophilaHemolytic activity (HUjml)Species1M CS CERabbit 1024 128 16Guinea-pig 128 32 64Human 128 32 32Eel 32 32 64Horse 8 128 8Sheep 8 16 8Table 4. Effect of pH on the stability of the intra-muscularly produced toxin of A. hydro-phila.*RemainedpH Buffer activity(HUjml)3.0 0.1 M glycine-HCI <164.0} 0.1 M2565.2acetate2566.8 0.1 M phosphate 256lO.O} 0.1 M bicarbonate-NaOH 25611.2 2561l.6} 0.1 M phosphate-NaOH6412.0 <16Fig. 3. Formation of hemolytic zones by hemolysins,incubated for 6 hr at 28°C in an agar platecontaining 2% rabbit erythrocytes. The in-tramuscularly produced toxin (1M) inducedan opaque zone, and sonic extract of cells(CE) and culture supernatant (CS) inducedclear zones.Table 3. Effect of heating on the stabilityintramuscularly produced toxinhydrophila.*Temperature(0C)3037455053576070of theof A.Remained activity(HUjml)5125125125123216<16<16* After dialyzing at each pH overnight, sampleswere further dialyzed to 0.1 M phosphate buffer,pH 6.8.extract both horse and rabbit erythrocytes weresensitive. On the contrary the culture supernatantwas active to the erythrocytes of guinea-pig andeel, but insensitive to those of horse and rabbit.Mode of hemolysis was studied in an agarplate containing 2% rabbit erythrocytes. As aresult, the partially purified toxin produced anopaque zone, i.e. incomplete hemolysis (a-type).On the other hand the cell-extract and the culturesupernatant produced transparent zones of he-molysis (ft-type) (Fig. 3).Hemolytic activity of the in vivo toxin wasstable to heat treatment at 50°C for 10 min., butabove 53°C reduction of the activity occurred(Table 3). The activity was stable at pH valuebetween 4.0 and 11.2 at 4°C (Table 4). Thetoxicity to carp was also heat-stable at 50°C, butinactivated by heating at 56°C for 5 min. Thetoxicity was stable at pH between 6.3 and 10.8,but dialysis of the toxin at pH 3.3 resulted in thereduction of the activity.Molecular weight estimated from the elutedposition in gel filtration was a value between40,000 and 50,000. EDTA and o-iodosobenzoicacid, an oxidizing agent of SH-group, inhibitedthe hemolytic activity. The hemolytic activitywas destroyed by trypsin and papain, but not bya-chymotrypsin (Table 5).Discussion* Sample (512 HUjml) was heated at each tem-perature for 10 min.Following observations suggested that hemo-lysin was implicated in the toxicity. First, hemo-Alpha-hemolytic Toxin of A. hydrophila 249Table 5. Effect of proteases and inhibitors on thestability of the intramuscularly producedtoxin of A. hydrophila.** Protease or inhibitor was added to sample in0.1 Mphosphate buffer, pH 6.8, and the mixturewas incubated for 1 hr at 3rC and dialyzed tothe buffer for two days.lysin activity was detected in accompany with thetoxicity throughout purification. Second, thetoxicity and the hemolytic activity showed re-semblant natures in heat and pH stability tests.Third, in vivo hemolysis was caused by an injec-tion of the toxin. Reddening of the body surface,one of the visible symptoms, would be due toexudation of hemoglobin from the capillaries.Mode of hemolysis of the toxin was a-type,because an opaque zone of hemolysis was formedin an agar plate containing erythrocytes. A.hydrophila was known to produce both a andj3-type hemolysins in vitro, which were designatedas a and j3-hemolysin, respectively (LJUNGH et al.,1981). The in vitro a-hemolysin was producedduring stationary phase of bacterial growth alongwith cell lysis (LJUNGH et a!., 1981; WERTLINDet al., 1973). This was different from the presenttoxin which was produced within 20 hr afterinoculation which corresponded to the logarithmicphase. But, because nutrient and growth con-dition were thought to be considerably differentbetween in vitro and in vivo, further study isnecessary to compare two toxins in detail, usingpurified materials.Beta-type hemolysin was produced duringlogarithmic phase intra- and extracellularly inBHI broth as described by LJUNGH et a!. (1981).However, the present a-hemolytic toxin wasProtease or inhibitorTrypsina-ChymotrypsinPapainEDTA 2Na0-PhenanthrolineN-Bromosuccinimidep-ChloromercuribenzoateMonoidooacetateo-Iodosobenzoic acidControlConcen-tration4 {tgjml4 {tgjml10 {tgjmlImMImMImM0.08mMImM1 roMRemainedactivity(HUjml)<16128<16<1612864128128<16128thought to be scarcely produced by this culturalmethod, because injection of the cell-extract orthe culture supernatant did not cause reddeningof the body surface. So a preliminary experimenton production of the toxin in vitro using carpmuscle was carried out. Cultivation techniqueswere as follows; a) fresh culture of A. hydrophilastrain AlO was inoculated into a fillet of muscleand incubated at 28°C for 24 hr in a sterile petridish, b) AID was inoculated into a fillet and in-cubated as a) except that a fillet was immersed inPBS in a petri dish, c) AlO was inoculated to thehomogenate of muscle and shaken at 28°C for24 hr, and d) the homogenate of muscle was cen-trifuged, and A10 was inoculated to the super-natant solution and shaken for 24 hr. As aresult, a-type, not j3-type, hemolysin was producedin vitro, but hemolytic titer was considerably dif-ferent among the four techniques, that is, in caseof a) the hemolytic titer was 16 HUjm/, b) was256 HUjm/, c) was 4 HUjm/, and d) was less than2 HUjm!. And upon injection to carp only theinjectant prepared by b) method caused reddeningof the body surface. These results suggest thatboth nutrient and cultural conditions influence theproduction of the toxin.Proteolytic or j3-hemolytic activity was hardlydetected in a crude extract of necrotic muscle, butthe present result did not negate the productionsof the protease and the j3-hemolysin in vivo,because there is a possibility that these substanceswere in inactive states as they had already adsorbedto substrates or inhibitors.AcknowledgmentsWe express our appreciation to Professor H.WAKABAYASHI, Department of Fisheries, Uni-versity of Tokyo, for reviewing the manuscript.This work was supported in part by a grant fromthe Ministry of Education, Science and Culture.ReferencesBERNHEIMER, A. W. and L. S. AVIGAD (1974): Partialcharacterization of aerolysin, a lytic exotoxin fromAeromonas hydrophila. Infect. Immun., 9, 1016-1021.BUCKLEY, J. T., L. N. HALASA, K. D. LUND and S.MAcINTYRE (1981): Purification and some pro-250 K. KANAI and Y. TAKAGIperties of the hemolytic toxin aerolysin. Can. J.Biochem., 59, 430-435.BURKE, V., J.ROBINSON, R. J. BERRY and M. GRACEY(1981): Detection of enterotoxin of Aeromonashydrophila by a suckling-mouse test. J. Med.Microbiol., 14, 401-408.DAVIS, B. J. (1964): Disc electrophoresis-II. Methodand application to human serum proteins. Ann.N. Y. Acad. Sci., 121, 404-427.EGUSA, S. (1978): Infectious Diseases of Fishes.Koseisha-Koseikaku, Tokyo, Japan, 554 pp.JOHNSON, W. M. and H. LIOR (1981): Cytotoxicityand suckling mouse reactivity of Aeromonas hydro-phila isolated from human sources. Can. J. Micro-bioi., 27, 1019-1027.KANAI, K. and H. WAKABAYASHI (1984): Purificationand some properties of protease from Aeromonashydrophila. Bull. Japan. Soc. Sci. Fish., 50, 1367-1374.KANAI, K., R WAKABAYASHI and S. EGUSA (1977):Comparison of intestinal microflora between healthyand diseased pond-cultured eels. Fish Path., 12,199-204.LIU, P. V. and H. C. HSIEH (1969): Inhibition ofprotease production of various bacteria by am-monium salts: Its effect on toxin production andvirulence. J. Bacteriol, 99, 406-413.LJUNGH, A., B. WRETLIND and R. MOLLBY (1981):Separation and characterization of enterotoxin andtwo haemolysins from Aeromonas hydrophila. ActaPhath. Microbiol. Scand. Sect. B, 89, 387~397.MIYAZAKI, T. (1980): Histopathological study onbacterial infections in fishes. Bull. Fac. Fish., MieUniv., 7, 63-149.MIYAZAKI, T. and Y. Jo (1985): A histopathologicalstudy of motile aeromonad disease in ayu. FishPath., 20, 55-59.THUNE, R. L., T. E. GRAHAM, L. M. RIDDLE and R.L. AMBORSKI (1982): Extracellular proteases fromAeromonas hydrophila: Partial purification andeffects on age-O channel catfish. Trans. Amer. Fish.Soc., 111, 749-754.WAKABAYASHI, H., K. KANAI, T. C. Hsu and S.EGUSA (1981): Pathogenic activities of Aeromonashydrophila biovar hydrophila (Chester) Popoff andVeron, 1976 to fishes. Fish Path., 15, 319-325.WRETLIND, B., L. HEDEN and T. WADSTROM (1973):Formation of extracellular haemolysin by Aeromonashydrophila in relation to protease and staphylolyticenzyme. J. Gen. Microbiol., 78, 57-65.

Aeromonas hydrophilaがコイの筋肉内で産生するα溶血性毒素

Kanai, Kinya

Takagi, Yasuki

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Aeromonas hydrophilaがコイの筋肉内で産生するα溶血性毒素

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