11 production and properties of a haemolytic toxin by vibrio anguillarum

8/13/2019 11 Production and Properties of a Haemolytic Toxin by Vibrio Anguillarum

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Production and Propertiesof aemolytic Toxin by Vibrio anguillarumC B MUNW

Introduction

Vibrio anguiliarum is an important pathogen of fish, especially marine fish rearedunder intensive conditions [2, 6]. The economic importance of this disease has

prompted considerable research into the development of vaccines for its control, andthese have met with partial success [3, 7]. However, very little is known of the biochemical basis of pathogenicity of V anguiliarum As well s the scientific value of understanding the fundamental mechanisms underlying the interactions between pathogensand their hosts [12], such knowledge can have practical importance, especially in thedevelopment of improved and more specific vaccines.

In those bacterial diseases of man and animals which have been well investigated,the production of a protein toxin or toxins by the pathogen has often been shown toplaya major role in pathogenesis [1].

The anaemic response of fish led Roberts [11] and Wolke [16] to postulate that ahaemolytic toxin played a key role in the pathogenesis of vibriosis. Most strains of Vanguiliarum are haemolytic when grown on blood agar, and the haemolysins and othermembrane-damaging toxins of many other bacteria have been isolated and proved tobe toxic [4, 9]. However, I would like to stress the difficultyofrelatingeffectsobserved in vitro with those in vivo [13], and considerable effort is required to prove a causal role for a toxin in the production of disease symptoms during natural infection.

In a previous paper [10] I described the isolation and partial purification of Vanguiliarum haemolysin. n this paper, I describe preliminary results of further studieson this protein toxin.

Materials and Methods

Isolation and Partial Purification of Haemolysin

V angui/iarum was cultivated in shake-culture for 22 h in a peptone-yeast extract medium, and crude haemolysin was prepared s previously described [10]. Gel chromatography was carried out on Sephacryl S-200 pharmacia) in sodium acetate 0.1 M),sodium chloride 0.2 M), pH 5.5 in a 90x1.5 cm column.

1 Plymouth Polytechnic, Plymouth PL4 BAA England

W. Ahne (ed.), Fish Diseases Springer-Verlag Berlin Heidelberg 1980


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70 C.B. Munn

Haemolysin Assays

Erythrocytes were washed thoroughly in phosphate-buffered saline (PBS) and resuspended to a density which gave an absorbance at 540 nm of 0.8 when lysed with an

equal volume of SDS (0.025%). Assays were performed in microtitre trays, 0.1 mloferythrocyte suspension being added to 0.1 ml of diluted haemolysin, incubated a t37C for 1 h, and the 50 endpoint estimated visually.

Kinetics of Lysis

For kinetic studies of lysis, equal volumes I.8 ml) of haemolysin and standardizederythrocyte suspension were mixed in 15-ml conical centrifuge tubes. Tubes were re

moved from incubation at various times and quickly centrifuged I min, 3000 r.p.m.).The absorbance of the supernatant was measured at 540 nm. Percentage haemolysiswas calculated relative to cells totally lysed with an equal volume of SDS.

In Vivo Effects

Dilutions of toxin (0.1 ml) were injected into the dorsal aorta of groups of 3 unanaesthetized eels (approximately 60 g) in 50% sea water. Control fish were injectedwith 0.1 ml PBS.

Results and Discussion

Isolation andPurification

A major problem in the isolation and purification of V nguillilrum haemolysin hasbeen its extreme lability. Even under cold-room conditions at a favourable pH [10]

losses during purification were considerable. t was thought that this mightbe due todegradation by contaminating proteolytic enzymes. However, incubation with chelatingagents (EDT A, nitrilotriacetate) and the protease inhibitor phenylrnethyl sulphonylfluoride did not reduce the loss. Partial purification by ammonium sulphate precipitation followed by gel chromatography has been achieved, but it has not yet beenpossible to purify the toxin further because of the low yield. Figure 1 shows a typicalseparation on Sephacryl S-200. Comparison of the elution characteristics of this peakwith standard marker proteins gave a molecular weight (very approximate) of 191,000.In some separations the peak showed considerable tailing reflecting degradation tolower molecular weight components. Although the purity of this material is satisfactoryfor many purposes, it is now recognised that for detailed study of the biochemicalproperties of toxins, very extensively purified proteins should be used [15]. Therefore,some caution is needed in the interpretation of the present results.


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Production and Properties of a Haemolytic Toxin by Vibrio anguillarum

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03

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0 ~ 5 4 0 ~ ~ ~ ~ 7 5 ~ ~ ~ ~ ~ ~ ~ 1 ~ O O

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Fig. 1 Separation of haemolysin from crude ammonium sulphate preparation on Sephacryl S-200.- absorbance, . . haemolytic activity against horse erythrocytes

Toxic Effects on Fish

Eels injected with the highest concentration of haemolysin used ca. 1,000 units/ml)died within 20 min of injection. Before death, the fish showed violent spasmic contractions of the body. Death was sudden and characterised by complete flaccidity ofthe body muscle. At lower doses, death occurred within 2 4 h. The symptoms observed suggest that this toxin preparation may possibly have neurotoxic activity.

Kinetics of Lysis

In order to determine whether the haemolysin behaves as a typical bacterial cytolytictoxin, the kinetics ofhaemolysis were followed. Horse erythrocytes were used for theseexperiments. The proportion of cells lysed at various times of incubation was followedby spectrophotometry of the haemoglobin released. Results show that haemolysis is a

two-stage process conSisting of an initial pre-lytic phase followed by a phase duringwhich actual cell lysis and release of haemoglobin occurs. Both the length of the prelytic phase, and the rate of haemoglobin release are dependent on haemolysin concentration Fig. 2) and on temperature Fig. 3). No haemolysis was detected within 4 hat temperatures below 10 C The pre-lytic phase also does not seem to occur at lowtemperatures, because erythrocytes incubated with haemolysin at 0 for 4 h beforetransferring to 37C still required the same pre-lytic period before haemoglobin reo

lease commenced. The results are rather unusual compared with other cytolytic toxinsand these effects should be reinvestigated with haemolysin of higher purity. The highoptimum temperature for haemolysis is perhaps surprising in view of the optimumtemperature of 20 22C for growth of V anguiliarum The nature of the pre-lyticphase was investigated by treating erythrocytes with haemolysin for various times,centrifuging quickly, resuspending in diluent and continuing incubation, so that cells


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o 20time,min

1.25

3

C.B. Munn

Fig. 2. Effect of haemolysin concentration units/ml) on rate ofhaemolysis of horse erythrocytes

were removed from contact with free haemolysin after various periods. Even after only5 min exposure to the haemolysin, most of the cells ultimately lysed. In another experiment, the supernatant fluid from cells treated with haemolysin for 10 min wasadded to fresh, untreated erythrocytes. The haemolytic activity of the supernatantfluid was considerably reduced, indicating that much of the haemolysin is either in-activated or bound irreversibly during the early part of the pre-lytic phase. The kinetics of lysis appear to be somewhat unusual and further analysis is required.

10

75

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25

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150

Fig. 3. Effect of temperature OC) on rate of haemolysis of horse erythrocytes


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Production and Properties of a Haemolytic Toxin by Vibrio anguillarum 7

Inactivation of Haemolysin by Gangliosides

The related species V cholerae [8] and V parahaemolyticus [14] both produce toxinswhich have been shown to bind to the membrane glycolipids known as gangliosides. I t

was therefore decided to determine whether V anguillarum behaved similarly. Variousconcentrations of haemolysin and bovine ganglioside mixture were mixed in microtit retrays in a chessboard titration. After incubation 37 e 10 min) horse erythrocyteswere added and incubation continued for 1 h Haemolysin was inactivated by gangliosides, and Fig. 4 shows that there is a linear relationship between the activity ofhaemolysin and the concentration of gangliosides required to inactivate it. Incubationof erythrocytes with gangliosides for 3 h followed by thorough washing before additionof haemolysin resulted in marked reduction in their susceptibility to lysis. This resultis rather surprising but might be explained if exogenous ganglioside becomes associatedwith the membrane and competes with the natural binding sites. Since only a crudemixture of gangliosides was used, it is not possible to identify at present the particularglycolipid structure with which haemolysin interacts. However, it does not appear tobe one of the neuraminidase-sensitive gangliosides, since pre-incubation of gangliosidemixture with this enzyme did not reduce the ability of the ganglioside to inactivatehaemolysin. In order to investigate the possibility that the ganglioside M t is the specific receptor, ganglioside mixture was incubated with excess cholera toxin before adding to haemolysin. The inactivating ability of ganglioside was not reduced by choleratoxin. Since cholera binds specifically and precipitates ganglioside M t [8] it seemsunlikely that this is the receptor for V anguillarum haemolysin. Future work will be

05

04

o 10 20Haemolysin units/ml

30 40 Fig. 4 Inactivation of haemolysinby ganglioside mixture


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74 C B Munn: Production and Properties of a Haemolytic Toxin by Vibrio anguilJarum

aimed at further elucidation of the mode of action of the haemolysin and the natureof possible receptors.

Role in Pathogenicity

I t is not yet possible to determine what role the haemolysin plays in pathogenicity. Wehave been unable to demonstrate a link between virulence and production of thehaemolysin in vitro. Also, haemolysin production is not coded by plasmid genes Munnand Pearcey, in preparation) whereas Crosa et al [5] claimed that virulence wasplasmid-coded. However, there is some evidence that haemolysis does occur in vivo,the haemolysin is lethal to eels, and some of the symptoms observed here are similar tothose seen in infected eels H. Chart, personal communication). However, the complexity of host-pathogen relationships is increasingly recognised and this toxin isprobably only one of several factors which determine pathogenicity.

Acknowledgements I thank Robert Pearcey for technical assistance and Henrik Chart for performing the in vivo experiments. The work was supported by the Science Research Council.

References1 Ajl SI Kadis S, Montie TC 1970) Microbial toxins vols I, 2A Academic Press, London New

York2 Anderson JW, Conroy DA 1970) Vibrio disease in fishes. In: Snieszko SF ed) Diseases of

fishes and shellfishes. American Fisheries Society, Washington, pp 266 -2723 Antipa R 1976) Field testing of injected Vibrio anguillarum bacterins in pen-reared Pacific

salmon. 1 Fish Res Board Can 33: 1291-12964. Bemheimer AW 1970) Cytolytic toxins of bacteria. In: Ajl SI Kadis S, Montie TC eds)

Microbial toxins, vol I Academic Press, London New York, pp 183 -2095. Crosa IH Schiewe MH, Falkow S 1977) Evidence for plasmid contribution to the virulence of

the fish pathogen Vibrio anguillarum Infect Immun 18:509-5136 Fryer lL Nelson IS Garrison RL 1972) Vibriosis in fish. In: Moore RW ed) Prog Fish Food

Sci 5:129-1337 Fryer lL Rohovec IS Garrison RL 1978) Immunization of salmonids for control of vibriosis.

Mar Fish Rev 40:20-238 Holmgren J 1978) Cholera toxin and the cell membrane. In: Jeljaszewicz J Wadstrom T eds)

Bacterial toxins and cell membranes. Academic Press, London New York, pp 333-3669. leljaszewicz J, Szrnigielski S, Hryniewicz W 1978) Biological effects of staphylococcal and

streptococcal toxins. In: leljaszewicz 1, Wadstrom T eds) Bacterial toxins and cell membrans.Academic Press, London New York, pp 185-219

10. Munn CB 1978) Haemolysin production by Vibrio anguillarum FEMS Microbiol L 3 :265 -26811. Roberts Rl 1976) Bacterial diseases of farmed fishes. In: Skinner FA Carr IG eds) Micro

biology in agriculture fisheries and food. Academic Press, London New York, pp 55 -6112. Smith H 1968) The biochemical challenge of microbial pathogenicity. Bacteriol Rev

32: 164-18413. Smith H, Taylor 1 1964) Microbial behaviour in vivo and in vitro, CUP, Cambridge14. Takeda Y, Takeda T, Honda T, Miwatani T 1976) Inactivation of the biological activities of

the thermostable direct haemolysin of Vibrio parahaemo[yticus by ganglioside GT 1. InfectImmun 14:1-5

15. Wadstrom T 1978) Advances in the purification of some bacterial protein toxins. In:

leljaszewicz J, Wadstrom T eds) Bacterial toxin s and cell membranes. Academic Press,London New York, pp 9-49

16. Wolke RE 1975) Pathology of bacterial and fungal disease affecting fish. In: Ribelin WE,Magaki G eds) Patholo gy of fishes. Univ Wisconsin Press, pp 33 -117

11 production and properties of a haemolytic toxin by vibrio anguillarum

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