© INSTITUTPASTEUR/ELsEVIER Paris 1992
Res. MicrobioL 1992, 143, 569-577
Purification and characterization of Kanagawa haemolysin from Vibrio parahaemolyticus J.P. Douet (1) (*), M. Castroviejo (2), A. Dodin (3) and C. B6b6ar (4) (/J Direction Gdndrale de la Concurrence de la Consommation et de la R~pression des Fraudes, Universitd de Bordeaux L 351 cours de la Liberation, 33405 Talence (France), ¢2) IBCN-CNRS, 1 rue Camille Saint-Sa~nx, 33077 Bordeaux Cedex (France), ¢~ Service du Cholera, D~partement Ecologie, Insiitut Pasteur, 75724 Paris Cedex 15, and ¢4) Laboratoire de Bact~riologie, Universit~ de Bordeaux I1, 146 rue L~o Saignat, 33076 Bordeaux Cedex (France). SUMMARY The haemolysin of a Kanagawa-phenomenon-positive Vibrio parahaemolyticus strain was purified to apparent homogeneity by acid precipitation, DEAE-Trisacryl, hydroxyapatite and FPLC (Mono-Q} columns: 1.4 Ezg of protein gave a single band on conventional SDS-PAGE with silver staining. The haemolysin was not inactivated by heating for 10 min at 1 0 0 ° C . It was a monomeric protein with a molecular weight estimated to be 29 kDa by PAGE under denaturing and non-denaturing conditions. The haemolysin caused fluid accumulation in the ligated mouse ileum, was cytolytic against cultured mammalian cells and also lysed erythrocytes of various animal species (equine erythrocytes being the most resistant).
Key-words: Haemolysin, Vibrio parahaemolyticus, Diarrhoea; Food poisoning, Kanagawa phenomenon, P:.,;:ication, Properties.
INTRODUCTION Vibrio parahaemolyticus is a halophilic marine bacteria. Isolated for the first time in 1950 in Osaka, this organism has been found to account for a b o u t 40 to 60 07o of all food poisoning cases in J a p a n (Fujino et al., 1953). In France, the first diarrhoeic syndrome produced by this Vibrio was described in 1983 by Boudon et aL It was also reported to be an important cause of traveller's diarrhoea. There is a close correlation between h u m a n pathogenicity and the production of the heat-stable direct haemolysin: 96.5 07o of the strains isolated from patient
Submitted March 27, 1992, accepted May 22, 1992. ,'~Correspondingauthor.
stools produce a thermostable haemolysin, while 99.0 °7o of those isolated from the marine environment do not (Miyamoto et aL, 1969). However, the exact role of this protein in the production of illness is not understood. A simple means of revealing this haemolysin is the Wagatsumn lnedium. On this medium, strains which show [3-haemolysis are called KP ÷ (Kanagawa phenomenon +) and those which give no haemolysis are called K P - (Miyamoto et al., 1980). Several workers have isolated and purified the thermostable direct haemolysin from culture filtrates of this organism (Zen-Yoji et al., 1971: H o n d a et al., 1976; Miyamoto et al.,
570
J,P. DOUET ET AL,
1980; C h e r w o n o g r o d z s k y and Clark, 1982). But reported techniques for purification o f this protein usually involved large initial volumes o f batch liquid culture ( H o n d a et aL, 1976; M i y a m o t o et al., 1980) or gave a low yield a n d relative activity ( C h e r w o n o g r o d z k y and Clark, 1982). To investigate its function in the infectious process, haemolysin must be highly purified. In this study, a new simple m e t h o d for the purification o f large quantities o f haemolysin to apparent h o m o g e n e i t y is described. S o m e physical and biological properties are also presented.
MATERIALS AND METHODS
Haemolysin purification
Preparation of crude haemolysin After an 18-h incubation, the medium in the 39 plates was homogenized. A solution of 0.01 M phosphate buffer pH 6.8 with 0.9 % NaCl was added and left for 30 rain to allow the diffusion of haemolysin into the buffer. The pieces of medium were discarded and the pH was adjusted to 6.8. The mixture was centrifuged at 28,000 g for 30 min at 4°C, and the pellet discarded. The ~,olume of the supernatant was 4,735 ml and its pH was decreased to 4.2 with 0.4 M acetic acid. Then, the haemolysin was precipitated at 28,000 g for 30 min at 4°C. The supernatant was discarded and the pellet homogenized with a French press in 0.01 M phosphate buffer pH 7. This solution was again centrifuged under the above conditions and the precipitate discarded. The supernatant o f 840 ml was used as crude haemolysin.
Bacterial toxins Commercial Kanagawa haemolysin from V. parahaemolyticus and cholera toxin, used as controls,
DEAE-Trisacryl column chromatography
were purchased from Sigma (L'Isle d'Abeau Chesnes, La Verpilliere, France). The reference o f the Kanagawa haemolysin purified by Cherwonogrodzky's method and employed in this study was H-3142, lot 97F-4027, and the reference of the cholera toxin was C-3012, lot 49F-0471.
The crude haemolysin was applied to a DEAETrisacryl column (IBF, Soc. Chim. Pointet Girard, Villeneuve-La-Garenne, France) (2.2 x 5.5 cm) equilibrated with 0.01 M phosphate buffer pH 7. Materials were eluted with the same buffer and then with 2 x 70 ml of a linear gradient from 0 to 0.7 M NaCl in the same buffer. Fractions with haemolytic activity were collected and pooled.
Bacterial strain and medium
V. parahaemolyticus (VP-7077) was originally isolated from a case of food poisoning in Abidjan. This strain belongs to a bacterial collection of the Cholera Unit of the Pasteur Institute. Four plates of Wagatsuma agar medium (12 x 12 cm) were inoculated with this strain. The components of the medium were as follows: 0.3 % yeast extract (Difco, Detroit, MI 48232, USA), 1 OTobacto-peptone (Difco), 7 °70 NaCI, 0.5 % K2HPO4, 1.5 °70 bacto-agar (Difco), distilled water added to a final volume of I 1. After dissolving by heating (heat sterilization should be avoided), D-mannitol was added to a concentration of 1 °70, 0 . 1 % crystal violet alcohol solution to 0.1 °70 and human defibrinated blood (O +) to 5 %. After 18 h at 37°C, the cells were collected and used to inoculate 39 other plates o f the same medium.
BSA DEAE FPLC HU
= = = =
bovine serum albumin. diethylaminoethyl. fast purificationliquid chromatography. baemolyticunit.
Hydroxyapatite column chromatography The pool obtained was applied to a hydroxyapatite bio-gel HTP Biorad column (Bio-Rad Laboratories, Richmond, CA, USA (2.2 x 6.0 cm). It was equilibrated with 0.01 M phosphate buffer pH 7. The column was eluted with 0.01 M phosphate buffer and then with 2 x 75 ml of linear gradient from 0.01 to 0.4 M phosphate buffer pH 7. Fractions with haemolysin were collected and pooled.
Mono-Qfast protein liquid chromatography (FPLC) One half of the sample was finally applied to a Mono-Q column (Pharmacia, Uppsala, Sweden)
KP = Kanagawaphenomenon. PAGE = polyacrylamidegel electrophoresis. SDS = sodium dodecy[sulphate.
K A N A G A WA H A E M O L YSIN F R O M VIBRIO PARAHAEMOLYTICUS equilibrated with 0.01 M phosphate buffer and eluted with 2 x 10 ml of linear gradient from 0 to 0.7 M NaCI in the same buffer. This preparation was used as purified toxin in this study.
Haemolytic assay Haemolytic activity was determined as follows. A mixture of 250 ~tl of haemolysin diluted or not with 0.01 M phosphate buffer pH 7 and an equal volume of 0.01 M phosphate buffer 1.8 070 NaCI was added to 500 izl of a 1 070 suspension of human (O +) erythrocytes (about 5.3 x 107 cells/ml). Before use, thes~ erythrocytes were washed 3 times with 0.9 070 NaCI buffered with 0.01 M phosphate pH 7. The reaction mixture was incubated at 37°C for 60 min and then centrifuged at 3,000 rpm for 5 min. The s,pernatant (200 ~tl) was transferred to a flat-bottom microtitre plate (Dynatech, boulevard de Jardy, Marnes-La-Coquette, France) and the haemolytic activity determined by measuring the absorbance at 540 nm. A control without erythrocytes was made for each fraction and treated in the same manner. One haemolytic unit (HU) was defined as the smallest dose causing 50 % haemolysis (Yamamoto et al., 1986). Erythrocytes from other animals were used in some experiments.
Determination of molecular weight
571
albumin, BSA and jack bean urease were used as molecular weight markers, purchased from Sigma. Staining o f the gels The gels were stained for protein by silver staining (Eschenbruch and Burk, 1982; Oakley et al., 1980).
Comparison of haemolysins by peptide mapping Peptide mapping was performed on purified and commercialized haemolysins from V. parahaemolyticus separately, and on a mixture of the two haemolysins. Haemolysins were dissolved at 0.5 mg/ml in the sample buffer which contained 0.125 M Tris-HCl pH 6.8, 0.5 070 SDS, 10 070 glycerol and 0.01 070 bromophenol blue (Cleveland et al., 1977). The samples were then heated to 100°C for 2 min. Proteolytic digestions were carried out at 37°C using 25 ~tg/ml of each protease. These enzymes were V8 protease, a-chymotrypsin and papain, and the durations of incubation were 30, 20 and 1 min, respectively. Following addition of t3-mercaptoethanol and SDS to final concentrations of 10 070 and 2 °70, respectively, proteolysis was stopped by boiling the samples for 2 rain. A total of 25 ~1 (12.5 ~g of protein) for each haemolysin alone, and 20 ~tl (10 ~tg of protein) for a mixture of both haemolysins were loaded into a sample well of the 20 070acrylamide gel. The gel was run in the normal manner and the peptide bands were detected by Coomassie blue staining.
SDS polyacrylamide slab gel electrophoresis (PAGE) According to Laemmli's method (1970), the gels were prepared with a 5 070 stacking gel and 12.5 070 separating gel. Samples were denaturated by boiling for 3 rain in 2 07oSDS, 5 070[3-mercaptoethanol sample buffer. Migrations were performed in Trisglycine-SDS buffer at a constant voltage of 60 V. Phosphorylase b, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and a-lactalbumin were used as molecular weight marker proteins purchased from Pharmacia. Non-denaturing P A G E The molecular weight of the protein was also determined by non-denaturing PAGE with a modification of Bryan's method (1977). The electrode buffer, the stacking and separating gels were without SDS and [3-mercaptoethanol. The samples were not heated before loading. The different polyacrylamide concentrations applied in separating gels were 6, 7, 8, 9 and 10 o7o.Migrations were performed at a constant voltage of 60 V. Bovine milk a-lactalbumin, bovine erythrocyte carbonic anhydrase, chicken egg
Cytolytic activity Cytolytic activity was evaluated on 3 cultured mammalian cells: Vero, HEp-2 and Intestine-407 cells. Vero and HEp-2 cells were grown in RPMI medium (Intermed S. A., Noisy-le-Grand, France) with 10 070 foetal calf serum and glutamine to a final concentration of 2 mM. Intestine-407 cells were grown in BME medium (lntermed S. A.) supplemented with 13 070 newborn calf serum, 1.7 mM glutamine, penicillin and s t r e p t o m y c i n to final concentrations of 87 IU/ml and 87 v.g/ml, respectively. Cytolytic activity was assayed in a 96-well flatbottomed microtitre plate (Dynatech) in duplicate. Samples of 100 rtl of suspension containing 2 x 104 cells were placed in the wells and incubated 24 h for Vero and Intestine-407 cells, and 48 h for the HEp-2 ceils. All cells were incubated at 37°C in a humidified atmosphere of 5 070 CO 2 in air. A 50-~tl volume of the haemolysin preparation, diluted to different concentrations with the same serum-free media as used in the cell culture, was added to each well and the microtitre plate was incubated at 37°C (Miyake et aL, 1988). Three controls
J.P. D O U E T E T A L .
572
without haemolysin were made. Cytotoxicity was assessed 10 h after the addition of haemolysin by the dye exclusion test with trypan blue. Alter discarding the supernatants, the wells were rinsed twice with 100 p.l of medium supplemented with serum for the first rinse and without serum for the second. Trypsinization was done with 50 ~1 of a solution of 0.05 °70 trypsin for 10 min at 37°C. It was stopped by adding 50 V.I of medium with 13 070 newborn calf serum. A volume of 100 V.I of 0 . 1 % trypan blue in phosphate-buffered saline was added and the cytotoxicity was read immediately in situ.
H e a t stability test
A purified haemolysin solution was heated at 100°C for l0 mln. This solution was loaded onto non-denaturing polyacrylamide gel (8 070 w/v) which was run under the conditions mentioned above. After migration, a plate of Wagatsuma agar medium (12 x 12 cm) was overlaid with the polyacry!am;.dc ~1 (i.e. the gel on the blood agar medium) and incubated at 37°C for 4 h. The remaining haemolytic activity was detected by a haemolysis zone on the Wagatsuma medium.
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Fig. 1. Purification of haemolysin. Crude haemolysin was chromatographed on DEAE-Trisacryl column (A), hydroxyapatite column (B) and Mono-Q column (C). For the DEAE column, the flow rate was 100 ml/h, and 4 ml were collected in each fraction. For the hydroxyapatite column, the flow rate was 30 ml/h, and 4 ml were collected in each fraction. For the Mono-Q column, the flow rate was 60 ml/h, and 0.25 ml were collected in each fraction. The haemolytic activity of the samples of each fraction was assayed as described in the text. --, absorbance at 280 rim; ©, haemolytic activity; ---, concentration of NaCI in (A) and (C), and phosphate in (B).
o.t
K A N A G A WA H A E M O L Y S I N F R O M V1BRIO PARAHAEMOLYTICUS
573
Table I. Purification of thermostable haemolysin produced by V. parahaemolyticus.
Total volume Total protein Total activity Specific activity Yield of (ml) (mg) (HU) (') (HU/mg) haemolytic activity %
Fraction Supernatant Acid precipitation DEAE-Trisacryl Hydroxyapatite Mono-Q (')
4,735 840 34 45 1.6
5,232.2 261.2 44.9 15.7 8.5
3,026 1,620 1,136 1,829 2,401
0,5 6,2 25.3 116.4 282.4
Relative activity
100 53.5 37.5 60.4 79.3
1.0 10.8 44.3 204.3 495.5
l HU was defined as the smallestdose causing 50 % haemolysis.
Molecular weight determination
Enteropathogenicity
The enteropathogenicity of the purified haemolysin was investigated in male mouse 0129SV ileal loops. This protein, diluted in physiological serum, was tested by a modification of the rabbit ileal loop technique (Brown et aL, 1977). Mice fasted for 24 h were anaesthetized intramuscularly with "Ketalar" (0.15 ml of 5 mg/ml), and 5 min after with "Vetranquil" (0.15 ml of 0.25 mg/ml). A volume of 0.2 ml of test preparations, or physiological serum for the control, was injected in the ligated individual loops. A test preparation was considered positive 18 h later if the ratio o f loop weight to loop length was 3-fold greater than that of the negative control. Cholera toxin was used as positive control.
C o m m e r c i a l K a n a g a w a haemolysin as reference and haemolytic pools f r o m the different purification steps were analysed by S D S - P A G E . T h e proteins f r o m this gel were silver-stained (fig. 2). In lane 6, 1.4 ~g o f the protein prepa-
I
2
3
4
5
6
7
$ 9
Protein assays .,
67
kDa
The amount of protein was determined by Bradford's method (1976). BSA was used as standard.
RESULTS
~ O
Purification
of haemolysin
~ 30 kDa O ~ g
~ 2Q.1 kDa ~ 14.4 kDa
T h e crude toxin o b t a i n e d by acetic acid precipitation at p H 4.2 was eluted f r o m the D E A E c o l u m n with 0.1 M NaC1 (fig. 1A) a n d f r o m hydroxyapatite and M o n o - Q columns with a p p r o x i m a t e l y 0.15 M p h o s p h a t e and 0.40 M NaC1, respectively (fig. 1B a n d IC). Typical results for purification are s u m m a r ; z e d in table I. A t the end o f the purification, the specific activity o f haemolysin was 282.4 H U / m g , i.e. a 496-fold purification.
Fig. 2. Analysis on SDS-PAGE of the diffe.ent steps of haemolysin purification and of commercial Ka~agawa haemolysin. Lanes 1, 7 and 9, molecular weight markers; lane 2, culture supernatant (1.1 ,t.tgof protein); 3, acid-precipitated fraction (1.24 ~tg); 4, eluate from the DEAE column (I.32 ptg); 5, eluate from the hydroxyapatite column (1.75 :~tg); 6, eluate from the Mono-Q column (I.4 ~g); 8, commercial haemolysin purified by Cherwonogrodzky's method (0.26 ~.g).
574
J.P. DOUET E T AL.
2
3
4
5
6
7
8
A8
C D
E.F
G H
I
J
K
L
'0 I T
-
Fig. 3. Non-denaturing PAGE of purified haemolysin.
The molecular weight markers were, respectively, lane 1, urease; lane 2, BSA; lane 3, chicken egg albumin ; lane
4, carbonic anhydrase; and lane 5, ct-laetalbumin. Lanes 6, 7 and 8 were the purified haemolysin (1.4 izg for each); lane 6 was silver-stained as were the markers; lanes 7 and 8 overlaid the Wagatsuma medium, and the whole was incubated at 37°C for 4 h; in addition, the haemolysin of lane S was heated for 10 min at 100°C, before being loaded on polyacrylamide gel. The gel contained 8 % acrylamide.
.q,.
-i
i
Fig. 4. Digestion patterns of purified and commercial haemolysin. These proteins were digested with V8 protease (lanes B to D), ct-chymotrypsin (lanes F to H) and papain (lanes J to L). The first lane of each digestion (B, F, J) was the commercial haemolysin pattern. The second lane of each digestion (C, G, K) was the purified haemolysin. The third lane (D, H, L) was a mixture of both haemolysins. Lanes A, E and I were molecular weight markers.
These d a t a indicate that h a e m o l y s i n is a m o n o m e r i c protein, with a m o l e c u l a r weight o f a b o u t 29 k D a . ration o b t a i n e d by F P L C ( M o n o - Q ) revealed a single b a n d . The molecular weight o f the haemolysin estimated under denaturating conditions was 28 k D A ; it did n o t vary in the presence or absence o f [3-mercaptoethanol a n d heating for 2 min 30 s at 100°C (data not shown). C o m m e r cial K a n a g a w a haemolysin gave a similar molecular weight (fig. 2, lane 8), but with one additional b a n d o f a b o u t 32 k D a (fig. 2, see arrow b e t w e e n lanes 7 a n d 8). N o n - d e n a t u r i n g P A G E with 1.4 ~tg o f purified haemolysin also revealed a single b a n d with the s a m e mobility as the b a n d with haemolytic activity (fig. 3, lanes 6 a n d 7). U n d e r these n o n denaturing conditions, the molecular weight was also 29 kDa.
Comparison of haemolysins by peptide mappinf~ P e p t i d e m a p s o f our h a e m o l y s i n a n d c o m mercial haemolysin prepared by limited proteolysis with V8 p r o t e a s e , a - c h y m o t r y p s i n a n d papain were c o m p a r e d . F r o m these 3 digestions, identical p e p t i d e m a p s a p p e a r e d for b o t h proteins (fig. 4). B o t h h a e m o l y s i n s were the s a m e protein.
Heat stability T h e heat stability o f the haemolytic activity was examined. This activity was not lost by heat-
K A N A G A WA H A E M O L Y S I N F R O M VIBRIO PARAHAEMOLYTICUS
575
Table !I. Specific activity (sp. act.) of purified haemolysin for lysis of various blood cells. Erythrocyte source
Sp. act. (HU/~tg of haemolysin) x l0 t*)
Monkey Chicken Human Sheep Horse
10.46 9.88 5.76 1.71 0.57
('~ Calculatedfrom two assays.
ing for 10 min at 100°C (fig. 3, lane 8). A control without thermal treatment was tested (fig. 3, lane 7). The haemolysis zones were similar in b o t h cases.
Biological activities The haemolytic activity o f haemolysin on erythrocytes from various animals was examined (table II). Haemolysin was the most active against monkey and the least active against equine erythrocytes. This protein also lysed cult u r e d m a m m a l i a n cells such as H E p - 2 , Intestine-407 a n d Vero. The amounts of haemolysin required for 99 070 lysis of cells were 1.0, 1.2 and 1.3 ng, respectively. Haemolysin caused fluid accumulation in the ligated mouse ileum (fig. 5). After injection of 9 ~tg of haemolysin, the ratio of loop weight to loop length 18 h later was at least 3-fold greater (1.25 g / 6 cm = 0.208) than that of the negative control (0.24 g / 6 cm = 0.04). A similar result was obtained with cholera toxin. The ratio obtained after injection of 9 ~g of cholera toxin was 3-fold greater (0.44 g/3.4 cm = 0.129) than that of the negative control.
DISCUSSION In this study, heat-stable haemolysin was purified from a V. parahaemolyticus KP + strain at a high yield and a degree of purity (1.4 ~.g of protein gave a single band in SDS-PAGE with
Fig. 5. Mouse ileal reactivity to haemolysin. Left intestine was negative control ; right intestine was obtained with 9 ~zg of purified haemolysin.
silver staining). All previous purifications of the Kanagawa haemolysin were made from bacteria grown in different liquid media (Zen-Yoji et aL, 1971 ; H o n d a et aL, 1976; Miyamoto et al., ! 980; Cherwonogrodzky and Clark 1982). They involved large initial volumes of batch liquid culture (Honda et aL, 1976; Miyamoto et al., 1980) or led to non-homogenous preparation as shown for commercial haemolysin purified by the Cherwonogrodzky's method. In the present study, solid Wagatsuma medium which contains erythrocytes (the target of haemolysin) was used. This fact could explain the variations in the total haemolytic activity during the different purification steps (see table I, column 3). Until the DEAE column step, the haemolysin and its target were co-purified together, and so the total haemolytic activity decreased. The increase in this total activity, after the hydroxyapatite and Mono-Q columns, resulted from the separation of the haemolysin from its target cells.
576
J.P. DOUET E T AL.
The fact that the peptide maps of our haemolysin and Kanagawa haemolysin, purified according to Cherwonogrodzky and Clark (1982), gave identical patterns had led us to believe that both haemolysins are the same protein. Moreover, in accordance with several workers who purified the haemolysin associated with the KP of V. parahaemolyticus (Zen-Yoji et aL, 1971; Honda et al., 1976; Miyamoto et aL, 1980), we found that this ,rotein had a highly thermostable activity and a weak activity against equine erythrocytes.
intestine. This fact could explain the diarrhoeic syndrome in a food poisoning produced by V. parahaemolyticus (Boudon et al., 1983). To investigate the function of this haemolysin in the infectious process, it would be interesting to know if the cholera-like effect was due to stimulation of adenylate cyclase, as for cholera toxin and enterotoxin LT of Escherichia coli, or to another mechanism, which is not specific to electrolyte transport but which follows cytolytic activity.
However, results from this study are in disagreement with the above authors on the deterrnination of the molecular organization and molecular weight of Kanagawa haemolysin, Data obtained by PAGE, under denaturing and non-denaturing conditions, demonstrated that haemolysin is a monomeric protein with a molecular weight of 29 kDa, whereas Zen-Yoji et aL (1974) and Miyamoto et al. (1980) showed by gel filtration that the active protein had a molecular weight of 44 kDa and, in contrast, by SDS-PAGE, a subunit size of 22 kDa, They did not specify whether the subunit alone presented haemolytic activity. A probable explanation for the apparent 44-kDa size of haemolysin is that this purified protein interacted with the dextran-based gels and so was retarded on the gel filtration columns. Similar results have been obtained in studies on the molecular weights of Clostridium perfringens delta-toxin (Alouf and Jolivet-Reynaud, 1981), V. vulnificus cytolysin (Gray and Kreger, 1985), and V. metschnikovii cytolysin (Miyake et aL, 1988). Haemolysin purified in the present study could be the subunit of the Kanagawa haemolysin; but in this case (1) the molecular weight of the haemolysin subanit would be underestimated (22 kDa in~.ead of 29 kDa), and (2) the subunit alone of the haemolysin would be active, like its dimer form. This dimer form was not found in nondenaturing gel electrophoresis.
Acknowledgements
In this study, the high cytolytic effect of the haemolysin on the different cultured mammalian ceils such as HEp-2, Intestine-407 and Vero was demonstrated. The haemolysin was also found to cause fluid accumulation in the ligated mouse
The authors wish to thank Jean Barr~re for producing the photographs.
Purification et caract~risation de I'h~molysine responsable du caract~re Kanagawa chez gibrio parahaemolyticus
L'h6molysine d'une souche de Vibrio parahaemolyticus KP ÷ (positive pour le ph6nom~ne de Kanagawa) a 6t6 hautement purifi6c par pr6cipitation /t l'acide ac6tique, par chromatographie sur DEAE-Trisacryl et hydroxyapatite et par FPLC (Mono-Q): 1,4 l~g de prot6ine donne une seule bande sur gel d'acrylamide en conditions d6naturantes (coloration par le nitrate d'argent). Cette prot6ine n'est pas inactiv6e par un chauffage de l0 min /l 100°C. Nous avons d6montr6 sur gel d'acrylamide en conditions d6naturantes et non d6naturantes que cette h6molysine est une prot6ine monom6rique de 29 kDa. Elle produit une accumulation de fluide dans l'anse ligatur6e d'il6on de souris, est cytotoxique pour des cellules de mammif/:re en culture et lyse les 6rythrocytes de diff6rents animaux (c~ux du cheval 6tant les plus r6sistants). Mots-clds: H~molysine, Vibro parahaemolyticus, Diarrh6e; Intoxications alimentaires, Caract&e Kangawa, Purification, Propri6t6s.
References
Alouf, J.E. & Jolivet-Reynaud,C. (1981), Purification and characterization of Clostridium perfringens deltatoxin. Infect. lmmun., 31, 536-546. Boudon, A., Richard, C., Le Corre, C. & Colombo, P.
K A N A G A WA H A E M O L Y S I N F R O M VIBRIO PARAHAEMOLYT1CUS (1983), Premier cas autochtone de syndrome diarrh6ique/l Vibrio parahaemolyticus en France, Donn6es b~ct6riologiques, cliniques et 6pid6miologiques. IF/6d. MaL infect., 13, 443-447. Bradford, M.M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem., 12, 248-254. Brown, D.F., Spaulding, P.L. & Twedt, R.M. (1977), Enteropathogenicity of Vibrio parahaemolyticus in the ligated rabbit ileum. Appl. Environ. Microbiol., 33, 10-14. Bryan, J.K. (1977), Molecular weights of protein multimers from po~yacrylamidegel electrophoresis. Analyt. Biochem., 78, 513-519. Cherwonogrodzky, J.W. & Clark, A.G. (1982), The purification of the Kanagawa haemolysin from Vibrio parahaemolyticus. FEMS Microbiol. Letters, 15, 175-179. Cleveland, D.W., Fischer, S.G., Kirschner, M.W. & Laemmli, U.K. (1977), Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel eiectrophoresis. J. biol. Chem., 252, 1102-1106. Eschenbruch, M. & Burk~ R.R. (1982), Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels. Analyt. Biochem., 125.96-99. Fujino, T., Okuno, Y., Nakada, D., Aoyama, A., Fukai, K., Mukal, T. & Veno, T. (1953), On the bacteriological examination of shirasu food poisoning. IVied. J. Osaka Univ., 4, 299-304. Gray, L.D. & Kreger, A.S, (1985), Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus. Infect. lmmun., 48, 62-72. Honda, T., Taga, S., Takeda, T., Hasibuan, M.A., Takeda, Y. & Miwatani, T. (1976), Identification of lethal toxin with the thermostabie direct hemolysin produced by Vibrio parahaemolyticus, and some
577
physicochemical properties of the purified toxin. Infect. Immun., 13, 133-139. Laemmli, U.K. (19701, Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680-685. Miyake, M., Honda, T. & Miwatani, T. (1988), Purification of Vibrio metschnikovii cytolysin. Infect. lmmun., 56, 954-960. Miyamoto, Y., Kato, T., Obara, Y., Akiyama, S., Takizawa, K. & Yamai, K. (1969), In vitro hemolytic characteristic of Vibrio parahaemolyticus: its close correlation with human pathogenicity. J. Bact., 100, 1147-1149. Miyamoto, Y., Obara, Y., Nikkawa, T., Yamai, S., Kato, T., Yamada, Y. & Ohashi, M. (1980), Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated hemolysin of Vibrio parahaemc'vticus. Infect. Immun., 28, 567-576. Oakley, B.R., Kirsch, D.R. & Morris, N.R. (1980), A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analyt. Biochem., 105, 361-363. Yamamoto, K., Ichinose, Y,, Nakasone, N., Tanabe, M., Nagahama, M., Sakurai, J. & lwanaga, M. (1936), Identity of hemolysins produced by Vibrio cholerae non-01 and Vibrio cholerae 01, Biotype El Tor. Infect. lmmun., 51, 927-931. Zen-Yoji, H., Kitokoto, H., Morozumi, S. & Le Clair, R.A. (1971), Purification and characterization of a hemolysin produced by Vibrio parahaemolyticus..L infect. Dis., 123, 665-667. Zer,-Yoji, H , Kudoh, ¥., lgarashi, H., Ohta, K. & Fukai, K. (1974), Purification and identification on enteropathogenic toxins "a" and " a ' " produced by Vibrio parahaernolyticus, and their biological and pathological activities, in "International Symposium on Vibrio parahaemolyticus'" (Fujino, T., Sakaguchi, G., Sakazaki, R., & Takeda, Y.) (pp. 237-243). Saikon Publ. Co, Tokyo.
Res. MicrobioL 1992, 143, 569-577
Purification and characterization of Kanagawa haemolysin from Vibrio parahaemolyticus J.P. Douet (1) (*), M. Castroviejo (2), A. Dodin (3) and C. B6b6ar (4) (/J Direction Gdndrale de la Concurrence de la Consommation et de la R~pression des Fraudes, Universitd de Bordeaux L 351 cours de la Liberation, 33405 Talence (France), ¢2) IBCN-CNRS, 1 rue Camille Saint-Sa~nx, 33077 Bordeaux Cedex (France), ¢~ Service du Cholera, D~partement Ecologie, Insiitut Pasteur, 75724 Paris Cedex 15, and ¢4) Laboratoire de Bact~riologie, Universit~ de Bordeaux I1, 146 rue L~o Saignat, 33076 Bordeaux Cedex (France). SUMMARY The haemolysin of a Kanagawa-phenomenon-positive Vibrio parahaemolyticus strain was purified to apparent homogeneity by acid precipitation, DEAE-Trisacryl, hydroxyapatite and FPLC (Mono-Q} columns: 1.4 Ezg of protein gave a single band on conventional SDS-PAGE with silver staining. The haemolysin was not inactivated by heating for 10 min at 1 0 0 ° C . It was a monomeric protein with a molecular weight estimated to be 29 kDa by PAGE under denaturing and non-denaturing conditions. The haemolysin caused fluid accumulation in the ligated mouse ileum, was cytolytic against cultured mammalian cells and also lysed erythrocytes of various animal species (equine erythrocytes being the most resistant).
Key-words: Haemolysin, Vibrio parahaemolyticus, Diarrhoea; Food poisoning, Kanagawa phenomenon, P:.,;:ication, Properties.
INTRODUCTION Vibrio parahaemolyticus is a halophilic marine bacteria. Isolated for the first time in 1950 in Osaka, this organism has been found to account for a b o u t 40 to 60 07o of all food poisoning cases in J a p a n (Fujino et al., 1953). In France, the first diarrhoeic syndrome produced by this Vibrio was described in 1983 by Boudon et aL It was also reported to be an important cause of traveller's diarrhoea. There is a close correlation between h u m a n pathogenicity and the production of the heat-stable direct haemolysin: 96.5 07o of the strains isolated from patient
Submitted March 27, 1992, accepted May 22, 1992. ,'~Correspondingauthor.
stools produce a thermostable haemolysin, while 99.0 °7o of those isolated from the marine environment do not (Miyamoto et aL, 1969). However, the exact role of this protein in the production of illness is not understood. A simple means of revealing this haemolysin is the Wagatsumn lnedium. On this medium, strains which show [3-haemolysis are called KP ÷ (Kanagawa phenomenon +) and those which give no haemolysis are called K P - (Miyamoto et al., 1980). Several workers have isolated and purified the thermostable direct haemolysin from culture filtrates of this organism (Zen-Yoji et al., 1971: H o n d a et al., 1976; Miyamoto et al.,
570
J,P. DOUET ET AL,
1980; C h e r w o n o g r o d z s k y and Clark, 1982). But reported techniques for purification o f this protein usually involved large initial volumes o f batch liquid culture ( H o n d a et aL, 1976; M i y a m o t o et al., 1980) or gave a low yield a n d relative activity ( C h e r w o n o g r o d z k y and Clark, 1982). To investigate its function in the infectious process, haemolysin must be highly purified. In this study, a new simple m e t h o d for the purification o f large quantities o f haemolysin to apparent h o m o g e n e i t y is described. S o m e physical and biological properties are also presented.
MATERIALS AND METHODS
Haemolysin purification
Preparation of crude haemolysin After an 18-h incubation, the medium in the 39 plates was homogenized. A solution of 0.01 M phosphate buffer pH 6.8 with 0.9 % NaCl was added and left for 30 rain to allow the diffusion of haemolysin into the buffer. The pieces of medium were discarded and the pH was adjusted to 6.8. The mixture was centrifuged at 28,000 g for 30 min at 4°C, and the pellet discarded. The ~,olume of the supernatant was 4,735 ml and its pH was decreased to 4.2 with 0.4 M acetic acid. Then, the haemolysin was precipitated at 28,000 g for 30 min at 4°C. The supernatant was discarded and the pellet homogenized with a French press in 0.01 M phosphate buffer pH 7. This solution was again centrifuged under the above conditions and the precipitate discarded. The supernatant o f 840 ml was used as crude haemolysin.
Bacterial toxins Commercial Kanagawa haemolysin from V. parahaemolyticus and cholera toxin, used as controls,
DEAE-Trisacryl column chromatography
were purchased from Sigma (L'Isle d'Abeau Chesnes, La Verpilliere, France). The reference o f the Kanagawa haemolysin purified by Cherwonogrodzky's method and employed in this study was H-3142, lot 97F-4027, and the reference of the cholera toxin was C-3012, lot 49F-0471.
The crude haemolysin was applied to a DEAETrisacryl column (IBF, Soc. Chim. Pointet Girard, Villeneuve-La-Garenne, France) (2.2 x 5.5 cm) equilibrated with 0.01 M phosphate buffer pH 7. Materials were eluted with the same buffer and then with 2 x 70 ml of a linear gradient from 0 to 0.7 M NaCl in the same buffer. Fractions with haemolytic activity were collected and pooled.
Bacterial strain and medium
V. parahaemolyticus (VP-7077) was originally isolated from a case of food poisoning in Abidjan. This strain belongs to a bacterial collection of the Cholera Unit of the Pasteur Institute. Four plates of Wagatsuma agar medium (12 x 12 cm) were inoculated with this strain. The components of the medium were as follows: 0.3 % yeast extract (Difco, Detroit, MI 48232, USA), 1 OTobacto-peptone (Difco), 7 °70 NaCI, 0.5 % K2HPO4, 1.5 °70 bacto-agar (Difco), distilled water added to a final volume of I 1. After dissolving by heating (heat sterilization should be avoided), D-mannitol was added to a concentration of 1 °70, 0 . 1 % crystal violet alcohol solution to 0.1 °70 and human defibrinated blood (O +) to 5 %. After 18 h at 37°C, the cells were collected and used to inoculate 39 other plates o f the same medium.
BSA DEAE FPLC HU
= = = =
bovine serum albumin. diethylaminoethyl. fast purificationliquid chromatography. baemolyticunit.
Hydroxyapatite column chromatography The pool obtained was applied to a hydroxyapatite bio-gel HTP Biorad column (Bio-Rad Laboratories, Richmond, CA, USA (2.2 x 6.0 cm). It was equilibrated with 0.01 M phosphate buffer pH 7. The column was eluted with 0.01 M phosphate buffer and then with 2 x 75 ml of linear gradient from 0.01 to 0.4 M phosphate buffer pH 7. Fractions with haemolysin were collected and pooled.
Mono-Qfast protein liquid chromatography (FPLC) One half of the sample was finally applied to a Mono-Q column (Pharmacia, Uppsala, Sweden)
KP = Kanagawaphenomenon. PAGE = polyacrylamidegel electrophoresis. SDS = sodium dodecy[sulphate.
K A N A G A WA H A E M O L YSIN F R O M VIBRIO PARAHAEMOLYTICUS equilibrated with 0.01 M phosphate buffer and eluted with 2 x 10 ml of linear gradient from 0 to 0.7 M NaCI in the same buffer. This preparation was used as purified toxin in this study.
Haemolytic assay Haemolytic activity was determined as follows. A mixture of 250 ~tl of haemolysin diluted or not with 0.01 M phosphate buffer pH 7 and an equal volume of 0.01 M phosphate buffer 1.8 070 NaCI was added to 500 izl of a 1 070 suspension of human (O +) erythrocytes (about 5.3 x 107 cells/ml). Before use, thes~ erythrocytes were washed 3 times with 0.9 070 NaCI buffered with 0.01 M phosphate pH 7. The reaction mixture was incubated at 37°C for 60 min and then centrifuged at 3,000 rpm for 5 min. The s,pernatant (200 ~tl) was transferred to a flat-bottom microtitre plate (Dynatech, boulevard de Jardy, Marnes-La-Coquette, France) and the haemolytic activity determined by measuring the absorbance at 540 nm. A control without erythrocytes was made for each fraction and treated in the same manner. One haemolytic unit (HU) was defined as the smallest dose causing 50 % haemolysis (Yamamoto et al., 1986). Erythrocytes from other animals were used in some experiments.
Determination of molecular weight
571
albumin, BSA and jack bean urease were used as molecular weight markers, purchased from Sigma. Staining o f the gels The gels were stained for protein by silver staining (Eschenbruch and Burk, 1982; Oakley et al., 1980).
Comparison of haemolysins by peptide mapping Peptide mapping was performed on purified and commercialized haemolysins from V. parahaemolyticus separately, and on a mixture of the two haemolysins. Haemolysins were dissolved at 0.5 mg/ml in the sample buffer which contained 0.125 M Tris-HCl pH 6.8, 0.5 070 SDS, 10 070 glycerol and 0.01 070 bromophenol blue (Cleveland et al., 1977). The samples were then heated to 100°C for 2 min. Proteolytic digestions were carried out at 37°C using 25 ~tg/ml of each protease. These enzymes were V8 protease, a-chymotrypsin and papain, and the durations of incubation were 30, 20 and 1 min, respectively. Following addition of t3-mercaptoethanol and SDS to final concentrations of 10 070 and 2 °70, respectively, proteolysis was stopped by boiling the samples for 2 rain. A total of 25 ~1 (12.5 ~g of protein) for each haemolysin alone, and 20 ~tl (10 ~tg of protein) for a mixture of both haemolysins were loaded into a sample well of the 20 070acrylamide gel. The gel was run in the normal manner and the peptide bands were detected by Coomassie blue staining.
SDS polyacrylamide slab gel electrophoresis (PAGE) According to Laemmli's method (1970), the gels were prepared with a 5 070 stacking gel and 12.5 070 separating gel. Samples were denaturated by boiling for 3 rain in 2 07oSDS, 5 070[3-mercaptoethanol sample buffer. Migrations were performed in Trisglycine-SDS buffer at a constant voltage of 60 V. Phosphorylase b, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and a-lactalbumin were used as molecular weight marker proteins purchased from Pharmacia. Non-denaturing P A G E The molecular weight of the protein was also determined by non-denaturing PAGE with a modification of Bryan's method (1977). The electrode buffer, the stacking and separating gels were without SDS and [3-mercaptoethanol. The samples were not heated before loading. The different polyacrylamide concentrations applied in separating gels were 6, 7, 8, 9 and 10 o7o.Migrations were performed at a constant voltage of 60 V. Bovine milk a-lactalbumin, bovine erythrocyte carbonic anhydrase, chicken egg
Cytolytic activity Cytolytic activity was evaluated on 3 cultured mammalian cells: Vero, HEp-2 and Intestine-407 cells. Vero and HEp-2 cells were grown in RPMI medium (Intermed S. A., Noisy-le-Grand, France) with 10 070 foetal calf serum and glutamine to a final concentration of 2 mM. Intestine-407 cells were grown in BME medium (lntermed S. A.) supplemented with 13 070 newborn calf serum, 1.7 mM glutamine, penicillin and s t r e p t o m y c i n to final concentrations of 87 IU/ml and 87 v.g/ml, respectively. Cytolytic activity was assayed in a 96-well flatbottomed microtitre plate (Dynatech) in duplicate. Samples of 100 rtl of suspension containing 2 x 104 cells were placed in the wells and incubated 24 h for Vero and Intestine-407 cells, and 48 h for the HEp-2 ceils. All cells were incubated at 37°C in a humidified atmosphere of 5 070 CO 2 in air. A 50-~tl volume of the haemolysin preparation, diluted to different concentrations with the same serum-free media as used in the cell culture, was added to each well and the microtitre plate was incubated at 37°C (Miyake et aL, 1988). Three controls
J.P. D O U E T E T A L .
572
without haemolysin were made. Cytotoxicity was assessed 10 h after the addition of haemolysin by the dye exclusion test with trypan blue. Alter discarding the supernatants, the wells were rinsed twice with 100 p.l of medium supplemented with serum for the first rinse and without serum for the second. Trypsinization was done with 50 ~1 of a solution of 0.05 °70 trypsin for 10 min at 37°C. It was stopped by adding 50 V.I of medium with 13 070 newborn calf serum. A volume of 100 V.I of 0 . 1 % trypan blue in phosphate-buffered saline was added and the cytotoxicity was read immediately in situ.
H e a t stability test
A purified haemolysin solution was heated at 100°C for l0 mln. This solution was loaded onto non-denaturing polyacrylamide gel (8 070 w/v) which was run under the conditions mentioned above. After migration, a plate of Wagatsuma agar medium (12 x 12 cm) was overlaid with the polyacry!am;.dc ~1 (i.e. the gel on the blood agar medium) and incubated at 37°C for 4 h. The remaining haemolytic activity was detected by a haemolysis zone on the Wagatsuma medium.
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Fig. 1. Purification of haemolysin. Crude haemolysin was chromatographed on DEAE-Trisacryl column (A), hydroxyapatite column (B) and Mono-Q column (C). For the DEAE column, the flow rate was 100 ml/h, and 4 ml were collected in each fraction. For the hydroxyapatite column, the flow rate was 30 ml/h, and 4 ml were collected in each fraction. For the Mono-Q column, the flow rate was 60 ml/h, and 0.25 ml were collected in each fraction. The haemolytic activity of the samples of each fraction was assayed as described in the text. --, absorbance at 280 rim; ©, haemolytic activity; ---, concentration of NaCI in (A) and (C), and phosphate in (B).
o.t
K A N A G A WA H A E M O L Y S I N F R O M V1BRIO PARAHAEMOLYTICUS
573
Table I. Purification of thermostable haemolysin produced by V. parahaemolyticus.
Total volume Total protein Total activity Specific activity Yield of (ml) (mg) (HU) (') (HU/mg) haemolytic activity %
Fraction Supernatant Acid precipitation DEAE-Trisacryl Hydroxyapatite Mono-Q (')
4,735 840 34 45 1.6
5,232.2 261.2 44.9 15.7 8.5
3,026 1,620 1,136 1,829 2,401
0,5 6,2 25.3 116.4 282.4
Relative activity
100 53.5 37.5 60.4 79.3
1.0 10.8 44.3 204.3 495.5
l HU was defined as the smallestdose causing 50 % haemolysis.
Molecular weight determination
Enteropathogenicity
The enteropathogenicity of the purified haemolysin was investigated in male mouse 0129SV ileal loops. This protein, diluted in physiological serum, was tested by a modification of the rabbit ileal loop technique (Brown et aL, 1977). Mice fasted for 24 h were anaesthetized intramuscularly with "Ketalar" (0.15 ml of 5 mg/ml), and 5 min after with "Vetranquil" (0.15 ml of 0.25 mg/ml). A volume of 0.2 ml of test preparations, or physiological serum for the control, was injected in the ligated individual loops. A test preparation was considered positive 18 h later if the ratio o f loop weight to loop length was 3-fold greater than that of the negative control. Cholera toxin was used as positive control.
C o m m e r c i a l K a n a g a w a haemolysin as reference and haemolytic pools f r o m the different purification steps were analysed by S D S - P A G E . T h e proteins f r o m this gel were silver-stained (fig. 2). In lane 6, 1.4 ~g o f the protein prepa-
I
2
3
4
5
6
7
$ 9
Protein assays .,
67
kDa
The amount of protein was determined by Bradford's method (1976). BSA was used as standard.
RESULTS
~ O
Purification
of haemolysin
~ 30 kDa O ~ g
~ 2Q.1 kDa ~ 14.4 kDa
T h e crude toxin o b t a i n e d by acetic acid precipitation at p H 4.2 was eluted f r o m the D E A E c o l u m n with 0.1 M NaC1 (fig. 1A) a n d f r o m hydroxyapatite and M o n o - Q columns with a p p r o x i m a t e l y 0.15 M p h o s p h a t e and 0.40 M NaC1, respectively (fig. 1B a n d IC). Typical results for purification are s u m m a r ; z e d in table I. A t the end o f the purification, the specific activity o f haemolysin was 282.4 H U / m g , i.e. a 496-fold purification.
Fig. 2. Analysis on SDS-PAGE of the diffe.ent steps of haemolysin purification and of commercial Ka~agawa haemolysin. Lanes 1, 7 and 9, molecular weight markers; lane 2, culture supernatant (1.1 ,t.tgof protein); 3, acid-precipitated fraction (1.24 ~tg); 4, eluate from the DEAE column (I.32 ptg); 5, eluate from the hydroxyapatite column (1.75 :~tg); 6, eluate from the Mono-Q column (I.4 ~g); 8, commercial haemolysin purified by Cherwonogrodzky's method (0.26 ~.g).
574
J.P. DOUET E T AL.
2
3
4
5
6
7
8
A8
C D
E.F
G H
I
J
K
L
'0 I T
-
Fig. 3. Non-denaturing PAGE of purified haemolysin.
The molecular weight markers were, respectively, lane 1, urease; lane 2, BSA; lane 3, chicken egg albumin ; lane
4, carbonic anhydrase; and lane 5, ct-laetalbumin. Lanes 6, 7 and 8 were the purified haemolysin (1.4 izg for each); lane 6 was silver-stained as were the markers; lanes 7 and 8 overlaid the Wagatsuma medium, and the whole was incubated at 37°C for 4 h; in addition, the haemolysin of lane S was heated for 10 min at 100°C, before being loaded on polyacrylamide gel. The gel contained 8 % acrylamide.
.q,.
-i
i
Fig. 4. Digestion patterns of purified and commercial haemolysin. These proteins were digested with V8 protease (lanes B to D), ct-chymotrypsin (lanes F to H) and papain (lanes J to L). The first lane of each digestion (B, F, J) was the commercial haemolysin pattern. The second lane of each digestion (C, G, K) was the purified haemolysin. The third lane (D, H, L) was a mixture of both haemolysins. Lanes A, E and I were molecular weight markers.
These d a t a indicate that h a e m o l y s i n is a m o n o m e r i c protein, with a m o l e c u l a r weight o f a b o u t 29 k D a . ration o b t a i n e d by F P L C ( M o n o - Q ) revealed a single b a n d . The molecular weight o f the haemolysin estimated under denaturating conditions was 28 k D A ; it did n o t vary in the presence or absence o f [3-mercaptoethanol a n d heating for 2 min 30 s at 100°C (data not shown). C o m m e r cial K a n a g a w a haemolysin gave a similar molecular weight (fig. 2, lane 8), but with one additional b a n d o f a b o u t 32 k D a (fig. 2, see arrow b e t w e e n lanes 7 a n d 8). N o n - d e n a t u r i n g P A G E with 1.4 ~tg o f purified haemolysin also revealed a single b a n d with the s a m e mobility as the b a n d with haemolytic activity (fig. 3, lanes 6 a n d 7). U n d e r these n o n denaturing conditions, the molecular weight was also 29 kDa.
Comparison of haemolysins by peptide mappinf~ P e p t i d e m a p s o f our h a e m o l y s i n a n d c o m mercial haemolysin prepared by limited proteolysis with V8 p r o t e a s e , a - c h y m o t r y p s i n a n d papain were c o m p a r e d . F r o m these 3 digestions, identical p e p t i d e m a p s a p p e a r e d for b o t h proteins (fig. 4). B o t h h a e m o l y s i n s were the s a m e protein.
Heat stability T h e heat stability o f the haemolytic activity was examined. This activity was not lost by heat-
K A N A G A WA H A E M O L Y S I N F R O M VIBRIO PARAHAEMOLYTICUS
575
Table !I. Specific activity (sp. act.) of purified haemolysin for lysis of various blood cells. Erythrocyte source
Sp. act. (HU/~tg of haemolysin) x l0 t*)
Monkey Chicken Human Sheep Horse
10.46 9.88 5.76 1.71 0.57
('~ Calculatedfrom two assays.
ing for 10 min at 100°C (fig. 3, lane 8). A control without thermal treatment was tested (fig. 3, lane 7). The haemolysis zones were similar in b o t h cases.
Biological activities The haemolytic activity o f haemolysin on erythrocytes from various animals was examined (table II). Haemolysin was the most active against monkey and the least active against equine erythrocytes. This protein also lysed cult u r e d m a m m a l i a n cells such as H E p - 2 , Intestine-407 a n d Vero. The amounts of haemolysin required for 99 070 lysis of cells were 1.0, 1.2 and 1.3 ng, respectively. Haemolysin caused fluid accumulation in the ligated mouse ileum (fig. 5). After injection of 9 ~tg of haemolysin, the ratio of loop weight to loop length 18 h later was at least 3-fold greater (1.25 g / 6 cm = 0.208) than that of the negative control (0.24 g / 6 cm = 0.04). A similar result was obtained with cholera toxin. The ratio obtained after injection of 9 ~g of cholera toxin was 3-fold greater (0.44 g/3.4 cm = 0.129) than that of the negative control.
DISCUSSION In this study, heat-stable haemolysin was purified from a V. parahaemolyticus KP + strain at a high yield and a degree of purity (1.4 ~.g of protein gave a single band in SDS-PAGE with
Fig. 5. Mouse ileal reactivity to haemolysin. Left intestine was negative control ; right intestine was obtained with 9 ~zg of purified haemolysin.
silver staining). All previous purifications of the Kanagawa haemolysin were made from bacteria grown in different liquid media (Zen-Yoji et aL, 1971 ; H o n d a et aL, 1976; Miyamoto et al., ! 980; Cherwonogrodzky and Clark 1982). They involved large initial volumes of batch liquid culture (Honda et aL, 1976; Miyamoto et al., 1980) or led to non-homogenous preparation as shown for commercial haemolysin purified by the Cherwonogrodzky's method. In the present study, solid Wagatsuma medium which contains erythrocytes (the target of haemolysin) was used. This fact could explain the variations in the total haemolytic activity during the different purification steps (see table I, column 3). Until the DEAE column step, the haemolysin and its target were co-purified together, and so the total haemolytic activity decreased. The increase in this total activity, after the hydroxyapatite and Mono-Q columns, resulted from the separation of the haemolysin from its target cells.
576
J.P. DOUET E T AL.
The fact that the peptide maps of our haemolysin and Kanagawa haemolysin, purified according to Cherwonogrodzky and Clark (1982), gave identical patterns had led us to believe that both haemolysins are the same protein. Moreover, in accordance with several workers who purified the haemolysin associated with the KP of V. parahaemolyticus (Zen-Yoji et aL, 1971; Honda et al., 1976; Miyamoto et aL, 1980), we found that this ,rotein had a highly thermostable activity and a weak activity against equine erythrocytes.
intestine. This fact could explain the diarrhoeic syndrome in a food poisoning produced by V. parahaemolyticus (Boudon et al., 1983). To investigate the function of this haemolysin in the infectious process, it would be interesting to know if the cholera-like effect was due to stimulation of adenylate cyclase, as for cholera toxin and enterotoxin LT of Escherichia coli, or to another mechanism, which is not specific to electrolyte transport but which follows cytolytic activity.
However, results from this study are in disagreement with the above authors on the deterrnination of the molecular organization and molecular weight of Kanagawa haemolysin, Data obtained by PAGE, under denaturing and non-denaturing conditions, demonstrated that haemolysin is a monomeric protein with a molecular weight of 29 kDa, whereas Zen-Yoji et aL (1974) and Miyamoto et al. (1980) showed by gel filtration that the active protein had a molecular weight of 44 kDa and, in contrast, by SDS-PAGE, a subunit size of 22 kDa, They did not specify whether the subunit alone presented haemolytic activity. A probable explanation for the apparent 44-kDa size of haemolysin is that this purified protein interacted with the dextran-based gels and so was retarded on the gel filtration columns. Similar results have been obtained in studies on the molecular weights of Clostridium perfringens delta-toxin (Alouf and Jolivet-Reynaud, 1981), V. vulnificus cytolysin (Gray and Kreger, 1985), and V. metschnikovii cytolysin (Miyake et aL, 1988). Haemolysin purified in the present study could be the subunit of the Kanagawa haemolysin; but in this case (1) the molecular weight of the haemolysin subanit would be underestimated (22 kDa in~.ead of 29 kDa), and (2) the subunit alone of the haemolysin would be active, like its dimer form. This dimer form was not found in nondenaturing gel electrophoresis.
Acknowledgements
In this study, the high cytolytic effect of the haemolysin on the different cultured mammalian ceils such as HEp-2, Intestine-407 and Vero was demonstrated. The haemolysin was also found to cause fluid accumulation in the ligated mouse
The authors wish to thank Jean Barr~re for producing the photographs.
Purification et caract~risation de I'h~molysine responsable du caract~re Kanagawa chez gibrio parahaemolyticus
L'h6molysine d'une souche de Vibrio parahaemolyticus KP ÷ (positive pour le ph6nom~ne de Kanagawa) a 6t6 hautement purifi6c par pr6cipitation /t l'acide ac6tique, par chromatographie sur DEAE-Trisacryl et hydroxyapatite et par FPLC (Mono-Q): 1,4 l~g de prot6ine donne une seule bande sur gel d'acrylamide en conditions d6naturantes (coloration par le nitrate d'argent). Cette prot6ine n'est pas inactiv6e par un chauffage de l0 min /l 100°C. Nous avons d6montr6 sur gel d'acrylamide en conditions d6naturantes et non d6naturantes que cette h6molysine est une prot6ine monom6rique de 29 kDa. Elle produit une accumulation de fluide dans l'anse ligatur6e d'il6on de souris, est cytotoxique pour des cellules de mammif/:re en culture et lyse les 6rythrocytes de diff6rents animaux (c~ux du cheval 6tant les plus r6sistants). Mots-clds: H~molysine, Vibro parahaemolyticus, Diarrh6e; Intoxications alimentaires, Caract&e Kangawa, Purification, Propri6t6s.
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