Antigenic and chromatographic identity of two apparently distinct toxins of Clostridium botirlinum type A

A. H. W. HAUSCHILD AND R. HILSHEIMER Research Laboratories of the Food and Drug Directorate, Department of National Health arid Welfare, Ottawa, Canada

Received May 5, 1969

HAUSCHILD, A. H. W. and HILSHEIMER, R. 1969. Antigenic and chromatographic identity of two appar- ently distinct toxins of C/ostridium botulinum type A. Can. J. Microbiol. 15: 1129-1132.

Two toxic components (I and 11) of Clostridium botirlinurn type A were separated by chromatography on Sephadex G-200. The relationship between these components was investigated. Antisera prepared against either component I or I1 cross-neutralized both toxins. The neutralizing values of each anti- serum were the same when tested at the L+/50 level against components I and 11, and unfractionated toxin.

Component I was associated with hemagglutinating activity. Toxin and hemagglutinin were separated by chromatography on diethylaminoethyl (DEAE) cellulose. Rechromatography of the toxin on Sepha- dex G-200 showed a reduction in molecular weight from 450 000 to 140 000. Component I1 was free of hemagglutinin, but its molecular weight shifted from 190 000 to 140 000 and its Stokes radius from 5.8 mp to 4.8 mp upon chromatography on DEAE cellulose.

It is concluded that components I and I1 have similar or identical antigenic sites, and it appears that their toxic moieties are also similar or identical in composition.

Introduction

The toxins of culture fluids (7, 8) and cells (8) of CIostridiuin botulinum type A have been separated into two distinct components by chromatography on Sephadex G-200. The present paper shows that these components possess common antigenic properties, and that both can be converted to new toxic forms with identical Kav values (9).

Materials and Methods Preparation of Crude Toxin

Clostridi~im botrrlinlrm type A, strain 62A, was grown as described previously (8). After 3 days of growth the cultures were centrifuged at 10 000 relative centrifugal force (r.c.f.) for 10 min. The supernatant fluids were treated with 450g of (NH4)2S04 per liter at pH 6.5, kept a t 5 "C for 4 h, and centrifuged at 8500 r.c.f. for 10 min. The sediments were resuspended to 3% of the original volume with 0.067 M sodium citrate buffer, pH 5.6, containing 0.1 MKC1, dialyzed against the same buffer for 16 h and centrifuged at 8500 r.c.f. for 10 min. The toxicities were assayed by intraperitoneal injections of mice (3).

Colirinn Chromatogr.~~plty Up to 2ml of the dialyzed (NH4)2S04 precipitate

were fractionated on a column of Sephadex G-200, 2.5 X 80 cm as described before (8), with the exception that elution was carried out with a Perpex peristaltic pump (LKB, Stockholm, Sweden) at a rate of 12 ml/h. The eluate was collected in volumes of 2.85 ml. The toxic fractions I and I1 were concentratcd by dialysis against polyethylene glycol 20 000 (Fisher Scientific Co.), dia- lyzed against 0.04 M Na-phosphate, pH 7.5, for 16 and 5 h, respectively, and adjusted to pH 7.9 with N NaOH. These steps preceded chromatography on diethylamino-

ethyl (DEAE) cellulose. The final pH adjustment to 7.9 was made after dialysis because about 70% of the toxicity was lost during dialysis at pH 7.9 while little or no loss occurred at pH 7.5.

Chromatography on DEAE cellulose (Matheson Co., Norwood, Ohio) was carried out essentially accord- ing to the method of DasGupta and Boroff (4). Volumes of 5 ml were applied to 2 X 15 cm columns of DEAE cellulose equilibrated with 0.04 M Na-phosphate, pH 7.9. Initial elution of the columns was carried out with the same buffer. This was followed by gradient elution with 0.1 M Na-phosphate in 0.1 M NaC1, pH 7.6, through a mixing vessel containing 50mI of 0.04 M Na-phosphate, pH 7.9. Occasionally, volumes of 1 ml were fractionated on a 1 X I5 cm column.

The fractions from the DEAE columns were dialyzed against citrate-KC1 buffer, pH 5.6 for 16 h, concentrated by dialysis against polyethylene glycol 20 000, and rechromatographed on Sephadex G-200.

Molecular weights were determined by the method of Andrews (I), which was modified by relating Knv values (9) of the toxic fractions to those of standard proteins.

Hernaggl~rtination The diluent for the hemagglutinin samples was 0.05 M

Na-phosphate in 0.5% NaC1, pH 7.0, with the following exceptions: (i) to neutralize fractions in citrate-KC1 buffer, pH 5.6, the first dilution was carried out by adding 0.1 ml of 0.2 M Na2HP04 and 0.3 ml of the phosphate-NaCl buffer, pH 7.0, to 0.4ml of sample; (ii) fractions in 0.04 M Na-phosphate, pH 7.9, were diluted 1:2 by adding equal volumes of 0.1 M Na- phosphate in 0.85% NaCl, pH 6.8. Mixtures of 0.4 ml of diluted samples and 0.2 ml of 1% rabbit red blood cells in 0.85% NaCl were kept at 5 OC for 4 11. One hemagglutinin unit (H.U.) was arbitrarily taken as the amount of hemagglutinin in the mixture producing 50% hemagglutination. With phosphate-NaC1 buffer, pH 7.0, as diluent, lower readings but sharper end points were obtained than with 0.85% NaC1.

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1130 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 15. 1969

Atztigenicity Components I and I1 (8) were purified further by

rechromatography on Sephadex G-200. The toxicities were 5 X 105 and 4 X 10s mouse minimum lethal dose (MLD) per ml, respectively. Toxoids were prepared by adding 1 ml of 37y0 formaldehyde per liter of toxin, and preserved with 1 ml of 17, penicillin G, 10 ml of 2% streptomycin, and 10 ml of parabens in 95Y0 ethanol containing 1.5 g methyl paraben (p-hydroxybenzoic acid methyl ester) and 0.15 g propyl paraben (Pivnick, personal communication). The preparations were in- cubated for about 4 weeks at 35 "C until the toxicities were reduced to 2 mouse MLD/ml, and subsequently stored at 5 "C. There was no measurable decrease in the concentration of formaldehyde during incubation at 35 "C as determined by the method of Nash (10).

Four- to five- month-old rabbits (New Zealand White) weighing 2.5-3 kg were used for the preparation of antitoxin. Rabbit 1 (Table I) received subcutaneous injections of 0.5 ml each of toxoid I on 3 successive days per week. The remaining three rabbits received weekly injections of 1.0 ml each of 1 :1 mixtures of toxoid and Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.) into the thigh muscle of the right hind leg. Rabbits 1 and 2 were immunized for 4 weeks, with a 10-day rest period after the last injection, and rabbits 3 and 4 for 6 weeks, with a subsequent 2-week rest period.

The animals were bled by cardiopuncture. The blood samples were kept at 20°C for 4 h in sterile 30-rnl test tubes, and the clots freed from the glass walls. The samples were then held at 5 OC for 20 h and centrifuged at 8500 r.c.f. for 10 min. The sera were stored at - 5 OC.

The toxins were neutralized at the L+/50 level. Unfractionated toxin of C. bot~rli t~~lw~ type A and com- ponents I and I1 were stored with 507, glycerol at - 5 "C, standardized with C. botlllinlrtn type A antitoxin (Con- naught Medical Research Laboratories, Toronto, Ont.), and diluted with gelatin-phosphate buffer (3) to contain 4/50 L+ doses/ml. The immune sera were diluted with the same buffer. The toxins were mixed 1:1 with diluted serum, and the mixtures kept at room temperature for

2 h. Volumes of 0.5 ml were injected intraperitoneally into mice. Three animals were used per dilution. The 50% end points were determined after 3 days.

Results Table I shows the antitoxin titers of the rabbit

sera against the three toxin preparations. Each preparation was neutralized by any of the four sera. The antitoxin titers of a given serum against the three toxin preparations were not significantly different.

Figure 1A shows tbe separation of the ammo- nium sulfate precipitate on Sephadex G-200 into the toxic components I and 11, and the association of component I with hemagglutinin. Attempts were made to separate the toxic and hemagglutinating activities of component I by treatment with 1 M NaCl, veronal buffer at pH 9, 3 M guanidine X HCI, and red blood cells, but with little or no success. However, the toxin was liberated from the hemagglutinin on a column of DEAE cellulose while the

TABLE I 0 ' 4 . lo

*.- ?; .,-.- - *.-. Neutralization of C. botallirl~rrn type A tox~ns with rabbit

scra prepared against components I and I1 15

An ti toxin m'

Toxoid Rabbit

Comp. I 1

Cornp. I1

*i.u. = international units.

Toxin content of

serum, i.u./ml*

Crude Comp. I Comp. I1 Crude Comp. I Comp. I1 Crude Comp. T Comp. I1 Crude Comp. I Comp. TI

FIG. 1. Chromatograplly of C. botulirz~~m type A toxin on Sephadex G-200. (A) Toxic and hemagglutinat- ing activities of the fractions derived from crude toxin. (B) Rechromatography of the toxic fractions of compo- nents I (A) and I1 (A) separated on DEAE cellulose.

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HAUSCHlLD AND HlLSHEIMER: TWO TOXlNS O F CLOSTRLDlUM BOTULlNUM 1131

hemagglutinin fraction remained associated with about 10yo of the total toxin (Fig. 2). In subsequent experiments the hemagglutinin was eluted with the same buffer, but without gradient.

Rechromatography of the main toxic fraction froin the DEAE column on Sephadex G-200 showed one distinct toxic peak, but its position did not coincide with either of the original toxic components (Fig. 1B). The molecular weight of the new toxic component (I-C) was calculated from five determinations of Kav values as M = 141 000 k 10 000 (S.D.). This value was significantly different (P < 0.001) from the molecular weight of component 11, which was calculated from six Kav determinations as M = 194 000 1 7000 (S.D.). The Stokes radii of components I-C and I1 were calculated as 4.8 mp and 5.8 mp, respectively (1 1).

Addition of the hemagglutinin fraction from the DEAE column to component I-C did not affect the molecular weight determination of the toxin on Sephadex G-200. Also, repeated passage of the toxin through columlls of DEAE cellulose did not further alter its position on the Sephadex column.

Although component I1 was not associated with hemagglutinin, it was also converted to a new toxic form (11-C) by treatment with DEAE cellulose. Figure 1B shows that compoilents

V O L U M E ( M L )

~ - ~ ' ~ ~ ~ - % ~ r a d elution wilh 01 hl Elc-P+Ol hl P ~ ~ C I , pH7.5

FIG. 2. Fractionation of component I on DEAE cellulose.

I-C and 11-C had identical positions on the Sephadex G-200 column.

Discussion

As in our previous work (8), we have avoided all purification procedures that might possibly have altered the original composition of the botulinum toxin, with the exception of the ammonium sulfate treatment. However, prelim- inary experiments showed that this procedure did not measurably alter the relative toxicities of the two components, or their Kav values.

Components I and I1 were converted by DEAE ion exchange to new toxic forms with identical Kav values on Sephadex G-200. This finding is consistent with the immunization experiment, which showed that the two com- ponents had similar or identical antigenic sites. I t is likely, therefore, that the new toxic forms derived from compoilents I and I1 are very similar or the same. Conclusive evidence will have to await further purification of the com- ponents, and determination of their composition.

The conversion of components I and I1 to a common toxic form may be explained by removal of hemagglutinin with a molecular weight of about 300 000 from component I, and of a 50 000 molecular weight moiety from component 11. DasGupta and Boroff (5) separated three forms of hemagglutinin from crystalliile toxin. The smallest had a molecular weight of 290 000. However, it is also possible that the conversion of component I1 as evidenced by a 25y0 red~~ct ion in molecular weight may involve only a coilfigurational change to a less spherical form. Such a change might be a pre- condition for the separatioil of component I into its toxic and hemagglutinating moieties.

We suggested previously (8) that coinponent I1 might be identical with the "a component" of type A toxin described by DasGupta et al. (4,6), who obtained the a component by DEAE ion exchange chromatography of high molecular crystalline toxin, and determined its molecular weight as 130 000 to 150 000 (2,5) and a Stokes radius as 4.8 mp (5). Our componellts I-C and 11-C were also obtained using DEAE ion exchange and had a molecular weight of about 140 000 and a Stokes radius of 4.8 mp. It seems more likely, therefore, that components I-C and 11-C are identical with the a component of DasGupta et al.

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1132 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 15, 1969

Wagman (12) converted crystalline toxin of C. botulinum type A at pH 9.2 to a 7 S com- ponent with a molecular weight of 158 000. This component cannot as yet be related t o any of the toxic forms described here.

Acknowledgments We thank Dr. F. S. Thatcher for his continued

support of this project, Dr. D. A. Boroff for his valuable advice. a n d Mr. 1. E. Erdman for performing cardiopuncture o n the immunized rabbits.

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5. DASGUPTA, B. R. and BOROFF, D. A. 1968. Separa- tion of toxin and hemagglutinin from crystalline toxin of Clostridium botulinum tvoe A bv anion exchange chromatography and &termination ~ of their dimensions by gel filtration. J. Biol. Chem. 243: 1065-1072.

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