Clostridium botulinum type D neurotoxin: Purification and detection

To.rlcm Vol. 27, Ha. 2, pp. 221-228. 1989. Printed in Great Britain.

0041-0)101/69 s3.00+.00 $3 1989FtIpmnRasple

CLOSTRIDIUM BOTULINUM TYPE D NEUROTOXIN: PURIFICATION AND DETECTION KARENS. DE JONGH,~C. LEIGH SCHWARTZKOFF~ and MERLINE. H. HOWDEN~* 5chool of Chemistry,MacquarieUniversity, North Ryde, 2109,Australia, %QthurWebsterPty Ltd. 23 Victoria Avenue, Castle Hill, 2154, Australia, and ‘Division of Biological and Health !Zcicnces,De&in University, Geelong, 3217, Australia (Accepted for publication

2 September 1988)

K. S. DE JONGHC. L. SCHWARTZKOFTand M. E. H. HO’WDEN.Clostridium botulinum type D neurotoxin: purification and detection. Toxicon 27,221-228, 1989.-A method is reported for the purification of type D botulinurn toxin using a combination of low and high pressure ion exchange chromatography. The procedure produced homogeneous toxin in its free form in 3 days, with a specific toxicity in mice of 5.4 x lo7 r_D%/mg protein. Polyclonal antibodies against the pure toxin were raised in rabbits and detected the toxin in both ELISA and western blotting. The antibodies also detected type Cl botulinum toxin using these techniques, confirming the presence of cross-reacting antigenic determinants in these two proteins. INTRODUCTION BOTULINUMtoxin, which is one of the most potent toxins known (SELL~N,1984), acts primarily on choline@ nerve terminals where it inhibits release of acetylcholine, although the precise site and mode of blockade is unknown. This neurotoxin, produced by the anaerobic, spore-forming bacterium Clostridium botulinum, has been classified into eight distinct types on the basis of immunological characteristics (A-G; for review see SUGIYAMA, 1980; SIMPSON,1981). Types A, B, E and F toxins have often been involved in human botulism, while types Cl and D are responsible for botulism in cattle. Although most botulinum cultures produce only one type of toxin, type C strains produce predominantly Cl toxin and minor amounts of C2 and D toxins, while type D strains afford mainly D toxin with minor amounts of Cl and C2 (GGUMAet al., 1980). Production of Cl and D toxins is governed by bacteriophages, since cultures cured of their phages cease to produce toxin and recommence toxin production when reinfected. C2 toxin production is not related to these phages since cured strains still continue to produce C2 toxin (OGUMAet al., 1980). In contrast to the other types of botulinum toxins, type C2 toxin affects cell function by ADP-ribosylation of actin, but lacks any neurotoxic effects (AKTORIESet al., 1986). All toxin types except G have been purified and found to possess a common type of structure with a molecular weight of approximately 150,000. Although some toxin types are composed of heavy and light chains, with molecular weights of approximately 100,000 and 50,000, respectively, linked by a disulphide bond, others are produced as a single

*To whom correspondenceshould be.addressed. 221

222

K. S. DE JONGH eral.

polypeptide chain which requires “nicking” by tryspin before two chains are detectable (SAKAGUCHI et al., 1984). In culture fluids the toxins form complexes (M, 350,00&900,000) with non-toxic components and haemagglutinin, although this association is dissociated in alkaline solutions (OGUMA et al. 1981). The exception to this is type C2 toxin, which differs entirely in molecular structure and biological activity from any of toxins A through to G and whose presence only becomes apparent after the cultures are activated with tryspin (OHISHIand OKADA, 1986). Although botulinum toxin is normally detected using a mouse biosassay, the recently developed enzyme-linked immunoassay (ELISA) has been used for the detection of botulinum toxins A, B and E (NOTERMANS et al.,1982) as well as of types C and D (NOTERMANS et al., 1982a). The purpose of the present study was to develop a simple ELISA for the detection of type D botulinum toxin in cultures which are used for the production of a toxoid vaccine for the protection of cattle against botulism. In order to assess accurately the quantity of botulinum toxin present in a crude culture it was necessary to use toxoid from pure botulinum toxin for the production of antibodies for use in the ELISA. Although procedures for the purification of type D botulinum toxin have been published previously (MIYAZAKI et al., 1977; MURAYAMA et al., 1984) a new purification method was developed. We report here this procedure as well as details of the ELISA developed using the purified toxin. A preliminary account of this work was reported by DE JONGH et al. (1987). MATERIALS AND METHODS Material.3 Type D C. botuhum cultures were grown by the dialysing cultivation method (S~~RNEand WENTZEL,1950). Commercial type. Cl botulinurn toxin, which was purchased from WAKO Pure Chemical Industries, Osaka, Japan, was supplied as a I mg/ml solution containing 0.4 x IO’Lo,/mg protein, and was assessed to be 8% pure based on SDS-polyacrylamide gel electrophoresis and densitometric scanning of the stained gel. DEAESepharose, CM-Trisacryl and electrophoresis marker proteins were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. High performance liquid chromatography (HPLC) columns were from LKB Producter. Sweden. CChloro-I-naphthol, 3,3.‘5,5’-tetramethylbenzidine and Tween-20 were obtained from Sigma Chemical Co., St Louis. MO, U.S.A.. aswere protein A-peroxidase and goat anti-rabbit IgG (whole molecule)-peroxidase conjugate. Protein Aperoxidase contained 100 units of peroxidase per mg of protein and goat anti-rabbit IgCi peroxidase contained 4.5 units of peroxidase per ml. where I .Ounit of peroxidase will form I .Omg of purpurogallin from pyrogallol in 20 set at pH 6.0 and 20°C. Bovine serum albumin (BSA) was purchased from BoehringerMannheim (Australia) Pty Ltd. Sydney, Australia. All other chemicals were of the highest grade available and were used without further purification. Distilled, deionised water or Milli-Q water were used throughout. Purificarion of type D boUrwn toxin Low pressure column chromatography was carried out at 4°C. Particulate matter was removed from a I liter culture of type D C. botulinurn by centrifugation for 30min at 7ooOg. The culture was then concentrated to approximately 100 ml in an Amicon DC2 hollow fibre concentrator using a 100,000 molecular weight cut-off fibre and dialysed against 20mM Tris. pH 8.0 in this same unit. The toxin was loaded onto a column of DEAESepharose (4.4; 8 cm) equilibrated with 20 mM Tris. pH 8.0. The column was then washed with this buffer until the absorbance of the eluate at 280 nm was less than 0.05. A 600 ml linear O-O.3M NaCl aradient in 20 mM Tris. pH 8.0 was used to elute the toxin. The flow rate was 120 ml/hr and 17 ml fractions were collected. Toxic fractions eluted in the gradient of the DEAE-Sepharose column were combined and concentrated by ultrafiltration in a 50ml Amicon unit using a YM 30 membrane. Following overnight dialysis at 4°C against 50 mM ammonium acetate, pH 4.8 the toxin was applied to a CM-Trisacryl column (2.6 x 30 cm) equilibrated to this same buffer and unbound protein eluted by washing with this buffer until the absorbance of the eluate at 280 nm was less than 0.05. Bound protein was eluted with a 600 ml gradient from 50 mM ammonium acetate, pH 4.8 to I60 mM ammonium acetate. pH 5.3. The flow rate was I50 ml/hr and I5 ml fractions were collected. Toxic fractions were combined and concentrated to approximately IO ml by ultrafiltration as above. Further purification was achieved by repetitive 1 ml injections onto a 7.5 x 75 mm HPLC TSK SP-SW column. The column was eluted with a gradient from 250 mM to 500 mM ammonium acetate, pH 5.0 over I5 min at a flow rate of I ml/min. The toxic material from each injection eluting between I5 and 20 min was collected, combined,

CIostridim borulinum Type D Toxin

223

concentrated to approximately 4 ml by ultrafiltration in a 10 ml Amicon unit using a YM 30 membrane and dialysed against 8 liters distilled water for 2 hr. High performance anion exchange chromatography was then carried out on a 7.5 x 75 mm TSK DEAE-SPW column by repeated 1ml injections of the toxic material from above. The column was eluted with a gradient from 25 mM to 1.0 M ammomium acetate, pH 8.0 over 30 mitt at a flow rate of 1 ml/mm. The toxic material from each injection eluting between 8 and 9 mitt was collected and combined. Determination

of toxicity by mouse bioassay

For routine testing of column fractions, toxicity was assessed by injecting 0.2 ml of fractions i.v. into mouse tail veins and monitoring the times to death. Column fractions were diluted from lOO-to lO,C@O-foldbefore injection in order to assess where the peak of toxic activity was located. For the determination of mouse LD~ values, 0.2 ml of two-fold dilutions of samples were injected i.v. into each of four mice. The dilution of samples which caused half the mice to die within 4 days was taken to be LD,. Production of antibodies to type D botulinurn toxin

Sixty-five microgrammes of pure toxin in 0.5 ml of 10 mM sodium phosphate, pH 8.0 containing 0.8% (v/v) glutaraldehyde was incubated at 25°C; after 4 days it was no longer toxic to mice. Two rabbits were immunised by subcutaneous injection with 10 pg each of this toxoid mixed with an equal volume of Fteund’s complete adjuvant. At 4 weeks and 6 weeks further 10 pg injections were given mixed with Freund’s incomplete adjuvant. Rabbits were bled 2 weeks later and 68 ml of serum obtained. The IgG from the above serum was isolated by the addition of 18% (w/v) sodium sulphate. After stirrin8 for I hr at room temperature the serum was centrifuged at 30,000~ for 15 min. at 20°C. The resulting precipitatewas dissolved in 20 ml of 0.15 M NaCl and dialysed overnight against 2 liters of 70 mM sodium phosphate, pH 7.0. Precipitated material was removed by 10 min centrifuga~on at 30,OOOg.The supematant was applied to a column of DEAE-Sepharose (4.4 x 8 cm) equilibrated with 70 mM sodium phosphate, pH 7.0. The column was washed with this buffer at a flow rate of 100 m&r. The IgG was collected as the major protein peak eluting just ahead of the dark red methaemoglobin peak. After overnight dialysis against 4 liters of distilled water, the IgG was freexe dried and stored at -20°C. Protein estimations The protein concentrations

of samples were estimated by the method of BRADFORD (1976) using bovine serum albumin as the protein standard. Electrophoretic

procedures

SDS-polyacrylamide gel electrophoresis was carried out in 8 x 8 cm, 0.5 mm thick, 8.5% acrylamide gels according to the method of LAEMMLI (1970). Gels were stained with silver bv the method of Mom (1981). Westem blotting using 0.45~ nit;oceliulose was carried out in a semi-dry apparatus which was tnade’as outlined by KHYSE-ANDERSEN (1984). Blotting was carried out at 20 V for 60 mitt using 1.3 mM SDS, 48 mM Tris, 39 mM glycine, 20% (v/v) methanol, pH 9.2 as transfer buffer (Bmaaust and SCHAFER-Ntntsq 1986). Total protein staining of western blots was achieved with a starchiodide stain (KUMARer al., 1985). Botulinum toxin was identified on western blots using the polyclonal antibodies raised in rabbits against pure type D botulinum toxin as outlined above. After electroblotting, the membrane was equilibrated with 0. I M sodium phosphate/O. 15 M sodium chloride, pH 7.4 (PBS) for 10 min. The membrane was blocked for 2 x 15 min with 3% (v/v) skimmed milk powder in PBS-O.l% (v/v) Tween-20, rinsed with PBS, incubated with O.lpg/ml purified antibody in 0.5% BSA-PBS for I hr and then washed for 5 x 5 min in PBS. Protein A-horseradish peroxidase was used for visualization of antibodies bound to the membrane at a concentration of 5&ml in 0.5% BSA-PBS. The membrane was incubated with this for 1 hr and washed for 5 x 5 min in PBS. The substrate was prepared by dissolving 6 mg of 4-chloronaphthol in 2 ml of methanol and adding 10 ml of PBS and 10~1of 30% (w/v) hydrogen peroxide. The membrane was incubated in this solution until colour was fully developed (515min) and then rinsed with water. ELISA for the detection of toxin The ELISA was carried out in flat-bottomed

PVC plates. The toxin was diluted in PBS, 0. I ml of solution was pipetted into each well and the plates were incubated at 28°C for 1 hr. The toxin was removed and unbound sites were. blocked with 0.15 ml/well of 2.5% (w/v) BSA-PBS containing 0.01% (v/v) Tween-20 at 28°C for 1 hr. Wells were then washed with PBSO.OS% (v/v) Tween-20 for 2 x I min and 4 x 4 min. The purified antibody to type D botuhnum toxin (0.1 ml per well), diluted in 0.5% (w/v) BSA-PBS to 1.0 pg/ml, was added and the plates were incubated at 28°C for 2 hr. WeUs were then washed as above before the addition of anti-rabbit IgG conjugated to horseradish peroxidase; 0.1 ml of a l/400 dilution in 0.5% (w/v) BSA-PBS was added to each well and the plates were incubated at 4°C overnight. After washing the plates as above, 0. I5 ml of substrate solution was added to each well. The reaction was allowed to proceed at room temperature in the dark. When the colour was sufficiently developed (approximately 5 min) the reaction was stopped by the addition of 0.05 ml of 2 M sulphuric acid to each well. The absorbance of each well was measured at 450 nm in a Titertek Multiskan instrument. The substrate

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et al.

solution was prepared by dissolving 350 mg of tetramethylbenzidine in 100 ml of methanol by stirring at room temoerature for several hr. Immediatelv before use 0.3 ml of this solution was added to 9.7 ml of I .O M sodium ace&titrate, pH 6.0 containing 1.Spi of 30% (v/v) hydrogen peroxide.

RESULTS

Previously published methods for the purification of type D botulinum toxin have used an initial ammonium sulphate precipitation for concentration of culture supematants followed by overnight dialysis before the first column chromatography. The use of hollow fibres as reported here allows these steps to be reduced to one rapid step and resulted in approximately 98% recovery of toxicity. Figure 1 shows the elution profile of type D toxin from the DEAE-Sepharose column. On some occasions not all of the toxin was bound to the DEAE column, with some toxicity being recovered in the wash fractions. Typically this toxin pool represented 25% of the bound fraction and was discarded. It may represent botulinum toxin which is still associated with the non-toxic protein component or with haemagglutinin, although such complexes may be expected to dissociate at alkaline pH. Only the toxic material that was bound to the DEAE column was applied to the CMTrisacryl column and the elution profile for this is shown in Fig. 2. Figures 3 and 4 show the elution profiles for the two HPLC chromatograms. The final preparation had a specific toxicity of 5.4 x lO’Lo,/mg protein. The preparation was assessed for purity by SDSpolyacrylamide gel electrophoresis. Under non-reducing conditions only one band was visible on a 0.5 mm, silver-stained gel containing 2.Opg protein, indicating the preparation was pure. This band had a relative mobility slightly higher than IgG, corresponding to a molwt of 140,ooO. Under reducing conditions two bands were visible with molecular weights of 50,000 and 90,000. This preparation was used to produce polyclonal antibodies in rabbits against type D botulinum toxin. The serum obtained neutralised 16 units of International Standard type D botulinum toxin per ml. After purification of the IgG, 160 pg of antibody was needed to neutral& 1 unit of type D toxin. The purified antibody was used for the detection of toxin in crude cultures following western blotting. Under reducing conditions two bands, corresponding to molecular weights of 90,000 and 50,000 were visible following immunostaining. This indicates the presence of antibodies in the polyclonal antiserum which recognise both the heavy and light chains of the toxin. 0.5 r

10

20

30

40

50

Fraction Number (17 ml fractions) FIG. 1. DEAE-SEPHAROSE CE~ROMATOGRAPHY OF TYPE D TOXIN. extract concentrated from 1 liter of type D C. botulinurn culture as outlined in Methods was dialysed against 29 mM Tris, pH 8.0 and applied to a DEAE-!Iepharose column (4.4 x 8 cm) which was equilibrated with 20 mM Tris, pH 8.0. A NaCl gradient was used to elute the bound protein using a flow rate of 120 ml/hr. The hatched area indicates toxic fractions. The

225

Closfridirunbot&rwn Type D Toxin

Fraction number (15 ml fractiona)

Fro. 2. CM-TRISACWLCHROMATOGRAPHYOF TYPE D TOXIN. Toxic fractions from the DEAE-Sepharose column (Fig. 1) were concentrated, dialysed to 50 mM ammonium acetate, pH 4.8 and applied to a CM-Trisacryl column (2.6 x 30 cm) which was equilibrated with this b&r. Unbound protein was eluted with this buffer at a flow rate of 150 ml/ hr. Sound protein was eluted with a gradient from 50 mM ammonium acetate, pH 4.8 to 160 mM ammonium acetate, pH 5.3 beginning at the arrow. Toxicity was determined by monitoring the hour till death after a 0.2 ml injection of a l/lo0 dilution of fractions i.v. into mice.

The antibodies were also used to develop an ELBA for toxin detection. An initial study involving variations in the concentrations of the antibody relative to botulinum toxin and to the anti-rabbit IgG-horseradish peroxidase conjugate, indicated that the optimal concentrations of these antibodies was 1.Opg/ml and a l/400 dilution, respectively. Using these concentrations it was found that the observed absorbance was directly proportional to toxin concentration between 25 and 700 LD, per well (Fig. 5). It was also observed that at very high concentrations of toxin (< l/250 dilution), absorbance in the ELBA was not proportional to the toxin concentration. Presumably this was due to a relatively high amount of toxin bound to the plate causing steric hindrance to antibody binding. The polyclonal antibody raised in rabbits against pure type D botulinum toxin was tested for cross-reaction with type Cl toxin. Figure 5 shows that the commercial type Cl botulinum toxin, purchased from WAKO chemicals, cross-reacted with the antibodies to pure type D botulinum toxin in ELBA. As was the case for D toxin, the absorbance

2

L 0

I

a

I

I

34 16 Elution Time (min)

1

32

FIG. 3. HIGH PERH~RMANCECATION EXCHANGECHROMATOGRAPHYOF TYPE D TOXIN. Toxic fractions from the CM-Trisacryl column (Fig. 2) were concentrated and applied to a 7.5 x 75 mm TSK SP-5W column which was eluted with a gradient from 250 mM to 5OOmM ammonium acetate, pH 5.0 over 15 min, followed by 500 mM ammonium acetate,pH 5.0 at a flow rate of 1 ml/mm. Toxicity was determined by monitoring the hour till death after a 0.2 ml injection of fractions i.v. into mice.

K. S. DE JONGH ef al.

16 8 El&on Time (min)

24

FIG. 4. HIGH PERFORMANCEANION EXCHANGECHROMATUGRAPHYOF TYPE D TOXIN. Toxic fractions from the TSK SP-SW column (Fig. 3) were concentrated, dialysed and applied to a 7.5 x 75 mm TSK DEAE-SPW column which was eluted with a gradient from 25 mM to 1.OM ammmonium acetate, pH 8.0 over 30min at a flow rate of 1 ml/min. Toxicity was determined by monitoring the hour till death after a 0.2 ml injection of fractions i.v. into mice and is indicated by the hatched area.

observed in the ELISA was proportional to the amount of Cl toxin used in the assay. Following electrophoresis and western blotting on nitrocellulose, type Cl toxin was detected immunochemically using the antibody to type D toxin and a protein Ahorseradish peroxidase conjugate as outlined in the methods section above. 1.6

FIG. 5. ELISA OF IYPES D AND cl TOXINS. Various dilutions (0.1 ml) of type D toxin(O), or type Cl toxin (0) were incubated in the wells of a PVC microtiter plate at 28°C for 1 hr and unbound sites on the plate were then blocked by incubation with 0.15 ml of 2.5% BSA-PBS at 28°C for 1 hr. Following washing of the plate, 0.1 ml of l.O&ml antibody to pure type D botulinurn toxin was added to each well and plates were incubated at 28’C for 2 hr. After washing of the plate, 0.1 ml of a l/400 dilution of anti-rabbit IgG horseradish peroxidase was added to each well and incubated overnight at 4’C. After a further washing, 0.15 ml of tetramethylbetuidine substrate solution was added to each well and the reaction was allowed to prcceed for approximately 5 min before being stopped by the addition of 0.05 ml of 2 M sulphuric acid to each well. The absorbance of each well was then measured at 450 nm.

Clostria%un bottdimm Type D Toxin

227

DISCUSSION

DASGUPTAand SATHYAMO~RTHY (1984) noted that the literature on botulinum neurotoxin included several claims of purifications and characterizations that were irreproducible, in agreement with our experience following published procedures for the purification of type D toxin, and thus a new purification procedure was developed. This procedure isolated homogeneous type D botulinum toxin in its uncomplexed form in 3 days. The use of HPLC enabled high chromatographic resolution to be obtained in a short period of time. The specific toxicity of the preparation (5.4 x 10’ q,Jmg protein) is similar to the value of 5.8 x 10’ q,Jmg protein reported for purified type D toxin by MURAYAMA et al. (1984). MNAZAKI et al. (1977) reported that the toxic component of type D botulinum toxin, although non-activatable, was a single polypeptide chain which could be converted to a two-chain molecule using tryspin. In contrast to this, MURAYAMA et al. (1984) purified type D toxin which had a two-chain structure. The toxin purified in the present study also has a two-chain structure, as evidenced by the two bands with molecular weights of 50,000 and 90,000 which were seen after SDS-polyacrykmide gel electrophoresis in the presence of /Imercaptoethanol. In addition, following electrophoresis under reducing conditions, westem blotting and immunodetection of a semi-purified toxin preparation, two bands which corresponded in molecular weights to the heavy and light chains of the reduced toxin were visible. Although the mouse test is the most commonly used assay for the detection of botulinum toxins, an alternative method which allows accurate estimation of toxin levels in cultures and samples without the need to use animals is desirable. The ELISA presented in this study could provide such an alternative at levels of sensitivity approaching those of the mouse assay. However, it is clear that the antiserum used in the assay must be prepared against toxoid generated from highly purified neurotoxin, from which the haemagglutinin and non-toxic components have been removed. Even though this was the case, the assay failed to distinguish between type Cl and D toxins. The ELISA of NOTFBMANSet al. (1982u) also failed to differentiate between these two toxin types. In their case the toxin used for antisera preparation may have been chemically impure. Their inability to distinguish between types Cl and D toxins may have been due to this last factor or to the existence of cross-reacting antigenic determinants in the two toxins. Indeed a number of reports have shown, by both ELISA and in vivo neutralization experiments, that D toxin is antigenically related to type Cl toxin (OGUMA et al., 1980, 1984; NOTERMANSet al., 1982a; OCHANDAet al., 1984). MURAYAMA et al., (1984) concluded that there are two kinds of antigenic sites in each of types Cl and D toxins, one of which is common between them while the other is toxin-type specific. Further evidence for this antigenic cross-reaction was provided by the fact that type Cl toxin was detected, following electrophoresis and western blotting, using an immunostain with antibodies raised against toxoid from pure type D toxin. Hence, although the ELISA reported here may be useful in detecting low levels of types Cl and D botulinurn toxins, it cannot be used to distinguish between them. AcknowIedgemenrs- K. S. De J. was supported by the National Research Fellowships Scheme of Australia. The authors thank A. F. Webster and M. Devine for producing the C. botulinum cultures and Dr A. Comis for special assistance. REFERENCES AKTORIES, K., BARMANN, M., Omm, I., TSUYASU,S., JAKOFIS, K. H. and HABERm, toxin ADP-ribosylatea actin. Nature 322,390.

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