FEMS MicrobiologyLetters 56 (1988) 331-336
Published by Elsevier
331
FEM 03391
Analysis of purity of Clostridium difficile toxin A derived by
affinity chromatography on immobilized bovine thyroglobulin
S. K a m i y a , P.J. R e e d a n d S.P. Borriello
Microbial Pathogenicity Research Group, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, U.K.
Received 17 August 1988
Accepted 19 August 1988
Key words: Clostridium difficile toxin A; Affinity chromatography; Bovine thyroglobulin
1. SUMMARY
Clostridium difficile toxin A separated by bovine
thyroglobulin affinity c h r o m a t o g r a p h y was
analysed for its purity by fast protein liquid anion-exchange chromatography (FPLC). By testing
each FPLC fraction for cytotoxicity the affinitypurified toxin A specimen was shown to contain
small amounts of contaminating toxin B. Coldhaemagglutination activity was detected in FPLC
fractions corresponding to toxin A but not in
those corresponding to toxin B. Toxin A obtained
from bovine thyroglobulin affinity chromatography should be further purified to remove traces of
toxin B.
2. I N T R O D U C T I O N
Clostridium difficile and its toxins are involved
in the aetiology of pseudomembranous colitis [1-3]
and a proportion of cases of antibiotic-associated
diarrhoea. Although two toxins of this organism,
designated toxin A (enterotoxin) and toxin B (cy-
Correspondenceto: Dr. S.P. Borriello, Microbial Pathogenicity
Research Group, MRC Clinical Research Centre, Watford
Road, Harrow, Middlesex HA1 3UJ, U.K.
totoxin), have similar biological activities; i.e. cytotoxicity, mouse lethality and ability to increase
vascular permeability, it has been reported that
only toxin A induces fluid accumulation with
haemorrhage in the rabbit ileal loop test [4-6].
Recently Krivan et al. [7] reported that toxin A
binds to cell-surface glycoconjugates containing
the non-reducing terminal sequence Gala 1-3
Galfl 1 - 4 GlcNAc, and that its binding is temperature dependent. Bovine thyroglobulin and rabbit
blood cells have also been shown to have such
sequences on their surfaces and to bind toxin A at
4 ° C and release it at 37 ° C [7,8]. By means of this
temperature-dependent binding between C. difficile toxin A and bovine thyroglobulin, a purification method for toxin A by affinity chromatography on immobilized thyroglobulin has recently
been developed [8]. In the present study, we
analysed toxin A obtained following bovine
thyroglobulin affinity chromatography for its purity, and found that it contained trace amounts of
toxin B.
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacterial strain and culture filtrate
Clostridium difficile VPI 10463 strain (originally
obtained from Dr. T.D. Wilkins) was kindly pro-
0378-1097/88/$03.50 © 1988 Federation of European MicrobiologicalSocieties
332
vided by Dr. M. Giuliano (Laboratorio di Batteriologia e Micologia Medica, Istituto Superior di
SanitS,, Rome, Italy). The organism was grown
anaerobically in 10 ml of brain heart infusion
broth (BHI, Oxoid) for 18 h at 37 ° C. The pelleted
bacterial cells were resuspended in 10 ml of phosphate buffered saline (pH 7.2), and 2 ml of this
suspension inoculated into dialysis tubing containing c. 100 ml PBSA, which had been equilibrated against BHI overnight, suspended in 2 1 of
BHI supplemented with 0.5% ( w / v ) yeast extract
(Beta Lab. Surrey), 0.05% ( w / v ) L-cysteine HC1
( B D H Chemicals) and 0.03% ( w / v ) sodium formaldehyde sulphoxylate. After anaerobic incubation for 5 days at 37 ° C, the content of the dialysis
tubing was centrifuged, the culture supernatant
retained and filtered (0.45 fire).
lobulin was bound to the beads. After blocking
the remaining active sites on the gel with 0.1 M
ethanolamine for 30 min at 4 ° C , the coupled
beads (approximately 20 mg of thyroglobulin/ml
of gel) were packed in a glass column (Pharmacia,
Uppsala, Sweden; column C 10/10, 10 by 100
mm) and washed at 3 7 ° C with 50 bed volumes
(250 ml) each of pre-warmed basic buffer (0.1 M
glycine-sodium hydroxide containing 0.5 M NaCl,
p H 10.0) and acidic buffer (0.1 M glycine-hydrochloride containing 0.5 M NaC1, p H 2.0). The
column was then equilibrated at 4 ° C with 0.05 M
Tris HC1, 0.15 M NaC1, p H 7.0 (TBS). C difficile
culture filtrate cooled to 4 ° C (100 ml) at a protein
concentration of 2,4 m g / m l , was applied to the
column at 4 °C. After washing the column with
250 ml or 1150 ml of TBS at 4 ° C , it was warmed
at 3 7 ° C for 2 h, and eluted with TBS at 3 7 ° C to
obtain toxin A. Fractions (5 ml) of the washings at
4 ° C and the ehiates obtained at 3 7 ° C were collected and monitored for absorbance at 280 nm
(A280) , cold-haemagglutination activity and cytotoxicity as described below.
3.2. Bovine thyroglobulin affinity chromatograph)'
The method used was as described by Krivan
and Wilkins [8]. Briefly, 25 mg of bovine thyroglobulin (Sigma) was dissolved in 50 ml of 0.1 M
morpholinepropane sulphonic acid buffer (pH 7.0),
centrifuged (8000 5< g) and filtered (0.2 ~m). The
glycoprotein solution was reacted with 10 ml of
activated Affi-Gel 15 (Bio-Rad Laboratories)
overnight at 4 ° C with shaking. 86% of thyrog-
3.3. Haemagglutination assay
Assay for cold-haemagglutination (HA) activity
of toxin A was based on the method described by
37c
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Fraction number
Fig. 1. Bovine thyroglobulin affinity chromatography of C. difficile culture filtrate. Culture filtrate of C. difficile was applied to the
affinity column, and 5 ml fractions collected at 4 ° C. From fraction 71 (arrowed) the elution was performed at 37 o C. (e) A2~0, (o)
reciprocal titre of cytotoxicity to Veto cells, (A) cold-haemagglutination activity. ND--below level of detection.
333
Krivan et al. [7]. Briefly, rabbit blood cells were
washed three times with isotonic buffer (0.1 M
Tris, pH 7.2 containing 50 mM NaC1), and diluted
to a 1.0% suspension. Two-fold serial dilution of
specimens (50/~1) was performed with the isotonic
buffer in V-bottom microplate wells (Sterilin,
Middlesex, England), 50 ~tl of blood cells were
added to each well, and the reaction performed at
4 °C for 3 h. The titre of HA was expressed as the
reciprocal of the highest dilution of the specimen
with macroscopically positive haemagglutination.
3.4. Cytotoxicity assay
Cytotoxicity was examined using African green
monkey kidney (Veto) cells as described previously [9] with the modification that all tests were
performed in microtitre wells. The titre (per 100
/~1) was expressed as a reciprocal of the dilution
which induced a 100% cytopathic effect (CPE)
after incubation overnight.
3.5. Anion-exchange chromatography
Anion-exchange chromatography was performed on a mono Q column HR 5/5 (50 × 5
mm, Pharmacia) incorporated into a fast protein
liquid chromatography (FPLC) apparatus (Pharmacia) as described previously for the analysis of
C. difficile toxins [10]. Sample (500/~1) was filtered
through a membrane filter (0.2/xm) before applying to the column. Each 1 ml fraction was examined for cytotoxicity and HA activity as described above.
Thyroglobulin affinity chromatography derived
fractions, which were expected to be free of toxin
B, were always run before fractions expected to
contain toxin B. A 'blank' FPLC run was
performed prior to and between each sample run
and buffer fractions collected during the 'blank'
runs examined for cytotoxicity.
3.6. Gel electrophoresis
S D S - p o l y a c r y l a m i d e gel electrophoresis
(SDS-PAGE) was performed as described by
Laemmli [11] on 5% resolving slab gel, 1.5 mm in
thickness, and the gels stained with Coomassie
blue R-250. Molecular weights were estimated by
comparison to high molecular weight standards
(BioRad Laboratories). Analysis by gradient polyacrylamide gel electrophoresis was done in a 4 to
30% gel in Tris-glycine buffer pH 8.8. Gradient
gels were silver stained (BioRad Laboratories), as
instructed by the manufacturers (BioRad Bulletin
1089), or stained with Coomassie blue. Molecular
weights were estimated by comparison to high
molecular weight standards (Pharmacia).
1.0--
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6
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24
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I i t 1 1 1 1 1
4 8 ]2 16 20 24 28 32
-ND
ND
Fraction number
Fig. 2. Anion-exchange chromatography profiles of fractions 71 (A) and 8 (B) obtained by thyroglobulin affinity chromatography.
Each 1 ml fraction collected was examined for cytotoxicity (©), and HA activity (A), as described in MATERIALSAND METHODS. Molar
concentration of NaC1 (11). ND--below level of detection.
334
3. 7. Protein assay
Protein was quantitated by the dye-binding
method of Bradford [12] with the Bio-Rad protein
assay kit. Bovine serum albumin was used as a
standard.
4. RESULTS
4.1. Affinity chromatography
Between eluted fractions 2 and 20, which are
unbound proteins, the peaks of A280 and cytotoxicity were 0.87 and 108 respectively (Fig. 1).
The value of A280 decreased thereafter to approximately zero. The cytotoxicity gradually decreased during washing of the column with TBS.
However, in fraction (fr) 70, eluted after washing
with 250 ml of TBS, a cytotoxic titre of 101 was
still detected. No haemagglutination activity was
detected with any of the fractions eluted at 4 ° C.
The column was warmed to 37 ° C, and elution at
37 ° C started from fr 71. The first fraction eluting
at 3 7 ° C had a high cytotoxic titre (104) and the
highest titre (1 : 64) of HA, although no significant
increase in A280 was observed (Fig. 1), unlike the
report described elsewhere [8]. By continuing elution at 3 7 ° C both HA activity and cyto,oxicity
decreased, and a low titre of cytotoxicity (10 ° i.e.
1 : 1) without HA activity was seen in fr 100.
Using the same column after washing with
acidic buffer and re-equilibrating with TBS [8],
100 ml of crude culture filtrate was applied to the
column, and an attempt to wash the column to the
point where eluted fractions would not exhibit any
cytotoxicity was made using 1150 ml TBS at 4 ° C.
The last fraction, fr 250, however, still had a low
titre of cytotoxicity (10°). When the temperature
of elution was changed to 37 ° C, the first fraction
eluting, fr 261, showed similar amounts of cytotoxicity (104) and HA activity (1 : 128) as previously without any significant increase of A 280.
4.2. Anion-exchange chromatography
The affinity-purified toxin A, fr 71, which was
expected to be pure toxin A, was analysed by
anion-exchange chromatography (FPLC) and each
1 ml fraction screened for cytotoxicity and HA
activity (Fig. 2A). Peaks of cytotoxicity were ob-
served in the fractions eluting between 0.32-0.36
M NaC1 (fractions 10 and 11), and 0.64-0.68 M
NaC1 (fractions 18 and 19) with titres of 103 and
102 respectively, indicating that the fr 71 specimen contained not only toxin A but a small
amount of toxin B. H A activity was detected in
fractions eluting at 0.32-0.36 M NaC1 (fractions
10 and 11), corresponding to toxin A (Fig. 2A).
FPLC analysis of fr 8 obtained from affinity
chromatography at 4 ° C also yielded two peaks of
cytotoxicity corresponding to toxins A and B (Fig.
Fig. 3. Analysis of toxin A preparations by native gradient
polyacrylamide gel electrophoresis (4 to 30%) in Tris-glycine
buffer pH 8.8 and developed by Coomassie blue. Lanes 1 and
3 high molecular weight markers, thyroglobulin (669 kDa),
ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase
(140 kDa), bovine serum albumin (67 kDa); lane 2 fraction 71
following affinity chromatography (15/~g).
335
2B). However, no HA activity was detected in
either fraction.
FPLC fractions collected from blank runs never
yielded cytotoxicity or H A activity corresponding
to either toxins A or B.
4.3. Gel electrophoresis
The first fraction, in this case fr 71, to be eluted
from the thyroglobulin affinity column at 3 7 ° C
was analysed directly by both native gradient
polyacrylamide gel electrophoresis (G-PAGE) and
S D S - P A G E . Only one protein band of molecular
weight corresponding to 520 kDa for G - P A G E
from a protein load of 15 ~g (Fig. 3) and to 240
kDa for S D S - P A G E from a protein load of 7 #g
(data not shown) was detected following Coomassie blue staining. Silver staining of 5 /~g of the
same fraction following G - P A G E yielded the presence of a contaminating protein (220 kDa; Fig. 4)
and two other faint bands (400 kDa, which may
represent toxin B; and 120 kDa which did not
reproduce at photography. None of these contaminating proteins were detected in the preparation following anion-exchange chromatography
(Fig. 4).
5. DISCUSSION
Fig. 4. Analysis of toxin A preparations by native gradient
PAGE (see Fig. 3) developed by silver stain. Lanes 1 and 4
high molecular weight markers (see Fig. 3); lane 2 fraction 71
followingboth affinity and anion-exchangechromatography(5
ktg); lane 3 fraction 71 following affinity chromatography (5
k~g). *, Contaminant of 220 kDa.
Affinity chromatography using bovine thyroglobulin for purification of C. difficile toxin A has
been recently reported [8]. This method depends
on the temperature dependent binding between
toxin A and thyroglobulin. Changing the temperature from 4 ° C to 37 ° C releases toxin A from the
glycoprotein molecule [7,8]. Similarly, haemagglutination of rabbit erythrocytes by toxin A is
temperature dependent [7]. The binding is thought
to be due to interaction with glycoconjugates containing the non-reducing terminal sequence Gala
1-3 Gal/3 1 - 4 GlcNAc.
In the present study we analysed thyroglobulin
affinity chromatography-purified toxin A for its
purity. A280 and HA activity elution profiles similar to those reported by Krivan and Wilkins [8]
were demonstrated following thyroglobulin affinity chromatography, with the notable exception
that no significant increase of A280 was observed
after raising the temperature to 37°C. It was
clearly shown by anion-exchange chromatography
followed by cytotoxicity assay that the affinitypurified toxin A was contaminated with small
amounts of toxin B. It is well known that cytoxicity of toxin B is at least 103 times greater than
that of toxin A [4,6] and that subpico-gram
amounts of toxin B are cytotoxic [13]. Therefore, a
toxin B titre of 101 to 10 2 represents a minute
amount of the toxin, but such a dose may be
significant if the specimen were to be used for
immunization or for studies on the mechanism of
action of cytotoxicity, especially if concentrated
336
before use. In addition other contaminating proteins could be seen in native gradient polyacrylamide gels following silver staining, though not
Coomassie blue staining.
Krivan and Wilkins [8] obtained purified toxin
A by thyroglobulin affinity-chromatography after
washing with 20 bed volumes of TBS before thermal elution and screening for the presence of
toxin A in the effluents by H A activity only. In
the present study, we checked both H A activity
and cytotoxicity. The last fraction eluting at 4 ° C
still had a low titre of cytotoxicity, shown to be
due to toxin A and low levels of toxin B, even
after washing with 1150 ml of TBS (230 bed
volumes). It is unlikely that the contaminating
toxin B in the first fraction eluted at 37 ° C represents such remaining toxin, as the last fraction
eluted at 4 ° C had a lower amount of toxin B than
that present in the affinity-purified toxin A fraction. This finding implies that small amounts of
toxin B may bind to the immobilized thyroglobulin and be preferentially released at 3 7 ° C . As
reported by Krivan and Wilkins [8] HA activity
was only detected in the chromatography. In addition, by H A assay of the fractions yielded by
F P L C analysis of the affinity-purified toxin A
sample, it was shown that only toxin A fractions
gave H A positive reaction. This result is concordant with the results described previously [8], but
not with the report by Thelestam and Florin [14]
that both toxins have H A activity.
The affinity-purified toxin A specimen was
analysed for its purity by native gradient polyacrylamide gel electrophoresis and crossed immunoelectrophoresis in the report described by
Krivan and Wilkins [8]. We analysed the first
fraction eluted at 3 7 ° C by both gradient and
S D S - P A G E and found that only one band, representing toxin A, was detected, following Coomassie blue staining, even with a protein load of 15/zg
as used by Krivan and Wilkins [8]. However, using
the more sensitive method of silver staining at
least one other protein, with a relative molecular
wt of 220 kDa was detected. From the present
study the indications are that purification of C.
difficile toxin A by thyroglobulin affinity chromatography should be followed by other procedures.
ACKNOWLEDGEMENTS
This work was supported by a grant from the
Wellcome Research Fellowship 1988. We wish to
thank Dr. P.E.H. Byfield for advice on preparing
the thyroglobulin column and Mr. A.R. Welch for
provision of Vero cells.
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