A new purification procedure for Clostridium difficile enterotoxin

Vol. 124, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

November 14, 1984

A NEW PURIFICATION Rihn

B.,

PROCEDURE FOR CLOSTRIDIUM DIFFICILE Scheftel

J.M.,

Girardot

R.,

690-695

ENTEROTOXIN

and Monteil

H.

Institut

de Bact&iologie de la Faculte de MBdecine - Universite 3, rue Koeberl6 - 67000 STRASBOURG - FRANCE

Received

September

cytotoxin. filtration, composed

Clostridium difficile produces two toxins, an enterotoxin and a The enterotoxin was purified using fast methods (tangential flow The purified enterotoxin is fast protein liquid chromatography). of two subunits (A1 = 41,500, A2 = 16,000) and its p1 is 3.5. 0 1984

Academic

Press,

17,

Pasteur

1984

Inc.

C.difficile pseudomembranous pathogeny

Louis

is

the This

colitis.

of APC : an enterotoxin The cytotoxin

toxin

has the property

toxin

named enterotoxin

ileal

loop

assay

or toxin of rounding or

toxin

and experimental

we report

a rapid

method

MATERIALS

AND METHODS

for

agent

causative organism

produces

of

antibiotic-associated

two toxins

implicated

in the

and a cytotoxin. B has been purified

by several

different

cells

in culture

A induces

fluid

accumulation

ileocecitis

the purification

in hamster

groups (10,15). in

(5,13).

of C.difficile

toxin

:

this

Another the

In this

rabbit work

A.

Bacterial strain, culture conditions and toxin concentration : C.difficile (strain 79685 was isolated in our laboratory from stool of a patient with pseudomembranous colitis). This strain was grown in an anaerobic Chamber (CO2 containing brain10 %, H 5 %, N 85 %) for 60 hours in three 5 liter-flasks heart br&h (In&itut Pasteur Production) at 37OC under stirring. The culture was then filtered using a sangential flow system in a microporoys filter HVLP (0,5 p; filter area : 5 ft ; recirculation rate : 100 ml/min/ft ; pressure : 10 psi; Millipore). Sixteen liters of filtrate (corresponding to 111 g of proteins) were ultrafiltered with the same system using a PTGC filter with a nominal molecular weight limit of 10,000 (Millipore). During this step the retentate (1,050 ml; 15.5 g of proteins) was equilibrated in 10 mM Tris-HCl buffer pH 7.4. Enterotoxic methods :

and cytotoxic assays : Toxic activities fluid accumulation in the isolated rabbit

were ileal

determined by two loop according to

Abbreviations: APC, antibiotic-associated pseudomembranous colitis; tridium; FPLC fast protein liquid chromatography; IEF, isoelectric MW, molecular weight; PAGE, polyacrylamid gel electrophoresis. 0006-291X/84 Copyright All rights

$1.50

0 I984 by Acudemic Press, Inc. of reproduction in any form reserved.

C, Closfocusing;

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the Pierce and Wallace method (9), cytopathic rounding of Mac Coy Cells (15). The rabbit method for enterotoxin detection.

effect consisting ileal loop assay

of complete a specific

is

Chromatography methods : A fast protein liquid chromatography (Pharmacia) a GP-250 gradient apparatus, was used. It was composed of two P-500 pumps, a FRAC-100 fraction collector and a programmer, two UV-1 and UV-2 monitors, REG482 recorder. Anion exchange chromatography was performed on a Mono Q column (100 x 10 mm, Pharmacia). In each run, 150 mg of protein from the retentate fraction were applied. Fractions containing the enterotoxic activity obtained after Mono Q run, were desalted on Pharmacia PD-10 column and lyophilized. The proteins were equilibrated with the starting buffer (50 NaCl 150 mM) used for chromatofocusing which was achieved mM Tris-HCl pH 8.3, with a Mono P HR column (200 x 5mm, Pharmacia). After chromatofocusing, polybuffer was removed from proteins by gel filtration with the FPLC apparatus using a spherogel TSK SW 3000 column (300 x 7.5 mm, Beckman). The purity was checked by gradient - PAGE (acrylamide 10 to 20 %) in denaturating conditions according to the method of Maize1 (8) and by IEF using a pH gradient ranging from 3 tc 10 (4). The molecular weight of the native protein was determinated by the Andrews's method (1) using the spherogel TSK column SW-3000 with the FPLC apparatus. At each step, protein concentration was measured by the method of Lowry and ~011. (7). RESULTS tangential

The

allowed the

a rapid

separation

filtrate

was

equilibrated

with fractions

cell

and the

rabbit

loop

ileal

loop

NaCl.

The

cytotoxic

(with

elution

at 0.4

both

(fig.1).

The

The pH variation

After

salt

of

elimination

chromatographied

in

separated

from small

composed

of

two

a

subunits

eluted

and with

whose

SW-3000

MW was 691

step

and

of

the

Mac

Coy

by fast

in

+ 0.2

showed

in the

and 0.32

0.95

then

M

M NaCl.

separation

of

submitted

to

enterotoxic

A was due to the buffer permitted

the elimination

at pH 5.0

enterotoxic

column. (fig.3).

estimated

the

0.13

were

2 M NaCl the

lyophilization,

of contaminants

0.55

an improvement

This

and

response

between

of toxin

Then,

in one hour

a positive

between

the elution

TSK

to

was performed

at pH 5.8

pH 8.3

in 30 min.

according

peak

spherogel

quantity

separation

A

fractions

at and

good

eluted

and polybuffer.

protein

ultrafiltration

enterotoxic

during

by protein

by

showing

eluted

culture

19-fold

allowed

NaCl

bacterial

medium

A) were

were M

The sharp

amount

assay

the

culture

fractions

the toxin

fractions

of

7.4.

The fractions

Discontinuous

of large

pH

enterotoxic

rabbit

composed

buffer

the

chromatography.

system

than

from

anion

activity.

more

Tris-HCl

ileal

chromatofocusing.

from

mM

assay

.system

of bacteria

liquid

toxins

filtration

concentrated 10

cytotcxic test

flow

by

A

(fig.2).

fraction

was

single

peak

was

The purified

toxin

was

to

20 %

PAGE in

10

Vol. 124, No. 3, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20 1.2 E

'fi

i

1A

iii

1.2

W %

1.0 om om

a OA 0.2

Figure

I-

FRACTION

1 : Anion-exchange separation : column Mono Q HR 100x10 mm ; buffer A : 10 mM buffer B : buffer A with NaCl 1 M ; discontinuous Tris-HCl pH 7.4 ; gradient from 0 to 1 M N&l ; flow rate : 1.0 ml/min ; sample : 147 mg of retentate fraction protein ; detection at 280 nm (2 absorbance units fractions. fill scale).: enterotoxic fractions I: cytotoxic

FRACTION Figure

No

No

Nacl2Y

2 : Chromatofocusing profile : column Mono P HR 200x5 ; starting buffer : mM Tris-HCl pH 8.3, 150mMNaCl . eluent : polybuffer 74/polybuffer 98/H 0:7/3/140 (v/v/v) pH 5.0, NaCi 150 mM . flow rate : 1.0 ml/min anion-exchange step samp 1 e : 9 mg of enterotoxic fractions from the detection at 280 nm (1 absorbance unit full scale). : enterotoxil fractions

692

50 ;

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984

0.3

E c 0.2 0

Fd

a

----\

/

, &-+

6

10

14

16

Ve (ml)

Figure 3 : Chromatographic profile of enterotoxin on spherogel TSK SW 3000 column; buffer 150 mM NaCl, 10 mM Tris-HCl, pH 7.4 ; absorbance at 260 nm ; flow rate 1 ml/min ; sample 500 ,ug (500 ~1) of enterotoxin A obtained after chromatofocusing. Only the major peak was collected.

acrylamide

gel

IEP showed

a single

native

protein

containing

0.1

% SDS:

band with

estimated

with

Al

(41,500)

a p1 of 3.5

(fig.5).

the spherogel

TSK

and A2 (16,000) The molecular

SW-3300

(fig.4).

weight

of the

was 52,000.

column

DISCUSSION Bacterial toxin

A

of

technics

buffer

affinity and buffer

the

the

system

been

(13). and toxin

in

the

consisting

in

and anion

exchange

with

classical

scheme

chromatography used gel

step

preparative

693

anion and

procedure was proposed

electrophoresis

chromatography.

With

The

were sulfate

or precipitation

Lijnnroth

method

toxin

ammonium

filtration,

fractionation

purification

the

in

(2,3)

(11).

chromatography of

consisted

electrophoresis.

a four Another

a FPLC system

characteristics

and co11.(14)

preparative

using

purified

exchange

Taylor

A in

purified

purification

anion

chromatography. coll.(lZ)

seldom

discrepancies

double

chromatography purified

some

has

Generally,

precipitation, acetate

were

C.difficile

but

reported.

toxins

in the

in exchange

Lange

(6)

including

an

by Stephen discontinuous

FPLC system,

the

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS A

A

B

6

C 3.5, 4.4, 4.7,

+ ._Q

2:;:

-

20.1+ cl6

0 4

05

14.4+

10.e

Ib

Figure 4 : SDS-electrophoresis in gradient ranging from 10 to 20 % of acrylamide gels according to a modified Maizel's method (8) ; A : low molecular weight B (94,000), bovine serum albumin (67,000), marker proteins : phosphorylase ovalbumin (43,01X)), carbonic anhydrase (3O,OCO), soybean trypsin inhibitor alpha-lactalbumin (14,400) ; 8 : pool of enterotoxic fractions (20,100), after ion-exchange chromatography ; C : pool of enterotoxic fractions after gel filtration. Figure 5 : Isoelectric focusing ; A : marker proteins kit : amyloglycosidase (p1 ferritin (p1 4.4), albumin bovine (p1 4.7), 13 lactoglobulin (p1 3.5), 5.3), conalbumin (p1 5.9), myoglobin horse (p1 7.3), myoglobin whale (p1 enterotcxic fraction (the cytochrome c (p1 10.6). B : purified 8.3), arrow indicates the migration of enterotoxin). Running conditions : 1,000 volthours, Pharmalyte 3 - 10 according to the instruction manual of Pharmacia Fine Chemicals (4).

anion

exchange

faster

than

chromatography classical

chromatofocusing

anion

and gel

a good recovery

of toxic

never

the

used

achieved

for with

conditions,

filtration

fixation

was unsuccessful.

was necessary

to perform for

molecular

weight

different

authors

have

of

(2,12,13).

But

toxin

in these

with

charge

the

In

of the

the

toxin. (8) Only

two, protein

bands weight

way,

and led

to was

chromatofocusing However,

These

toxins.

694

same

chromatofocusing

occured

method

molecular

was lo-fold

FPLC apparatus

NaCl-free.

toxin.

Maizel's

on C.difficile

cases,

and

but

its

of 150 mM NaCl to the

of the

either

hour

respects,

A on the column Addition

showing

one

Usually,

buffer

A with

working

been performed,

elution

ionic

toxin

faster

In other

the elution

a adequate

in

purification.

and of the

out

chromatography.

were

activities.

a pH gradient

be required

exchange

enterotoxin

starting

carried

was

in elution

buffer seemed to

Determination was

not

such with

elution

conditions

is

of the

used

by the

non-denaturating

gels

(14)

band

or a single

of these

bands

was not

BIOCHEMICAL

Vol. 124, No. 3, 1984 reported,pI

of toxin

molecular the

weight

Andrews's

conditions,

of

A was not toxin

method

(1).

corresponding

chromatography) The most

were

interesting

reliability

determined

or not

A was estimated Toxin

techniques easy-to-use characteristics

to

A showed

to a dimeric

Purification

AND BIOPHYSICAL RESEARCH COMMUNICATIONS published

550,000 52,000

configuration

reported methods of

here for these

and

the

- 600,000 molecular (Al)1

methods

according weight

to

in

our

(A2)l.

(tangential isolation

apparent

filtration, of C.difficile were

their

FPLC toxin. rapidity,

and accuracy.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Andrews P. (1965) Biochem. J. 96, 595-606. Kobayashi T., Banno Y., Kono H., Watanabe K., Ueno K., and Nozawa Y. (1984) Rev. Infect. Dis. 6, Sll-S20. Banno Y., Kobayashi T., Watanabe K., Ueno K., and Nozawa Y. (1981) Biochemistry International 2, 629-635. Isoelectric focusing : principles and methods (1982) published by Pharmacia Fine Chemical AB, UPPSALA, Sweden. Libby J.M., Jortner B.S., and Wilkins T.D. (1982) Infect. Imun. 36, 822-829. Lijnnroth I., and Lange S. (1983) Acta Pathol. Microbial. Stand. Sect. B Microbial. Immunol. 91, 395-400. Lowry O.H., Rosebrough N.J., Farr A.L., and Randall R.J. (1951) J. Biol. Chem. 193, 265-275. Maize1 Jr. J.V. (1970) Nature 227, 680-686. Pierce N.F., and Wallace C.K. (1972) Gastroenterology 63, 439-448. Rolfe R.D., and Finegold S.M. (1979) Infect. Inmun. 25, 191-201. Rijnnberg B., Wadstrijm T. (in press) Prep. Biochem. Stephen J., Redmond S.C., Mitchell T.J., Ketley J., Candy D.C.A., Burdon D.W., and Daniel R. (1984) Biochem. Sot. Trans. 12, 194-195. Sullivan N.M., Pellett S., and Wilkins T.D. (1982) Infect. Imnun. 35, 1032-1046. Taylor N.S., Thorne G.M., and Bartlett J.G. (1981) Infect. Imnun. 34, 1036-1043. Tytgat F. (1980) Ann. Microbial. (Paris) 131 B, 11-20.

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