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Production, purification and characterization of Clostridium difficile toxic proteins different from toxin A and from toxin B
Biochimica et Biophysica Acta, 998 (1989) 151-157
151
Elsevier BBAPRO 33448
Production, purification and characterization of Clostridium d6fficile toxic proteins different from toxin A and from toxin B Javier F. Torres 1,2 and Ivar LiSnnroth 1 t Department of Medz'cal Microbiology, Gothenburgh University, Giiteborg(Sweden) and 2 Unidad de lnvex'tigaci6n en Enfermedades Infecciosas y Parasitarias, Subdireccion General Medica, Instituto Mexicano dei Seguro Social, M$xico, D.F. (M$xico)
(Received29 December1988) (Revisedmanuscriptreceived21 April 1989)
Key words: Enterotoxin;Cytotoxin; Intestinalsecretion; (C. difficile) The purification and characterization of three new proteins called CI, C2 and C3 from CIostridium difficile are described. Their estimated molecular mass were about 350 (C1), 270 (C2) and 140 (C_.3) kDa, consisting of subunits of 39 (Cl), 43 (C2) and 41 (C3) kDa, respectively. Immnnediffnsion revealed that the three proteins contained similar but not identical antigenic determinants to toxin A. Each protein induced a cytotonic effect on hamster ovaric cells; the combined proteins, had a specific activity on cells 5-times higher than that of toxin A. In rat intestinal loops, they induced a dear fluid secretion, while toxin A elicited a haemorrhagic fluid response. The cytotonic ac~vities of ~! three proteins were abolished by antiserum against toxin A, while antisennn against toxin B inhibited only the activity of the 270 kDa protein. In contrast to toxin A, the cytotoxieity of the three proteins was inactivated by trypsin. Thus, the chemical, antigenic and biological properties of these proteins differed from those of toxin A and toxin B.
Introduction Clostridium dtffi'cile has been associated with cases of antibiotic-associated diarrhea, colitis and pseudomembranous colitis in humans [1,2]. The offending antibiotic disrupts the normal intestinal flora and suppresses organisms which might compete against C. difficile [3,4]. Studies on animal models suggest that exotoxins cause most of the symptoms during the disease [5,6]. These toxins are active on most cells in culture, and therefore various cell tests have been employed as diagnostic tools [7,8]. Two toxins, known as A and B. have been described, each of them being lethal to mice in nanogram amounts [9]. Toxin A is an enterotoxin, inducing a haemorrhogic fluid accumulation in intestinal loops of rats [10], rabbits and hamsters [11,12]. Toxin A causes elongation of Chinese hamster ovary cells (CHO) [13], an effect similax to that of the enterotoxins from V. cholerae, E. coil and C. jejuni. T h e toxin seems normally to consist of two noncovalently bound peptides, each with a moleculax mass of about 200-300 kDa [11,14]; but it might also form aggregates of larger molecules depending on
Correspondence: I. LSnnroth, Departmentof Medical Microbiology, Guldhedsgatan 10, 413 46 G~teborg, Sweden.
the conditions during the growth of the bacteria [10]. Toxin B is a cytotoxin causing damage to cells in culture in amounts of less than one picogram [9,11]. The cytotoxin exists in two forms, one consisting of two subunits of about 250 kDa, or one consisting of several subunits each about 43 kDa [15]. Recent evidence suggests that toxin A and toxin B have at least one epitope in common [16]. This paper describes the purification to homogeneity of three toxic proteins which are separated from toxin A by high resolution ion-exchange chromatography. The chemical, antigenic and biological properties of these proteins, called fraction C, are compared with those of toxin A and toxin B.
Material and Methods
Strain and culture conditions Clostridium difficile CCUG 19126 (VPI 10463, ob-
tained from DM Lyerly/TD Wilkins, VPI Blacksburg, U.S.A.) was used for production of toxins. The organism was grown at 37 °C for 72 to 170 h in 2 litre brain heart infusion dialysis flasks, as described elsewhere [17]. After centrifugation at 6000 rpm for 20 rain, the toxincontaining supernatant was sterilised by filtration through a 0.45 ltm (pore size) membrane.
0167-4838/89/$03.50 © 1989 ElsevierSciencePubfishersB.V.(BiomedicalDivision)
152
Purification of toxins The crude preparation was concentrated on a XM100A ultrafiltration membrane (Amicon, U.S.A.) and washed three times with 0.1 M "Iris (pH 8.0) containing 0.1 M NaC1. The final concentrate was applied onto a column of DEAE-Sephadex A-25 (2.6 × 14 cm) and eluted by a step-wise gradient of NaCI from 0.25 to 0.4 M as described by Banno et al. [11]. The fractions obtained were assayed for protein at 280 nm and for cytotonic activity in tissue culture cells. The active fractions eluting at 0.1 M NaCl were pooled and dialysed against 0.01 M acetate buffer (pH 5.5) for an acid precipitation, as described by Sullivan et al. [9]. After centrifugation at 5000 rpm for 15 rain, the precipitate was washed in acetate buffer, and dissolved in 20 mM triethanolamine (pH 7.5) (buffer 1). 4 mg of this solution were applied to a Mono Q HR 5/5 column coupled to equipment for Fast Protein Liquid Chromatography (FPLC, Pharmacia, Sweden). Separation was achieved at 0.75 m l / m i n by a combination of discontinuous and continuous NaCI gradient in buffer 1 (see Fig. 1). The column was first washed with 3 ml of 0.15 M NaCI, and then a linear NaC1 gradient from 0.15 to 0.30 M NaC! during 20 ml was applied. The concentration of NaCI was then raised in one step ~o 1.0 M. Fractions (1 ml) were collected and screened for cytotonic activity. The fractions corresponding to pe~k C, referred from now on as fraction C (Fig. 1) were equilibrated in buffer 1 and reapplied to the Mono Q column. In this second FPLC separation, the column was first flushed with 3 ml 0.27 M NaC1, thereafter a linear gradient from 0.27 to 0.47 M NaCI during 20 ml was applied (see Fig. 2).
buffered saline, containing 0.15 M NaCI, 0.1~ sodium thiosulphate and 0.01 M sodium phosphate (pH 7.2). Non-denaturating PAGE was run in a 4 to 30~ gradient in 0.09 M "Iris, 0.08 M boric acid, 0.0025 M Na-EDTA (pH 8.4). The proteins were dissolved in the same buffer supplied with 1/10 of its volume with glycerol. The migration of the toxic proteins were compared with the migrations of molecular mass marker proteins (Pharmacia). Denaturing PAGE was done in a 2 to 16~ gradient gel in 0.04 M "Iris, 0.02 M sodium acetate, 0.002 M EDTA, 0.2~ SDS (pH 7.4). Before the application, the samples were incubated at 100 °C for 10 min in 0.01 M Tris-HCl, 0.001 M EDTA, 1~ SDS (pH 8.0) with or without 5~ /~-mercaptoethanol. The gels were stained with Coomassie blue R-250 or with a silver reagent kit according to the manufacturer (Bio-Rad, U.S.A.).
Preparation of antitoxins New Zealand rabbits were used to prepare antisera against toxin A and against fraction C. Animals were initially inoculated with 20/~g of each protein in complete Freund's adjuvant by multiple intradermal dorsal injection. Ten days later, they were inoculated with 20 /~g of protein in incomplete Freund's adjuvant by multiple intradermal dorsal injection. This process was repeated every 10 days until a significant neutralising level was reached (as measured in CHO cells). Eight days after the last injection, animals were bled to death. The blood was incubated overnight at 4°C; and the sera separated by centrifugation and freeze-dried. Antiserum against to~Jn B was obtained as previously described [15]. C. sordellii antitoxin was obtained from the Bureau of Biologics (Rockville, MD, U.S.A.).
Toxin B preparation Purified toxin B was prepared for comparative analysis; it was produced and purified as previously described [16].
Assay of toxins in cells
Neutralisation of toxins The toxins were diluted 2-fold in tissue culture medium and each dilution was mixed 1 : 1 with antisera against either toxin A, toxin B or fraction C. The mixtures were incubated for 1 h at room tempera~re and then inoculated in CHO cells. The cells were read for elongation as described above.
A modification of the Chinese hamster ovary cell (CHO) test by Katoh et al. [13] was used. 200/~1 of a 3.10 3 ceils/m[ suspension were distributed in a microtiter plate; after 45 min, 20 t~l of the samples Were tested in 2- or 5-fold dilutions. After incubation overnight, the cells were fixed with 80% methanol and stained with Giemsa. The cytotonic activity was expressed as elongation units (EU), i.e., the inverse of the maximal dilution which induces elongation of at least 25~ of the cells.
Double diffusion-in-gel was done in 5 × 5 cm plates covered with agar, as previously described [19]. 20 #1 of antigen or antiserum were applied per hole, and diffusion was allowed to proceed in a humidified chamber for 48 h. Diffused plates were washed with phosphatebuffered saline and stained with Coomassie blue R-250.
PolyacJylamide gel electrophoresis
Digestion of toxins with t~psin
Samples were analysed in precast gradient gel electrophoresis in accordance to the manufacturer's instructions (Pharmacia, Sweden). Some gels were sliced into sections, and active toxin eluted by dialyses against
20/~g of each toxin (A o~ C) was digested for 7 h with 500/~g of trypsin (type XI, DPCC treated from Sigma, MO, U.S.A.) dissolved in 0.5 m[ 0.2 M sodium phosphate buffer (pH 7.0).
Immunodiffusion
153
Intestinal loop test Sprague-Dawley rats, 2-3 months old, were used to measure the effect of toxin A and fraction C on the small intestine [20]. One loop of about 10 cm was made in the middle of the small bowel and the sample, diluted in buffer 1, was injected in a volume of 1.0 ml. After 6 h, fluid accumulation was estimated from the weight/ length ratio (mg/cm) of the test loop and compared with that of control loops injected with buffer 1 alone. Each sample was assayed in triplicate.
1.°1
1
-->" 0.3
~ Z
0.2
0 l ' ..3" 0.6 a
Results
600
After 5 days of growth of C difficile, the crude cell-free culture contained a cytotonic activity of 14 elongation units per /~g of protein (EU//~g). Purification by ion-exchange chromatography and acid precipitation increased the specific activity about 30-times with a yield of 37~ (Table I). During further parification on high resolution ion-exchange gel, the cytotonic material was split into two peaks (Fig. 1). One peak corresponding to toxin A, eluted at 0.19 M NaC1; the other eluted at about 0.3 M NaCl, having a specific activity which was 5-times higher than that of toxin A (Table I). This second peak, called fraction C, was further separated into four peaks by recycling on the mono Q column and eluting with a gradient of NaC1 (Fig. 2). Peak I to 3 (CI, C2 and C3) exerted activity on CHO cells (Table I), while the fourth peak contained no activity. The purified toxins A and fraction C were analysed on gradient PAGE. When run under non-denaturing conditions (Fig. 3), toxin A gave a f-7~7 band migrating as a protein of molecular weight 400-600 kDa (row 2); fraction C separated into three proteins. Their apparent molecular masses were: 350 (C1), 270 (C2) and 140 kDa
TABLE I
Purification of proteins from C difficile causing elongation of CHO cells Purification step a
Cell free culture D E A E Sephadex A-25
Acid precipitation Mono Q, FPLC: toxin A fraction C Fraction C on Mono Q: C1 C2 C3
Protein (mg)
Activity on cells total EU
EU/Fg
7.6-10 s 4.1- l 0 s
14 b 100
6.6
2.8-106
420
5.0 1.3
7.5.105 9.5.105
150 760
0.14 0.16 0.72
8.0.10 2 1.6.103 1.0-104
6 10 14
540 41
a The production and purification procedure was repeated twice with similar results. b Elongation activity in crude culture-filtrate was determined by neutralizing the activity of toxin B with toxin B antiserum.
E ¢= 0
=L
0.4=
Cq
I
400 e-. o
,Q
~ 0.2,
e-.
200
,ID
<
o U.I
O,
4O 6O number Fig. 1. Fast prote~ liquid c~omatography of toxic proteins after acid precipitation. A total of 4 mg of protein was applied to a Mono Q column. Upper panel, NaCI gradient used for elution. Lower panel, elution profile: denotes absorbance ~.t 280 rim, and - - cytotonic activity on CHO cells. The first peak of cytotonic activity corresponds to toxin A and the second to fraction C. 0
20
Fraction
(C3) as compazed to reference proteins~ The toxic activity recovered from these regions, when they were eluted from slices of the unstained gel, was weak (100 to 1000 E U / m l ) but present only in the three zones corresponding to C1, C2 and C3. By recycling on Mono Q (Fig. 2), the C1 and C2 proteins were purified to homogeneity as shown by PAGE in Fig. 3 (rows 4 and 5). The subunit composition of the fraction C proteins was analysed in SDS-PAGE (Fig. 4a). In this gel, toxin A migrated as one band of molecular mass around 260 kDa (row 2); fraction C migrated in three bands of molecular mass 43, 41 and 39 kDa (row 3). In order to determine their subunit composition, C1, C2 and C3 were first separated by running fraction C under nondenaturing conditions, each protein band electroeluted and then run again on SDS-PAGE. As shown in Fig. 4b, the 39 kDa s;ubunit (row 1) originated from C1 (350 kDa), the 41 kDa subunit (row 3) from C3 (140 kDa), and the 43 kDa subunit (row 2) from C2 (270 kDa). The toxins were also run in SDS-PAGE under non-reducing conditions. However, toxin A and fraction C proteins migrated in a pattern similar to that obtained in the presence of fl-mercaptoethanol (not shown). These re-
154
,.o!
_1"
0.5
[ ~0.3 Z
0.1'
.I'
[3)
E ,- .10,
,20
0 CO
e=)
e-
,10
m .05'
cO
.Q
e0 UJ
< 0o
F r a c t i o n number Fig. 2. Separation of the three proteins of fraction C by fast protein liquid chromatography. 200 Fg of fraction C separated from toxin A (Fig. 1) were applied to a Mono Q column. Upper panel, NaCI gradient used for elution; lower panel, elution profile: ~ denotes absorbance at 280 nm, and - - - cytotonic activity on CHO cells. The first peak of cytotonic activity corresponds to C3, the second to C2 and the third to C1.
Activity, 1
232
2
3
~
140 -=,-,
4-
EU 10
20
4
5
4.-
~ll
"-
i TABLE II
qlm
67 .,m
~-
s.ults suggest that their peptide chains are non-covalently linked (i.e., not kept together by S-S bridges). Cultures of different ages were assayed for their content of fraction C and toxin A (Table II). Fraction C was detected after three days of growth, became maximal on day 5 (1250 Fg) and decreased after 7 days. Toxin A reached a high level after 3 days was increased further on day 5 (5000 Fg) and decreased again at day 7. Antitoxins against fraction C and toxin A and B were evaluated for their ability to neutralise the action of fraction C on CHO cells (Table III). All the elongation, activity of fraction C was neutralised by serum against either toxin A or fraction C, while toxin B antiserum had a partial neturalisation effect. When the separated C1, C2 and C3 proteins were tested, each of them was completely neutralised by the antiserum against toxin A. In contrast, toxin B antiserum had a pronounced effect on C2 only (Table III). The activity of toxin A was neutralised by antiserum against toxin C but not by antiserum against toxin B. Double-immunodiffusion analysis of fraction C was performed with antisera against fraction C, toxin A, toxin B and C. sordelii culture filtrate (Fig. 5). Fraction C antisera (Fig. 5a and b) produced one precipitin line with toxin A and three with fraction C, two of which spured in partial identity with toxin A. With toxin A antisera (Fig. 5a), fraction C precipitated in one line, showing complete identity with toxin A and partial identity with one of the precipitates formed between fraction C and fraction C antisera. Toxin B was not precipitated by fraction C antisera (Fig. 5b). Toxin B antiserum (Fig. 5c) precipitated with toxin B, but it did not precipitate fraction C. Antiserum against C. sordellii recognised toxin A, toxin B as well as fraction C (Fig. 5d); the precipitates formed with toxin A and fraction C showed partial identity, while the lines formed with toxin B and fraction C showed no identity. The effect of treatment with trypsin on toxin A and fraction C was tested. Digestion of 20 Fg of fraction C with 5 0 F g of trypsin completely destroyed the activity of fraction C on CHO cells. In contrast, toxin A was
7"~
Production of fraction C and toxin A by C. difficile Day
Fig. 3. Polyacrylamide gel electrophoresis of cytotoaic proteins under non-denaturating conditions (4 to 30~ gel); row I, molecular mass markers, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (67 kDa); row 2, toxin A after FPLC; row 3, fraction C after first FPLC (Fig. 1) and to the right the toxic activity of the same material (diluted 1/10) eluted from sliced pieces (arrows); row 4 and 5, C2 and C1, respectively, after second FPLC (Fig. 2). The gel was stained with Coomassie blue R-250.
2 3 5 7
Fraction C
Toxin A
O=g)
O,g)
0a 200 1 250 500
n.d. b 2100 ~ 5 000 3 800
a Yield of the protein after purification from two litre of culture filtrate as described in Table I. b n.d., not determined.
155 1
2
3
1
2
3
TABLE III Decrease of the activity of C. difficile toxins after treatment with antiserum or trypsin
U
220
Toxin
Toxin A Fraction C C proteins b: C1
67 I=, 60 q~
36
No treatment a
Effect on CHO cells (EU/ml) toxin A antiserum
6.2.10 4 3.1.105
0 0
2.0-10 2
0
C2
2.0- l0 2
0
C3
8.0-102
0
toxin B antiserum
fraction C antiserum
6.2-104 1.2-104
0 0
1.0.10 2
0
50
8.0-102
0
0
~
Fig. 4. Polyacrylamide gel electrophoresis of cytotonic proteins in the presence of SDS (2 Tto 16~ gel). (a) Row 1: molecular mass markers, ferritin (subunit 220 kDa), bovine serum albumin (67 kDa), catalase (subunit 60 kDa) anti lactate dehydrogenase (subunit 36 kDa); row 2, toxin A after FPLC; row 3, fraction C after first FPLC (Fig. 1). (b) SDS-electrophoresis of fraction C proteins previously run in non-denaturating conditions and electroeluted; row 1, protein C1; row 2 protein C2; row 3, protein C3. (a) was stained with Coomassie blue R-250 and (b) was silver-stained.
resistant to the proteolytic activity of even 500 pg of the enzyme. The enterotoxic effects of toxin A and fraction C were evaluated in small intestinal loops of rats. 2 pg of
a-C
A
a Values are expressed as EU/ml, e.g., the inverse of the maximal dilution which caused elongation of CHO cells. b Proteins are single peptides from fraction C separated in Mono Q column.
toxin A elicited a strong haemorrhagic fluid response (322 + 27 mg/cm). Fraction C at the same concentration elicited no response, whereas 15 /~g induced a pronounced fluid accumulation (320 + 40 mg/cm) without bleeding or macroscopical evidence of tissue damage. Discussion T h r e e toxic p r o t e i n s f r o m C. difficile w h i c h a r e diff e r e n t f r o m t h e a l r e a d y k n o w n toxins A a n d B a r e
a-C
a-A
C
A
A
C
a-sord.
a-B
C
B
B
A
C
B
Fig. 5. Comparison of fraction C, toxin A and toxin B in double immunodiffusion. The proteins were analysed after FPLC purification. (a) A, 8/tg of toxin A; C, 8/tg of fraction C; a-C, 20/~1 of fraction C antiserum; a-A, 20/tl of toxin A antiserum. (b) A, 8/tg of toxin A; C, 8/tg of fraction C; B, 20/tg of toxin B; a-C, 20/tl of fraction C antiserum. (e) C, 20/tg of fraction C; B, 8/tg of toxin B; a-B, 20 lgl of toxin B antiserum. (d) A, 25/~g of toxin A; C, 20/~g of fraction C; B, 20/tg of toxin B; a-sord., 20 ttl of C. sordelli antiserum.
156 described here; they were designated C1, C2 and C3 or taken together fraction C. All three were cytotoxic and dissociated into subunits of similar size (around 40 kDa). None of them seemed to contain any interchain S-S bridge. When fraction C was separated on PAGE, the various activities and protein bands migrated to the same positions as those of the C proteins, which previously had been purified on the mono Q column. These results suggest that the effect of fraction C is due to the combined action of the three C proteins. However, since the separations of fraction C on polyacrylamide and mono Q led to significant losses of activity, there may be a still unidentified component which stabilise the fraction C complex in its active form. The intestinal effect of the fraction C complex resembled that of cholera toxin or E. coil LT [21] rather than that of toxin A [11,12]. Thus, a clear fluid was secreted without the haemorrhagic response seen after exposure to toxin A. The specific enterotoxic activity of fraction C was 5- to 10-fold less than that of toxin A. This difference might be due to the different sensitivity to trypsin, which enzyme inactivated fraction C but not toxin A. In previous studies toxin A has been reported to contain subunits of a size similar to that of the fraction C proteins [22,23]. These subunits might in fact belong to fraction C proteins, which copurifies with toxin A during ion-exchange chromatography and precipitation with acid. There have also been reports of partially purified toxins of smaller size which induce a nonhaemorrhagic fluid response in the small intestine [11,24,25]. These toxic activities might be caused by fraction C; however, since the preparations were not pure and immunological data were lacking, no meaningful comparison can be made. Although toxin A and B have epitopes in common [16], no significant neutralisation is achieved with the heterologous antiserum. In contrast, fraction C was totally neutralised by toxin A antiserum and partially neutralised by toxin B antiserum. The immunodiffusion experiments confirmed the cross-reactivity with toxin A but not that with toxin B. The lack of precipitation of fraction C with toxin B antiserum might be due to the insensitivity of the method with respect to the quantity and affinity of the antigen-antibody complex formed. Of the separated proteins, C2 was neutralised by A antise~m as well as B antiserum, suggesting that it had epitopes in common with both the toxins. The precipitation of toxin A, toxin B as well as fraction C with C. sordellii antiserum confirm that this bacterium produces similar toxins [26,27]. Among them, the so-cal, M lethal toxin has been purified and shown to have subunits of similar size to those of fraction C [28]. It cross-reacts with C difficile toxin B and exerts a moderate response in the intestinal loop test. Further, a low molecular
mass form of toxin B was recently described which has subunits of similar size to those of toxin C [15,29]. Large peptide chains are mostly made up of repetitive sequences, and this concept seems to be true also for toxin A [14,30]. Toxin A is probably endocytosed and hydrolysed lysozomally before its action is expressed [31] and the C proteins might represent the active fragments of toxin A. A variety of bacterial toxins are processed by cell wall endopeptidases, e.g., tetanus, botulinum, diphtheria and cholera toxin [32-35]. These so called 'nicked' molecules are supposed to represent the active form of the toxins. Thus, some toxin A molecules might be processed into C proteins already during the transport across the bacterial cell wall.
Acknowledgements Financial support was provided by the Swedish Medical Council (grant 7481) and the Medical Faculty of the Gothenburgh University. References 1 Barlett, J.G. (1979) Rev. Infect. Dis. 1, 530-539. 2 Gerding, D.N., Olson, M.M, Peterson, L.R., Teasley, D.G., Gebhard, R.L., Schwarz, M.L. and Lee, J.T. (1986) Arch. Intern. Med. 146, 95-100. 3 Lusk, R.H., Fekety, R., Silva, J., Browne, R.A., Rigler, D.H. and Abrams, G.D. (1978) J. Infect. Dis. 137, 464-475. 4 Wilson, K.H., Silva, J. and Fekety, F.R. (1981) Infect. Immun. 34, 626-628. 5 Bartlett, J.G., Onderdonk, A.B., Cisneros, R.L. and Kasper, D.L. (1977) J. Infect. Dis. 136, 701-705. 6 Rifkin, G.D., Silva, J. Jr., Fekety, F.R. and Sack, R.B. (1977) Lancet ii, 1103-1106. 7 Chang, T.W., Lauermann, M. and Bartlett, J.G. (1979) J. Infect. Dis. 140, 765-770. 8 Donta, S.T., Sullivan, N. and Wilkins, T.D. (1982) J. Clin. Microbiol. 15, 1157-1158. 9 Sullivan, N.M., Pellet, S. and Wilkins, T.D. (1982) Infect. Immun. 35, 1032-1040. 10 Tones, J.F. and l.~nnroth, I. (1988) FEMS Microbiol. Lett. 52~ 41-46. 11 Banno, Y., Kobayashi, T., Kono, H., Watanabe, K., Ueno, IL and Nozawa, Y. (1984) Rev. Infect. Dis. 6, Sll-$20. 12 Lyedy, D.M., Lockwood, D.E., Richardson, S.H. and Wilkins, T.D. (1982) Infect. Immun. 35, 1147-1150. 13 Katoh, T., Higaki, M., Honda, T. and Miwatani, T. (1986) FEMS Microbiol. Lett. 34, 241-244. 14 Lyerly, D.M., Krivan, H.C. and Wilkins, T.D. (1988) Clin. Microbiol. Rev. 1, 1-18. 15 Tortes, J.F. and Lt~nnroth, I. (1988) FEBS Lett. 233, 417-420. 16 Lyerly, D.M., Phelps, C.J., Toth, J. and Wilkins, T.D. (1986) Infect. Immun. 54, 70-76. 17 Tayler, N.S., Thorne, G.M. and Bartlett, J.G. (1981) Infect. Immun. 34, 1036-1043. 18 Vesterberg,O. (1971) Methods Enzymol. 22, 387-412. 19 Wadsworth, C. (1957) Int. Arch. Allergy Appl. Immunol. 10, 355-360. 20 Lange, S. (1982) FEMS Microbiol. Lett. 15, 239-242.
157 21 Richard, ICL. and Douglas, S.D. (1978) Microbiol. Rev. 42, 592-613. 22 Rilm, B., Scheftel, J.M., Girardot, 1L and Moteil, H. (1984) Biochem. Biophys. Res. Commun. 124, 690-695. 23 Rautenberg, P. and Stender, F. (1986) FEMS Microbiol. Lett. 37, 1-7. 24 Giuliano, M., Piemonte, F. and Gianffilli, P.M. ,1,~o,,, ~.oo~ FEMS Miorobiol. Lett. 50, 191-194. 25 l.iSnnroth, I. and Lange, S. (1983) Aeta Pathol. Microbiol. Immunol. Stand. 91, 395-400. 26 Martinez, R.D. and Wilkin.q, T.D. (1988) Infect. Immun. 56, 1215-1221. 27 Nakamura, S., Tanabe, N., Yamakawa, K. and Nishida, S. (1983) Microbiol. Immunol. 27, 495-502.
28 Poppoff, M.R. (1987) Infect. hranun. 55, 35-43. 29 Pothoulakis, C., Barone, L.M., Ely, R., Faris, B., Clark, M.E., Franzblau, C. and LaMont, J.T. (1986) J. Biol. Chem. 261, 1316-1321. 30 Price, S.B., Phelps, CJ., Wilkins, T.Do and Johnson, J.L. (1987) Curr. Microbio!_ 16, 55-60. 31 Henriques, B., Florin, I. and Thelestam, M. (1987) Microbial Pathogenesis 2, 455-463. 32 Bizzini, B. (1979) Microbial Rev. 43, 224-240. 33 Das Gupta, B.R. and Sugiyama, H. (1972) Infect. Immun. 6, 587-590. 34 Collier, RJ. and Kaplan, D.A. (1984) Sci. Am. 251, 44-52. 35 Betley, M.J., Miller, V.L. and Mekaianos, J.J. (1986) Ann. Rev. Microbiol. 40, 577-605.
151
Elsevier BBAPRO 33448
Production, purification and characterization of Clostridium d6fficile toxic proteins different from toxin A and from toxin B Javier F. Torres 1,2 and Ivar LiSnnroth 1 t Department of Medz'cal Microbiology, Gothenburgh University, Giiteborg(Sweden) and 2 Unidad de lnvex'tigaci6n en Enfermedades Infecciosas y Parasitarias, Subdireccion General Medica, Instituto Mexicano dei Seguro Social, M$xico, D.F. (M$xico)
(Received29 December1988) (Revisedmanuscriptreceived21 April 1989)
Key words: Enterotoxin;Cytotoxin; Intestinalsecretion; (C. difficile) The purification and characterization of three new proteins called CI, C2 and C3 from CIostridium difficile are described. Their estimated molecular mass were about 350 (C1), 270 (C2) and 140 (C_.3) kDa, consisting of subunits of 39 (Cl), 43 (C2) and 41 (C3) kDa, respectively. Immnnediffnsion revealed that the three proteins contained similar but not identical antigenic determinants to toxin A. Each protein induced a cytotonic effect on hamster ovaric cells; the combined proteins, had a specific activity on cells 5-times higher than that of toxin A. In rat intestinal loops, they induced a dear fluid secretion, while toxin A elicited a haemorrhagic fluid response. The cytotonic ac~vities of ~! three proteins were abolished by antiserum against toxin A, while antisennn against toxin B inhibited only the activity of the 270 kDa protein. In contrast to toxin A, the cytotoxieity of the three proteins was inactivated by trypsin. Thus, the chemical, antigenic and biological properties of these proteins differed from those of toxin A and toxin B.
Introduction Clostridium dtffi'cile has been associated with cases of antibiotic-associated diarrhea, colitis and pseudomembranous colitis in humans [1,2]. The offending antibiotic disrupts the normal intestinal flora and suppresses organisms which might compete against C. difficile [3,4]. Studies on animal models suggest that exotoxins cause most of the symptoms during the disease [5,6]. These toxins are active on most cells in culture, and therefore various cell tests have been employed as diagnostic tools [7,8]. Two toxins, known as A and B. have been described, each of them being lethal to mice in nanogram amounts [9]. Toxin A is an enterotoxin, inducing a haemorrhogic fluid accumulation in intestinal loops of rats [10], rabbits and hamsters [11,12]. Toxin A causes elongation of Chinese hamster ovary cells (CHO) [13], an effect similax to that of the enterotoxins from V. cholerae, E. coil and C. jejuni. T h e toxin seems normally to consist of two noncovalently bound peptides, each with a moleculax mass of about 200-300 kDa [11,14]; but it might also form aggregates of larger molecules depending on
Correspondence: I. LSnnroth, Departmentof Medical Microbiology, Guldhedsgatan 10, 413 46 G~teborg, Sweden.
the conditions during the growth of the bacteria [10]. Toxin B is a cytotoxin causing damage to cells in culture in amounts of less than one picogram [9,11]. The cytotoxin exists in two forms, one consisting of two subunits of about 250 kDa, or one consisting of several subunits each about 43 kDa [15]. Recent evidence suggests that toxin A and toxin B have at least one epitope in common [16]. This paper describes the purification to homogeneity of three toxic proteins which are separated from toxin A by high resolution ion-exchange chromatography. The chemical, antigenic and biological properties of these proteins, called fraction C, are compared with those of toxin A and toxin B.
Material and Methods
Strain and culture conditions Clostridium difficile CCUG 19126 (VPI 10463, ob-
tained from DM Lyerly/TD Wilkins, VPI Blacksburg, U.S.A.) was used for production of toxins. The organism was grown at 37 °C for 72 to 170 h in 2 litre brain heart infusion dialysis flasks, as described elsewhere [17]. After centrifugation at 6000 rpm for 20 rain, the toxincontaining supernatant was sterilised by filtration through a 0.45 ltm (pore size) membrane.
0167-4838/89/$03.50 © 1989 ElsevierSciencePubfishersB.V.(BiomedicalDivision)
152
Purification of toxins The crude preparation was concentrated on a XM100A ultrafiltration membrane (Amicon, U.S.A.) and washed three times with 0.1 M "Iris (pH 8.0) containing 0.1 M NaC1. The final concentrate was applied onto a column of DEAE-Sephadex A-25 (2.6 × 14 cm) and eluted by a step-wise gradient of NaCI from 0.25 to 0.4 M as described by Banno et al. [11]. The fractions obtained were assayed for protein at 280 nm and for cytotonic activity in tissue culture cells. The active fractions eluting at 0.1 M NaCl were pooled and dialysed against 0.01 M acetate buffer (pH 5.5) for an acid precipitation, as described by Sullivan et al. [9]. After centrifugation at 5000 rpm for 15 rain, the precipitate was washed in acetate buffer, and dissolved in 20 mM triethanolamine (pH 7.5) (buffer 1). 4 mg of this solution were applied to a Mono Q HR 5/5 column coupled to equipment for Fast Protein Liquid Chromatography (FPLC, Pharmacia, Sweden). Separation was achieved at 0.75 m l / m i n by a combination of discontinuous and continuous NaCI gradient in buffer 1 (see Fig. 1). The column was first washed with 3 ml of 0.15 M NaCI, and then a linear NaC1 gradient from 0.15 to 0.30 M NaC! during 20 ml was applied. The concentration of NaCI was then raised in one step ~o 1.0 M. Fractions (1 ml) were collected and screened for cytotonic activity. The fractions corresponding to pe~k C, referred from now on as fraction C (Fig. 1) were equilibrated in buffer 1 and reapplied to the Mono Q column. In this second FPLC separation, the column was first flushed with 3 ml 0.27 M NaC1, thereafter a linear gradient from 0.27 to 0.47 M NaCI during 20 ml was applied (see Fig. 2).
buffered saline, containing 0.15 M NaCI, 0.1~ sodium thiosulphate and 0.01 M sodium phosphate (pH 7.2). Non-denaturating PAGE was run in a 4 to 30~ gradient in 0.09 M "Iris, 0.08 M boric acid, 0.0025 M Na-EDTA (pH 8.4). The proteins were dissolved in the same buffer supplied with 1/10 of its volume with glycerol. The migration of the toxic proteins were compared with the migrations of molecular mass marker proteins (Pharmacia). Denaturing PAGE was done in a 2 to 16~ gradient gel in 0.04 M "Iris, 0.02 M sodium acetate, 0.002 M EDTA, 0.2~ SDS (pH 7.4). Before the application, the samples were incubated at 100 °C for 10 min in 0.01 M Tris-HCl, 0.001 M EDTA, 1~ SDS (pH 8.0) with or without 5~ /~-mercaptoethanol. The gels were stained with Coomassie blue R-250 or with a silver reagent kit according to the manufacturer (Bio-Rad, U.S.A.).
Preparation of antitoxins New Zealand rabbits were used to prepare antisera against toxin A and against fraction C. Animals were initially inoculated with 20/~g of each protein in complete Freund's adjuvant by multiple intradermal dorsal injection. Ten days later, they were inoculated with 20 /~g of protein in incomplete Freund's adjuvant by multiple intradermal dorsal injection. This process was repeated every 10 days until a significant neutralising level was reached (as measured in CHO cells). Eight days after the last injection, animals were bled to death. The blood was incubated overnight at 4°C; and the sera separated by centrifugation and freeze-dried. Antiserum against to~Jn B was obtained as previously described [15]. C. sordellii antitoxin was obtained from the Bureau of Biologics (Rockville, MD, U.S.A.).
Toxin B preparation Purified toxin B was prepared for comparative analysis; it was produced and purified as previously described [16].
Assay of toxins in cells
Neutralisation of toxins The toxins were diluted 2-fold in tissue culture medium and each dilution was mixed 1 : 1 with antisera against either toxin A, toxin B or fraction C. The mixtures were incubated for 1 h at room tempera~re and then inoculated in CHO cells. The cells were read for elongation as described above.
A modification of the Chinese hamster ovary cell (CHO) test by Katoh et al. [13] was used. 200/~1 of a 3.10 3 ceils/m[ suspension were distributed in a microtiter plate; after 45 min, 20 t~l of the samples Were tested in 2- or 5-fold dilutions. After incubation overnight, the cells were fixed with 80% methanol and stained with Giemsa. The cytotonic activity was expressed as elongation units (EU), i.e., the inverse of the maximal dilution which induces elongation of at least 25~ of the cells.
Double diffusion-in-gel was done in 5 × 5 cm plates covered with agar, as previously described [19]. 20 #1 of antigen or antiserum were applied per hole, and diffusion was allowed to proceed in a humidified chamber for 48 h. Diffused plates were washed with phosphatebuffered saline and stained with Coomassie blue R-250.
PolyacJylamide gel electrophoresis
Digestion of toxins with t~psin
Samples were analysed in precast gradient gel electrophoresis in accordance to the manufacturer's instructions (Pharmacia, Sweden). Some gels were sliced into sections, and active toxin eluted by dialyses against
20/~g of each toxin (A o~ C) was digested for 7 h with 500/~g of trypsin (type XI, DPCC treated from Sigma, MO, U.S.A.) dissolved in 0.5 m[ 0.2 M sodium phosphate buffer (pH 7.0).
Immunodiffusion
153
Intestinal loop test Sprague-Dawley rats, 2-3 months old, were used to measure the effect of toxin A and fraction C on the small intestine [20]. One loop of about 10 cm was made in the middle of the small bowel and the sample, diluted in buffer 1, was injected in a volume of 1.0 ml. After 6 h, fluid accumulation was estimated from the weight/ length ratio (mg/cm) of the test loop and compared with that of control loops injected with buffer 1 alone. Each sample was assayed in triplicate.
1.°1
1
-->" 0.3
~ Z
0.2
0 l ' ..3" 0.6 a
Results
600
After 5 days of growth of C difficile, the crude cell-free culture contained a cytotonic activity of 14 elongation units per /~g of protein (EU//~g). Purification by ion-exchange chromatography and acid precipitation increased the specific activity about 30-times with a yield of 37~ (Table I). During further parification on high resolution ion-exchange gel, the cytotonic material was split into two peaks (Fig. 1). One peak corresponding to toxin A, eluted at 0.19 M NaC1; the other eluted at about 0.3 M NaCl, having a specific activity which was 5-times higher than that of toxin A (Table I). This second peak, called fraction C, was further separated into four peaks by recycling on the mono Q column and eluting with a gradient of NaC1 (Fig. 2). Peak I to 3 (CI, C2 and C3) exerted activity on CHO cells (Table I), while the fourth peak contained no activity. The purified toxins A and fraction C were analysed on gradient PAGE. When run under non-denaturing conditions (Fig. 3), toxin A gave a f-7~7 band migrating as a protein of molecular weight 400-600 kDa (row 2); fraction C separated into three proteins. Their apparent molecular masses were: 350 (C1), 270 (C2) and 140 kDa
TABLE I
Purification of proteins from C difficile causing elongation of CHO cells Purification step a
Cell free culture D E A E Sephadex A-25
Acid precipitation Mono Q, FPLC: toxin A fraction C Fraction C on Mono Q: C1 C2 C3
Protein (mg)
Activity on cells total EU
EU/Fg
7.6-10 s 4.1- l 0 s
14 b 100
6.6
2.8-106
420
5.0 1.3
7.5.105 9.5.105
150 760
0.14 0.16 0.72
8.0.10 2 1.6.103 1.0-104
6 10 14
540 41
a The production and purification procedure was repeated twice with similar results. b Elongation activity in crude culture-filtrate was determined by neutralizing the activity of toxin B with toxin B antiserum.
E ¢= 0
=L
0.4=
Cq
I
400 e-. o
,Q
~ 0.2,
e-.
200
,ID
<
o U.I
O,
4O 6O number Fig. 1. Fast prote~ liquid c~omatography of toxic proteins after acid precipitation. A total of 4 mg of protein was applied to a Mono Q column. Upper panel, NaCI gradient used for elution. Lower panel, elution profile: denotes absorbance ~.t 280 rim, and - - cytotonic activity on CHO cells. The first peak of cytotonic activity corresponds to toxin A and the second to fraction C. 0
20
Fraction
(C3) as compazed to reference proteins~ The toxic activity recovered from these regions, when they were eluted from slices of the unstained gel, was weak (100 to 1000 E U / m l ) but present only in the three zones corresponding to C1, C2 and C3. By recycling on Mono Q (Fig. 2), the C1 and C2 proteins were purified to homogeneity as shown by PAGE in Fig. 3 (rows 4 and 5). The subunit composition of the fraction C proteins was analysed in SDS-PAGE (Fig. 4a). In this gel, toxin A migrated as one band of molecular mass around 260 kDa (row 2); fraction C migrated in three bands of molecular mass 43, 41 and 39 kDa (row 3). In order to determine their subunit composition, C1, C2 and C3 were first separated by running fraction C under nondenaturing conditions, each protein band electroeluted and then run again on SDS-PAGE. As shown in Fig. 4b, the 39 kDa s;ubunit (row 1) originated from C1 (350 kDa), the 41 kDa subunit (row 3) from C3 (140 kDa), and the 43 kDa subunit (row 2) from C2 (270 kDa). The toxins were also run in SDS-PAGE under non-reducing conditions. However, toxin A and fraction C proteins migrated in a pattern similar to that obtained in the presence of fl-mercaptoethanol (not shown). These re-
154
,.o!
_1"
0.5
[ ~0.3 Z
0.1'
.I'
[3)
E ,- .10,
,20
0 CO
e=)
e-
,10
m .05'
cO
.Q
e0 UJ
< 0o
F r a c t i o n number Fig. 2. Separation of the three proteins of fraction C by fast protein liquid chromatography. 200 Fg of fraction C separated from toxin A (Fig. 1) were applied to a Mono Q column. Upper panel, NaCI gradient used for elution; lower panel, elution profile: ~ denotes absorbance at 280 nm, and - - - cytotonic activity on CHO cells. The first peak of cytotonic activity corresponds to C3, the second to C2 and the third to C1.
Activity, 1
232
2
3
~
140 -=,-,
4-
EU 10
20
4
5
4.-
~ll
"-
i TABLE II
qlm
67 .,m
~-
s.ults suggest that their peptide chains are non-covalently linked (i.e., not kept together by S-S bridges). Cultures of different ages were assayed for their content of fraction C and toxin A (Table II). Fraction C was detected after three days of growth, became maximal on day 5 (1250 Fg) and decreased after 7 days. Toxin A reached a high level after 3 days was increased further on day 5 (5000 Fg) and decreased again at day 7. Antitoxins against fraction C and toxin A and B were evaluated for their ability to neutralise the action of fraction C on CHO cells (Table III). All the elongation, activity of fraction C was neutralised by serum against either toxin A or fraction C, while toxin B antiserum had a partial neturalisation effect. When the separated C1, C2 and C3 proteins were tested, each of them was completely neutralised by the antiserum against toxin A. In contrast, toxin B antiserum had a pronounced effect on C2 only (Table III). The activity of toxin A was neutralised by antiserum against toxin C but not by antiserum against toxin B. Double-immunodiffusion analysis of fraction C was performed with antisera against fraction C, toxin A, toxin B and C. sordelii culture filtrate (Fig. 5). Fraction C antisera (Fig. 5a and b) produced one precipitin line with toxin A and three with fraction C, two of which spured in partial identity with toxin A. With toxin A antisera (Fig. 5a), fraction C precipitated in one line, showing complete identity with toxin A and partial identity with one of the precipitates formed between fraction C and fraction C antisera. Toxin B was not precipitated by fraction C antisera (Fig. 5b). Toxin B antiserum (Fig. 5c) precipitated with toxin B, but it did not precipitate fraction C. Antiserum against C. sordellii recognised toxin A, toxin B as well as fraction C (Fig. 5d); the precipitates formed with toxin A and fraction C showed partial identity, while the lines formed with toxin B and fraction C showed no identity. The effect of treatment with trypsin on toxin A and fraction C was tested. Digestion of 20 Fg of fraction C with 5 0 F g of trypsin completely destroyed the activity of fraction C on CHO cells. In contrast, toxin A was
7"~
Production of fraction C and toxin A by C. difficile Day
Fig. 3. Polyacrylamide gel electrophoresis of cytotoaic proteins under non-denaturating conditions (4 to 30~ gel); row I, molecular mass markers, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (67 kDa); row 2, toxin A after FPLC; row 3, fraction C after first FPLC (Fig. 1) and to the right the toxic activity of the same material (diluted 1/10) eluted from sliced pieces (arrows); row 4 and 5, C2 and C1, respectively, after second FPLC (Fig. 2). The gel was stained with Coomassie blue R-250.
2 3 5 7
Fraction C
Toxin A
O=g)
O,g)
0a 200 1 250 500
n.d. b 2100 ~ 5 000 3 800
a Yield of the protein after purification from two litre of culture filtrate as described in Table I. b n.d., not determined.
155 1
2
3
1
2
3
TABLE III Decrease of the activity of C. difficile toxins after treatment with antiserum or trypsin
U
220
Toxin
Toxin A Fraction C C proteins b: C1
67 I=, 60 q~
36
No treatment a
Effect on CHO cells (EU/ml) toxin A antiserum
6.2.10 4 3.1.105
0 0
2.0-10 2
0
C2
2.0- l0 2
0
C3
8.0-102
0
toxin B antiserum
fraction C antiserum
6.2-104 1.2-104
0 0
1.0.10 2
0
50
8.0-102
0
0
~
Fig. 4. Polyacrylamide gel electrophoresis of cytotonic proteins in the presence of SDS (2 Tto 16~ gel). (a) Row 1: molecular mass markers, ferritin (subunit 220 kDa), bovine serum albumin (67 kDa), catalase (subunit 60 kDa) anti lactate dehydrogenase (subunit 36 kDa); row 2, toxin A after FPLC; row 3, fraction C after first FPLC (Fig. 1). (b) SDS-electrophoresis of fraction C proteins previously run in non-denaturating conditions and electroeluted; row 1, protein C1; row 2 protein C2; row 3, protein C3. (a) was stained with Coomassie blue R-250 and (b) was silver-stained.
resistant to the proteolytic activity of even 500 pg of the enzyme. The enterotoxic effects of toxin A and fraction C were evaluated in small intestinal loops of rats. 2 pg of
a-C
A
a Values are expressed as EU/ml, e.g., the inverse of the maximal dilution which caused elongation of CHO cells. b Proteins are single peptides from fraction C separated in Mono Q column.
toxin A elicited a strong haemorrhagic fluid response (322 + 27 mg/cm). Fraction C at the same concentration elicited no response, whereas 15 /~g induced a pronounced fluid accumulation (320 + 40 mg/cm) without bleeding or macroscopical evidence of tissue damage. Discussion T h r e e toxic p r o t e i n s f r o m C. difficile w h i c h a r e diff e r e n t f r o m t h e a l r e a d y k n o w n toxins A a n d B a r e
a-C
a-A
C
A
A
C
a-sord.
a-B
C
B
B
A
C
B
Fig. 5. Comparison of fraction C, toxin A and toxin B in double immunodiffusion. The proteins were analysed after FPLC purification. (a) A, 8/tg of toxin A; C, 8/tg of fraction C; a-C, 20/~1 of fraction C antiserum; a-A, 20/tl of toxin A antiserum. (b) A, 8/tg of toxin A; C, 8/tg of fraction C; B, 20/tg of toxin B; a-C, 20/tl of fraction C antiserum. (e) C, 20/tg of fraction C; B, 8/tg of toxin B; a-B, 20 lgl of toxin B antiserum. (d) A, 25/~g of toxin A; C, 20/~g of fraction C; B, 20/tg of toxin B; a-sord., 20 ttl of C. sordelli antiserum.
156 described here; they were designated C1, C2 and C3 or taken together fraction C. All three were cytotoxic and dissociated into subunits of similar size (around 40 kDa). None of them seemed to contain any interchain S-S bridge. When fraction C was separated on PAGE, the various activities and protein bands migrated to the same positions as those of the C proteins, which previously had been purified on the mono Q column. These results suggest that the effect of fraction C is due to the combined action of the three C proteins. However, since the separations of fraction C on polyacrylamide and mono Q led to significant losses of activity, there may be a still unidentified component which stabilise the fraction C complex in its active form. The intestinal effect of the fraction C complex resembled that of cholera toxin or E. coil LT [21] rather than that of toxin A [11,12]. Thus, a clear fluid was secreted without the haemorrhagic response seen after exposure to toxin A. The specific enterotoxic activity of fraction C was 5- to 10-fold less than that of toxin A. This difference might be due to the different sensitivity to trypsin, which enzyme inactivated fraction C but not toxin A. In previous studies toxin A has been reported to contain subunits of a size similar to that of the fraction C proteins [22,23]. These subunits might in fact belong to fraction C proteins, which copurifies with toxin A during ion-exchange chromatography and precipitation with acid. There have also been reports of partially purified toxins of smaller size which induce a nonhaemorrhagic fluid response in the small intestine [11,24,25]. These toxic activities might be caused by fraction C; however, since the preparations were not pure and immunological data were lacking, no meaningful comparison can be made. Although toxin A and B have epitopes in common [16], no significant neutralisation is achieved with the heterologous antiserum. In contrast, fraction C was totally neutralised by toxin A antiserum and partially neutralised by toxin B antiserum. The immunodiffusion experiments confirmed the cross-reactivity with toxin A but not that with toxin B. The lack of precipitation of fraction C with toxin B antiserum might be due to the insensitivity of the method with respect to the quantity and affinity of the antigen-antibody complex formed. Of the separated proteins, C2 was neutralised by A antise~m as well as B antiserum, suggesting that it had epitopes in common with both the toxins. The precipitation of toxin A, toxin B as well as fraction C with C. sordellii antiserum confirm that this bacterium produces similar toxins [26,27]. Among them, the so-cal, M lethal toxin has been purified and shown to have subunits of similar size to those of fraction C [28]. It cross-reacts with C difficile toxin B and exerts a moderate response in the intestinal loop test. Further, a low molecular
mass form of toxin B was recently described which has subunits of similar size to those of toxin C [15,29]. Large peptide chains are mostly made up of repetitive sequences, and this concept seems to be true also for toxin A [14,30]. Toxin A is probably endocytosed and hydrolysed lysozomally before its action is expressed [31] and the C proteins might represent the active fragments of toxin A. A variety of bacterial toxins are processed by cell wall endopeptidases, e.g., tetanus, botulinum, diphtheria and cholera toxin [32-35]. These so called 'nicked' molecules are supposed to represent the active form of the toxins. Thus, some toxin A molecules might be processed into C proteins already during the transport across the bacterial cell wall.
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28 Poppoff, M.R. (1987) Infect. hranun. 55, 35-43. 29 Pothoulakis, C., Barone, L.M., Ely, R., Faris, B., Clark, M.E., Franzblau, C. and LaMont, J.T. (1986) J. Biol. Chem. 261, 1316-1321. 30 Price, S.B., Phelps, CJ., Wilkins, T.Do and Johnson, J.L. (1987) Curr. Microbio!_ 16, 55-60. 31 Henriques, B., Florin, I. and Thelestam, M. (1987) Microbial Pathogenesis 2, 455-463. 32 Bizzini, B. (1979) Microbial Rev. 43, 224-240. 33 Das Gupta, B.R. and Sugiyama, H. (1972) Infect. Immun. 6, 587-590. 34 Collier, RJ. and Kaplan, D.A. (1984) Sci. Am. 251, 44-52. 35 Betley, M.J., Miller, V.L. and Mekaianos, J.J. (1986) Ann. Rev. Microbiol. 40, 577-605.