Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B

Microbial Pathogenesis 1991 ; 11 : 337-346

Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B Inger Florin Department of Bacteriology, Karolinska Institutet, S- 104 01 Stockholm, Sweden

(Received May 13, 1991 ; accepted in revised form July 3, 1991)

Florin, I . (Dept of Bacteriology, Karolinska Institutet, S-104 01 Stockholm, Sweden) . Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B . Microbial Pathogenesis 1991 ;11 :337-346 .

A mutant of Chinese hamster lung fibroblasts (Don cells), resistant against Clostridium difficile toxins A and B, was isolated after mutagenization with ethylmethanesulphonate and a two-step selection with toxin B . The mutant, termed Cdt R -Q, was 10 4 times more resistant to toxin B than wild-type cells and cross-resistant to toxin A (10 3 times more resistant) . The resistance was overcome by increasing the dose of toxin . The resistance has been stable after cultivation for 40 generations in the absence of toxin . The morphology of the mutant was more epithelial-like than that of the fibroblast parental cells . The plating efficiency was about half that of the wild-type, whereas the growth rate was the same . The mutant was significantly less sensitive than the wild-type to the microfilament-interacting cytochalasins B and D . It was as sensitive as the wild-type to endocytosed toxins (diphtheria, pertussis, ricin), to microtubuleinteracting agents (colchicine, gossypol, nocodazole, taxol, vinblastine), and to membranedamaging toxins with different mechanisms of action, with one exception ; the mutant was more highly sensitive to the action of phospholipase C (with broad substrate-specificity) than the wild-type . The results suggest that the mutant has a normal endocytosis, and that the mutation does not affect the microtubuli . The results are consistent with a mutation affecting the microfilaments in the cytoskeleton . Key words: Clostridium difficile toxins ; cytopathogenicity ; Chinese hamster fibroblasts ; toxin-

resistant mutant .

Introduction The bacterium Clostridium difficile produces two toxins, toxin A and toxin B, which are involved in the aetiology of antibiotic-associated diarrhoea and colitis ." Both toxins are cytotoxic to cultured cells, inducing a characteristic actinomorphic change visible in the light microscope ." Toxin B is 1000 times more potent as a cytotoxin than toxin A, but toxin A also has an enterotoxic activity . After binding of the toxins to the surface of cultured cells, the toxins are taken up into the cells via endocytotic vesicles ." In the case of toxin B, the vesicles fuse with the lysosomes, where the toxin is processed, probably enzymatically,' whereas toxin A might be processed in the Golgi region .' Both toxins (or active fragments?) are supposed to be translocated to the cytosol, where the final effect of the intracellular steps is a disorganization of the microfilament system, 9-14 which results in the actinomorphic change . Whereas the internalization of both toxins has been the subject of several studies," detailed information is still lacking about the binding and enzymatic processing of the toxins and the molecular mechanism for the effect of the toxins on the microfilaments . 0882-4010/91/110337+10 s03 .00/0

© 1991 Academic Press Limited



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Cells defective at different steps in the intoxication processes would facilitate the study of these steps at the molecular level, and would also be useful for investigating the normal cellular functions which are directly involved in the intoxication processes . No naturally resistant cell type has been found for any of the toxins, although toxin B has been tested in more than 30 different cell types from different animal species and tissues 15,16 and toxin A in approximately 20 different cell types .'"" The aim of this study was to isolate mutant cells resistant to the action of toxin A and/or toxin B from C. difficile .

Results Mutagenization procedure and selection of resistant cells In most of our previous work,"" 1-2' human lung fibroblasts (MRC-5) were used . These have a finite life span in culture and thus are unsuitable for mutagenization purposes . Chinese hamster lung fibroblasts (Don cells) were therefore chosen for these studies because of their similarity to human fibroblasts . Chinese hamster lines also possess relatively stable karyotypes . 22 Don cells were mutagenized with ethylmethanesuIphonate and toxin B-resistant clones selected . Several different modifications of mutagenization and selection procedures were used in parallel . Resistant mutants were obtained first by the procedure described in Materials and methods, but this may be a coincidental result and does not necessarily imply that the procedure was optimal . After one-step selection with toxin B, many low-resistant mutants were obtained which were 2-100 times less sensitive than the wild-type cells to toxin B . After reselection with higher doses of toxin B, a highly resistant mutant, termed Cdt R -Q, was obtained . This mutant was 104 times more resistant to toxin B than the wild-type . General characteristics of the mutant The morphology of the mutant, as viewed under the light microscope, was clearly different from that of the wild-type (Fig . 1) . The wild-type is a typical fibroblast [Fig . 1 (A)], whereas the mutant cells more closely resemble epithelial cells ; they are polygonal and flattened [Fig . 1 (C)] . When detached by trypsin treatment from the substratum the cells rounded, but whereas the wild-type cells became completely round, the mutant cells became slightly more irregular . When treated with very high doses of toxin B, the mutant develops a cytopathogenic effect (CPE), but not the actinomorphic change characteristic of fibroblasts [Fig . 1 (B)], or the typical rounding of epithelial cells . 23 The mutant cells contract at one side only, resembling a fan [Fig . 1 (D)] The mutant cells have so far retained the resistance for up to 7 months when cultivated in the absence of toxin . The growth rate was the same for both the wild-type and the mutant, plating efficiency for the mutant was about half that of the wild-type efficiency of the wild-type was approximately 50% . Both the mutant cells and the wild-type cells were free of mycoplasma

(40 passages) although the . The plating infection .

Dose-response and time-course relationships of C . difficile toxin B in wild-type and mutant cells Cells were exposed to serial 10-fold dilutions of toxin for 24 h to determine the 50% tissue culture dose (TCD 50 ) .'a The TCD 50 of the wild-type was 10 4 times lower than that of the mutant . The dose-response and time-course for the development of toxin B-induced CPE in the wild-type Don cells were similar to human fibroblasts, 18 the

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Fig . 1 . Wild-type Don cells : (A) control cells ; (B) toxin-treated cells . Mutant Don cells : (C) control cells ; (D) toxin-treated cells . The cells were stained by Giemsa . Magnification 400x .

CPE starting to develop after a latency period, and reaching 100% after several hours, depending on the dosage (Fig . 2) . In the mutant, no CPE developed until after 36 hours or more, depending on the dose (Fig . 2) . However by exposing the mutant cells to 10 ° times higher doses than the wild-type, similar time-course relationships as for the wild-type were obtained .

100

w a v

50

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Time ( h) Fig . 2 . Dose-response and time-course relationships for toxin B in wild-type and mutant cells . Toxin was added to the cells in different doses and the development of the CPE at 37'C followed . Note : the scale on the x-axis is logarithmic . A, wild-type 100 TCD 5o ; El, wild-type 200 TCD 50 ; /, wild-type 1000 TCD00 : 0, mutant 100 TCD50; 0, mutant 200 TCD50; A, mutant 1000 TCD5o .



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100

w 0 0 0

50

•1

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o I 10 Time ( h )

100

Fig . 3 . Dose-response and time-course relationships for toxin A in wild-type and mutant cells . Toxin was added to the cells in different doses and the development of the CPE at 37'C followed . Note : the scale on the x-axis is logarithmic . E], wild-type 1 TCD 50; 9, wild-type 10 TCD 50 ; A, wild-type 100 TCD 50 ; E, wildtype 1000 TCD50; 0, mutant 100 TCD 50 ; A, mutant 1000 TCD5o.

Cross-sensitivity to other toxins (a) Other microfilament-interacting agents . Human fibroblasts dose-response and time-course relationships of toxin A produced by C . difficile are very similar to those obtained with toxin B . 6 In wild-type Don cells, the same was true when comparing the response to toxin A (Fig . 3) with that to toxin B (Fig . 2) . Furthermore, the toxin B-resistant mutant Don cells were found to be cross-resistant to toxin A (Fig . 3) . No CPE developed in the mutant until after exposure to toxin A for 5 h or more, depending on the dose (Fig . 3) . On a TCD 50 basis, the mutant was 10 3 times less sensitive to toxin A than the wild-type . As can be seen in Fig . 3 by comparing 1 TCD 50 in the wild-type with 1000 TCD 50 in the mutant, a similar time-course to the wild-type was obtained by exposing the mutant to 10 3 times higher doses . The mutant was also significantly less sensitive against cytochalasins B and D . 24 With a dose of cytochalasin B (2,ug/ml) inducing 80% CPE in the wild-type after 2 h, no CPE was developed in the mutant after 2 h, and after 24 h with the same dose the CPE was only 20% (Fig . 4) . The dose-response results were similar with cytochalasin D (data not shown) . For both cytochalasins B and D the values for 50% effective dose (ED 50 ) (determined as described in Materials and methods) were 0 .2 yg/ml in the wild-type and 20 ug/ml in the mutant after 4 h ; the same values were obtained after 24 h . (b) Microtubule-interacting agents . The development of the specific morphological effects induced in cells by five microtubule-interacting agents (colchicine, gossypol, nocodazole, taxol and vinblastine) with different mechanisms of action, 24 followed the same time-course in the wild-type and the mutant, when comparing each agent . The ED 50 values after 4 and 24 h were the same in the wild-type and the mutant for each agent and corresponded to effective doses previously reported .25,2' These results indicate that the microtubules in the mutant were not affected by the mutation . (c) Endocytosed toxins . The ED 50 values after 24 h for the specific morphological effects induced in cells by the three toxins (diphtheria, ricin and pertussis) which require endocytosis in order to intoxicate cells, 27 were the same for the mutant and



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Time( h ) Fig . 4 . Dose-response and time-course relationships for cytochalasin B in wild-type and mutant cells . Cytochalasin B was added to the cells in different concentrations and the development of the CPE at 37 °C followed . Note : the scale on the x-axis is logarithmic . 0, wild-type 2 pg/ml ; 0, wild-type 5 pg/ml ; /, mutant 2 Mg/ml ; 9, mutant 5 pg/ml .

28,29 the wild-type and comparable to effective doses reported in the literature . Since the morphological effect of these toxins is not detectable until after 24 h, it was not possible to obtain ED 50 values for any earlier time . The results suggest that the process of endocytosis in the mutant was not affected by the mutation . (d) Membrane-damaging toxins . The development of the specific morphological effects induced in cells by three membrane-damaging toxins with different mechanisms of action 30 (Staphylococcus aureus alpha-toxin, Clostridium perfringens theta-toxin, C. perfringens phospholipase C) was followed . The effects of alpha-toxin and thetatoxin were developed with the same time-course in wild-type and mutant cells (as shown in Table 1 with ED 50 values after 4 h), although the ED 50 value after 24 h for theta-toxin was consistently 10-fold lower in the wild-type than in the mutant (Table 1) .

Table 1 toxins

Sensitivity of mutant and wild-type cells to membrane-damaging

ED 50 (U/ml) 4h Toxin S . aureus alpha-toxin C. perfringens theta-toxin Phospholipases A2 B C (C. perfringens) C (B . cereus) C, sphingomyelin-specific C, phosphatidylinositol-specific D, type 1 D, type VI

24h

Wild-type

Mutant

Wild-type

Mutant

650 8

650 8

650 0.8

650 8

(a) (a) 40 (a) (a) 25 250 250

(a) (a) 0 .04 (a) (a) 25 250 250

10 2 .5 40 (a) 330 2 .5 250 25

10 2 .5 0 .00004 120 330 2 .5 250 25

Cells were exposed at 37°C to serial 10-fold dilutions of the agents and the ED 50 values determined after 4 and 24 h . (a) No detectable effect in the highest concentration available, which was 1020 U/ml for phospholipase A2, 25 U/ml for B, 120 U/ml for C (B . cereus) and 3300 U/ml for sphingomyelinspecific C .



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In contrast to the results with alpha- and theta-toxins, the effect of phospholipase C was induced with significantly lower doses in the mutant than in the wild-type (Table 1) . The ED 50 values after 24 h showed that the mutant was 10 6 times more sensitive to phospholipase C from C . perfringens . To find out whether this hypersensitivity of the mutant was general to all phospholipases, 31 phospholipases A 2 , B and D as well as phospholipase C from other sources were tested (Table 1) . The increased sensitivity was verified with phospholipase C from Bacillus cereus, although the difference was not as large as for phospholipase C from C. perfringens . In contrast to the findings with these two phospholipases, the mutant was as sensitive as the wild-type to sphingomyelin-specific and phosphatidylinositol-specific phospholipase C as well as to phospholipases A 2 , B and D (Table 1) . The results show that the mutant has obtained a specific hypersensitivity against phospholipase C with broad substrate-specificity, although it was not generally more sensitive to other membrane-damaging toxins .

Discussion This is the first report of a cell mutant resistant to the action of toxins A and B from C. difficile . The mutant was obtained from mutagenized Don cells by a two-step selection procedure with toxin B and was 10' times more resistant to toxin B than the parental cell . Surprisingly, it was cross-resistant (10 3 times more resistant) to toxin A . These resistant cells will be a valuable tool in clarifying the part of the intoxication processes of toxins A and B which are affected by the mutation, since there is no known cell type which is naturally resistant to the action of these toxins . The intoxication processes of toxins A and B may be divided into four major parts, each consisting of several substeps ; 17• 2 3 (i) toxin binding to the cell surface, (ii) toxin uptake, (iii) intracellular toxin processing and (iv) toxin-induced intracellular biochemical changes resulting in disorganization of the microfilaments . Theoretically the mutation in the isolated mutant may affect any of these parts . When increasing the dose of toxin B 10' times and the dose of toxin A 10 3 times, a CPE was developed in the mutant with the same time-course as with lower doses in the wild-type . Since the mutant cells were able to develop a CPE, the mutation cannot have caused a complete loss of any protein . More probably the mutation has induced a change in the affinity of either a structural protein or a regulatory protein, or an increased/decreased level of such a protein . Since cross-resistance studies with mutants often provide valuable information regarding the nature of the genetic lesion, the mutant was characterized with respect to sensitivity to other toxins . The mutant was cross-resistant to the microfilamentinteracting cytochalasins, but as sensitive as the wild-type to microtubule-interacting toxins and to endocytosed toxins . The mutant was as sensitive as the wild-type to membrane-damaging toxins, with one exception ; the mutant was hypersensitive to phospholipase C with broad substrate-specificity . Of the agents tested the mutant was resistant only to toxins A and B and to cytochalasins B and D . Toxins A and B somehow disorganize the microfilament system,9-14 whereas cytochalasins induce a similar disorganization by binding to actin filaments ." The resistance of the mutant would easily be explained by supposing a mutation affecting some structure in the microfilament system, which is required for intoxication with these toxins . Other observations in favour of a mutated cytoskeleton are the changed morphology and the lower plating efficiency of the mutant cells, as well as the changed appearance of the CPE in toxin-treated mutant cells .



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Toxins A and B are not assumed to affect the two other parts of the cytoskeleton, the microtubules and the intermediate filaments .' 1,12,14 To verify that the microtubules were intact in the mutant, five microtubule-interacting agents with different mechanisms of action 24 were tested ; the mutant did not have a different sensitivity to any of these compared to the wild-type . Unfortunately the same approach to intermediate filaments was not possible, since agents interfering specifically with these filaments are lacking . 24 To explain the results by supposing a mutation in any of the other parts of the intoxication processes would be more difficult . Concerning endocytosis of the toxins, we have shown earlier that toxins A and B are taken up by the same endocytotic route as diphtheria toxin ."' Mutant Chinese hamster ovary cells defective in endocytosis, selected to be resistant against diphtheria toxin, 32 were cross-resistant to toxins A and B ." Since the toxin B-resistant mutant described here responded as the wild-type to diphtheria, ricin and pertussis toxins, which all require endocytosis in order to intoxicate cells, 27 it is concluded that the endocytosis of the toxin B-resistant mutant appears normal ; this observation is supported by cross-resistance to cytochalasins . Since cytochalasins penetrate the cell membrane directly without requiring endocytosis, 33 it is not possible to explain the decreased sensitivity to these agents by a mutation affecting endocytosis. This resistance would then have to be explained by supposing a second mutation . It is not probable that two different mutational events would have occurred simultaneously within the same cell, conferring resistance by different mechanisms to agents inducing the same morphological effect . Neither is it possible to explain the resistance of the mutant being caused by a mutation affecting the intracellular enzymatic processing of the toxins, since toxins A and B seem to require different types of enzymatic processing," and cytochalasins do not require any processing . The mutation would then have to affect the different enzymes needed for processing of toxins A and B, and as above the resistance to cytochalasins would have to be explained by a second mutation . Toxins A and B are assumed to have different cell membrane receptors ." Although the nature of the receptor for toxin B has not yet been identified, the suggested carbohydrate receptor for toxin A has been shown to be specific for toxin A . 34 Cytochalasins are lipophilic and penetrate directly through the cell membrane without involvement of any high affinity binding proteins in the membrane ." In order to explain the resistance of the mutant as caused by defective receptors in the cell membrane, several different mutations would thus have to be implied . However, a mutation changing some membrane component near the different receptors for toxins A and B, leading to steric hindrance for toxin binding, and to decreased permeability of the membrane and thus reduced penetration of the cytochalasins, cannot be excluded at this stage . Nevertheless, a change in some component of the membrane surface does not usually lead to a totally different morphology . The hypersensitivity of the mutant to phospholipase C with broad substratespecificity shows that the cell membrane in the mutant was indeed affected in some way, although the mutant did not have any generally increased sensitivity to other membrane-damaging toxins . The hypersensitivity may be due to a change in the content or the exposition of the membrane phospholipids, leading to steric hindrance for binding of toxins A and B . It may also be the consequence of a mutation affecting the microfilaments . Since the microfilaments are connected to the cell membrane via membrane proteins in the adhesion plaques, 35 a mutation in the microfilaments might lead to a changed topology of the surface proteins so that the phospholipases more easily reach the membrane phospholipids . At this stage a mutation affecting the binding of the toxins to the cell surface cannot



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be completely ruled out, but the hypothesis which fits best with all available experimental data is that the mutation in the resistant mutant affects an intracellular structure involved in disorganization of the microfilaments . In order to identify the mutated structure, biochemical characterization using two-dimensional electrophoresis of membrane and cytoskeletal proteins of the mutant, and comparison with the wildtype, is in progress, as well as ultrastructural characterization of the mutant using electron microscopy . However, whether the mutation will turn out to affect the binding or the cytoskeletal target of the toxins it will be a very useful tool to clarify this part of the intoxication processes of toxins A and B .

Materials and methods

Chemicals and toxins . Ethyl methanesuIphonate, cytochalasin B, cytochalasin D, N-deacetylN-methylcolchicine (demecolcine), gossypol (2,2'-bis(8-formyl-1,6,7-trihydroxy-5-isopropyl3-methylnaphthalene)), ricin agglutinin and vinblastine sulphate were purchased from Sigma Chemical, St. Louis, Missouri . Nocodazole was obtained from Janssen Chimica, Beerse, Belgium, and taxol kindly provided by Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland . Stock solutions of these agents were prepared in dimethylsulphoxide, except ethyl methanesuIphonate and ricin, which were obtained as a liquid and as a solution in sodium phosphate, respectively . Diphtheria toxin (10 mg/ml) and pertussis toxin (64 pg/ml) were kindly provided by Dr Per Askelof, National Bacteriological Laboratory, Stockholm, Sweden . Highly purified S . aureus alpha-toxin, C . perfringens theta-toxin, phospholipase C from C. perfringens and B . cereus, and sphingomyelin-specific phospholipase C from S . aureus were kindly donated by Dr Monica Thelestam, Department of Bacteriology, Karolinska Institutet . Lyophilized phospholipases A 2 (bee venom), B (Vibrio sp .), phosphatidylinositol-specific C (B . cereus), D type I (cabbage) and D type VI (Streptomyces chromofuscus) were purchased from Sigma Chemical . Stock solutions of the membrane-damaging toxins were prepared in distilled water, except phosphatidylinositol-specific phospholipase C, which was obtained as a solution in Tris-HCI with 50% glycerol . The activity of all membrane-damaging toxins was measured in units (U) as defined by Sigma, except S . aureus alpha-toxin, C. perfringens theta-toxin and sphingomyelinspecific phospholipase C, the activity of which was measured as haemolytic units according to Thelestam and Mollby . 30 Purification of toxins . Toxin B was purified from dialysis cultures of C. difficile VPI 10463 according to Meador and Tweten 36 with some modifications . 37 In short, the filtered and concentrated culture supernatant was applied to an anion-exchange column of Accell QMA (Millipore, Gothenburg, Sweden) and toxin B eluted with a linear gradient of NaCl in 20 mm Tris-HCI, pH 8 .0, containing 50 mm CaC1 2 . Cytotoxic fractions were pooled and applied to a column of MonoQ (Pharmacia, Uppsala, Sweden) and eluted with NaCl (in the same buffer as above) using FPLC equipment (Pharmacia) . When analysed by native polyacrylamide gel electrophoresis, the cytotoxic fractions showed one major protein band (385 kD), where the cytotoxic activity was located . Toxin A was purified as previously described .' When analysed by native polyacrylamide gel electrophoresis, the major band was located at approximately 550 kD . Cultivation of cells . Eagle minimal essential medium, fetal bovine serum and trypsin were obtained from Flow Laboratories, Irvine, Scotland . Diploid Chinese hamster lung fibroblasts (Don cells ; ATCC no . CCL 16) (Flow) were cultivated in Eagles medium supplemented with 10% fetal bovine serum, 5 MM L-glutamine, penicillin (100 U/ml) and streptomycin (100 Mg/ml), in a humid atmosphere containing 5% CO 2 . The cells were free of mycoplasma infection as determined by Hoechst staining 38 and cultivation . The unit of toxin activity was the 50% tissue culture dose (TCD 50 ), i .e. the toxin dilution inducing, within 20 h, a characteristic actinomorphic cytopathogenic effect in 50% of wildtype cells exposed under standardized conditions ."' The amount of protein corresponding to one TCD 50 was approximately 5 x 10 -3 pg for toxin B and approximately 1 ng for toxin A . Dose-response curves and cross-sensitivity studies. Unless otherwise stated, the cells were



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seeded in polystyrene 96-well plates (A/S Nunc, Roskilde, Denmark) with 50000 to 100000 cells per plate (the mutant cells were seeded with twice the amount of the wild-type due to the lower plating efficiency) and cultivated for 3 to 4 days before exposure to toxin or other agent. To obtain the dose-response curves with toxins A, B and cytochalasin B, the development of the CPE at 37°C was followed by counting the number of affected and unaffected cells in three randomly selected fields of view (in the light microscope) per well with approximately 500 cells per field, and the percentage CPE calculated . The values are averages of duplicate samples . For cross-sensitivity studies, cells were exposed at 37°C to serial 10-fold dilutions (in 200 hl Eagle medium per well) of the agent to be tested, starting with the highest concentration available . The development of the specific cytotoxic effect of the agent was followed at regular intervals from 15 min until 8 h after addition of the agent, and a final scoring was performed after 24 h . The dilution of agent causing a morphological effect closest to 50% (50% effective dose; ED 50) was determined . All experiments were performed at least three times . Mutagenization procedure and selection of resistant cells . Exponentially growing Don cells in cell culture bottles (75 cm' ; Greiner Labortechnik, Nurtingen, Germany) (700000 cells seeded/bottle) were exposed to ethyl methanesu I phonate in varying doses (500-800 pg/ml), principally according to Moehring and Moehring . 39 With 800 ,ug/ml, the survival of the cells was approximately 30-50%. After 18-20 h, the mutagen was replaced by medium and after a further 24 h, the bottles were passaged by trypsinization and all cells from each bottle seeded into one 96-well plate . Cells surviving after the mutagen treatment were allowed a recovery period from sublethal effects of the mutagen, and for fixation of the mutation and expression of the mutant phenotype . After 3 days further cultivation in non-selective medium, mutagenized cells were exposed to toxin B in doses which intoxicated all non-resistant cells (20 to 50 TCD 50 ) . (Under these conditions, no cells survived 50 TCD 50, whereas a few survived 25 TCD 50 .) After 1 week, the toxin was replaced with fresh medium . Clones of resistant cells were detected after approximately 6 weeks . Several low-resistant clones were expanded, the amplitude of the resistance tested by exposure to serial 10-fold dilutions of toxin B, and the most resistant ones frozen . To improve the resistance of these low-resistant clones, some of them were exposed to a reselection treatment . The clones were seeded into 96-well plates with 1, 10 and 100 cells per well, respectively, and toxin B added directly (50 and 100 TCD 50 , respectively) . After approximately 4 weeks, one highly resistant mutant, termed Cdt R -Q, appeared in a plate where 100 cells per well were seeded and exposed to 100 TCD5O .

The patient technical assistance of Kerstin Andreasson and Lena Norenius in isolation of the mutant cells is acknowledged . Diphtheria and pertussis toxins were kindly provided by Dr Per Askelof, National Bacteriological Laboratory, Stockholm, and several membrane-damaging toxins by Dr Monica Thelestam, Karolinska Institutet, Stockholm, who also contributed valuable information concerning this topic . This project was supported by grants from the Swedish Medical Research Council grant no . 16X-05969, the foundations of Magnus Bergvall and of Ake Wiberg, Hvitfeldtska saliskapet and the Swedish Society of Medicine .

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Rolfe RD, Finegold SM, eds . Clostridium difficile : its role in intestinal disease . San Diego : Academic Press, 1988 . 4. Florin I, Thelestam M . Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts . Biochim Biophys Acta 1983; 763 : 383-92 . 5 . Shahrabadi MS, Bryan LE, Lee PWK . Interaction of Clostridium difficile toxin A with L cells in culture . Can J Microbiol 1984 ; 30 : 874-83 . 6 . Henriques B, Florin I, Thelestam M . Cellular internalization of Clostridium difficile toxin A. Microb Pathogen 1987 ; 2 : 455-63 . 7 . Kushnaryov VM, Sedmak JJ . Clostridium difficile enterotoxin A on ultrastructure of Chinese hamster ovary cells . Infect Immun 1989 ; 57 : 3914-21 . 8 . Florin I, Thelestam M . Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B . Microb Pathogen 1986 ; 1 : 373-85 . 3.



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Fiorentini C, Malorni W, Paradisi S, Giuliano M, Mastrantonio P, Donelli G . Interaction of Clostridium difficile toxin A with cultured cells : cytoskeletal changes and nuclear polarization . Infect Immun 1990 ; 58 :2329-36 . 15 . Donta ST, Sullivan N, Wilkins TD . Differential effects of Clostridium difficile toxins on tissue-cultured cells . J Clin Microbiol 1982 ; 15 : 1157-8 . 16 . Taylor GR, Carter GI, Crow TJ . A comparison of the effects of cytotoxic cerebrospinal fluid on cell cultures with other cytopathogenic agents . Exp Mol Pathol 1985 ; 42 : 401-10 . 17 . Fiorentini C, Thelestam M . Clostridium difficile toxin A and its effects on cells . Toxicon 1991 ; 29 : 54367 . 18 . Florin I, Thelestam M . Intoxication of cultured human lung fibroblasts with Clostridium difficile toxin . Infect Immun 1981 ; 33 : 67-74 . 19 . Ahlgren T, Florin I, Jarstrand C, Thelestam M . Loss of surface fibronectin from human lung fibroblasts exposed to cytotoxin from Clostridium difficile . 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