Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin

Microbial Pathogenesis 1987 ; 3 : 279-286

Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin Masami Miyake and Iwao Ohishi* University of Osaka Prefecture, College of Agriculture, Sakai, Osaka 591, Japan

(Received May 18, 1987 ; accepted in revised form July 14, 1987)

Miyake, M . (University of Osaka Prefecture, College of Agriculture, Sakai, Osaka 591, Japan) and I . Ohishi . Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C 2 toxin . Microbial Pathogenesis 1987 ; 3 : 279-286 . C 2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two nonlinked protein components, designated components I and II . The toxin, a mixture of untrypsinized component I and trypsinized component II, induced marked morphological changes of tissue-cultured cynomolgus monkey kidney cells; the characteristic response of the cells to the toxin was rounding, which increased proportionally to log dose of the toxin . The components alone and a combination of untrypsinized components I and II showed little activity . The rounding of the cultured cells was not accompanied by inhibition of protein and nucleic acid syntheses of the cells, although the rounded cells ultimately lost viability . Immunofluorescence studies showed that component II, either trypsinized or untrypsinized, bound to the cell surface, whereas component I bound to the cells only in the presence of trypsinized component II . The present results support the previously proposed idea concerning the mode of action of C2T, that components I and II of C2T act together as a molecule with dual functions ; component I I as the recognizer of the receptor site on the cell surface membranes and component I as the effector in the cytoplasm by preferential inactivation of cytoskeletal actin, which results in alteration of cell morphology, and subsequently in cellular damage . Clostridium botulinum ; botulinum C 2 toxin ; tissue-cultured cells ; immunofluorescence ; staining . Key words :

Introduction Clostridium botulinum is known to produce an extremely potent neurotoxin . In 1935,

Mason and Robinson reported that C . botulinum type C produces two immunologically distinct toxins and designated them C, and C 2 toxins, respectively .' Later on, it was found that certain strains of C . botulinum type D also produce C 2 toxin (C2T) . 2 4 In 1980, C2T was purified and characterized .' The toxin, as well as being lethal, has novel biological activities, including enterotoxic and vascular permeability activities, which are not displayed by the neurotoxin elaborated by C. botulinum types A through F ; 6. ' C2T has no inhibitory activity upon the release of acetylcholine at peripheral cholinergic junction, which is specifically inhibited with botulinum neurotoxin . $ These facts show that, although C2T has potent lethal activity, the mode of action of the toxin differs entirely from that of botulinum neurotoxin . Recent studies have revealed that C2T is composed of two separate protein components, designated components I and II, which are linked with neither covalent 'Author to whom correspondence should be addressed . 0882-4010/87/010279+08 $03 .00/0

© 1987 Academic Press Ltd



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Fig . 1 . Morphological changes of CMK cells with C2T . The cells in 98-wells plate were exposed to 40 ng of the toxin per well for : (a) 30 min ; (b) 1 h ; (c) 2 h ; (d) 4 h ; (e) 24 h and (f) the cells not exposed to the toxin . Magnification of all photographs is the same . The bar represents 10 µm .

nor noncovalent bonds . The components alone have very little activity, but, together, act as a toxin of high activity. Previously, we have reported that C2T induced rounding of tissue-cultured cells of various mammalian cell lines, although the minimum dose of the toxin required to induce the response varied depending on the cell line .' In the present study, we attempted to investigate this cytopathic effect of C2T on tissuecultured cynomolgus monkey kidney (CMK) cells in more detail . In addition, the binding of components I and II of C2T to the cells was examined by immunofluorescence staining with specific antibody to the two components of the toxin .

Results Morphological changes of CMK cells with C2T The time course of morphological change of the cells exposed to C2T was observed under phase contrast microscope (Fig . 1) . With the toxin dose used, no marked change was observed within 30 min incubation . The shape of cells exposed to the toxin for 1 h altered from a polygonal to actinomorphic form, and some of the cells rounded . After 2 h incubation, the cells rounded completely, but still attached to the culture plate with fibrous branches . The cells appeared to have shrunk around the nuclei . After 4 h incubation, most of the fibrous branches had disappeared and some of the nuclei in the cells located close to the cell membrane and had protruded to the outside



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Fig . 2 . Time course of rounding response of CMK cells to C2T . The cells in 98-wells plate were incubated with : •, 1000 ng per well ; A, 40 ng ; A, 8 ng ; E, 1 .6 ng and 0, no toxin . Each point represents the mean + the standard error of the mean for four values .

of the cell . At 24 h incubation, some of the cells disrupted and detached from the culture plates (Fig . 1 (e) ) . Time course of rounding of CMK cells As described above, the rounding of the cells exposed to C2T was the characteristic response and could easily be observed under phase contrast microscope . Therefore, the rounding response of the cells to the toxin was examined by counting the rounded cells as described in 'Materials and methods' . Figure 2 shows the time course of the rounding after exposing the cells to different doses of C2T . The cells rounded gradually after exposure to the toxin, although the initial rate varied depending on the toxin dose . The rounding reached 100% within 2 h with the toxin doses of 40 and 1000 ng per well, whereas those cells exposed to lower toxin doses did not round even after incubation for 8 h . At 8 ng toxin per well rounding reached 100% after 18 h incubation (data not shown in the figure) . Rounding activity of components l and // of C2T As we have reported previously, either component I or II alone is not lethal to mice and the lethal activity of C2T, a mixture of components I and II, is enhanced by trypsinization . 5 Figure 3 shows the rounding activities of components I and II alone and the mixtures of the two components before and after trypsinization . Each component alone before and after trypsinization and the mixture of untrypsinized components I and II showed very low activity, whereas the mixture of untrypsinized component I and trypsinized component II induced rounding of the cells at a very low toxin dose . The 50% rounding activity of C2T, a mixture of untrypsinized component I and trypsinized component II, was 400 times higher than that of a mixture of untrypsinized components I and II . Effect of C2T on macromolecular synthesis To examine if the marked morphological changes of CMK cells with C2T are accompanied by inhibition of macromolecular syntheses, the cells were incubated with [ 14 C]leucine, [ 3 H]Uridine or [ 3 H]thymidine in the presence of 120 ng C2T, and the incorporation of the radioactive compounds was measured as a function of incubation time . During 3 h incubation, no inhibitory effect of C2T on the incorporation



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Fig . 3 . Rounding response of CMK cells as a function of concentrations of two components of C2T before and after trypsinization . The CMK cells in 98-well plate were incubated with the toxin component for 18 h and the rounding was determined as described in 'Materials and methods' . Each point represents the mean ± the standard error of the mean for four values . Symbols: 0, a mixture of untrypsinized component I and trypsinized component 11 ; Q, a mixture of untrypsinized components I and 11 ; A, trypsinized component 11 ; Q, untrypsinized component II and N, untrypsinized component I .

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Fig . 4. Time courses of viability and detachment of CMK cells exposed to C2T . The cells in culture dishes were incubated with 357 ng of C2T and the viability and detachment of the cells were determined as described in 'Materials and methods' . Each point represents the mean +the standard error of the mean for four values . Symbols: A, viability ; A, control for viability ; 40, detachment and 0, control for detachment .

rates of the compounds into the cells was observed, while the cells rounded completely after 2 h incubation with the toxin dose used (data not shown) . This indicates that C2T has no effect on macromolecular synthesis from these precursors under the experimental conditions used . Time courses of viability and detachment of the cells Figure 4 shows the time courses of the viability and the detachment of the cells with a toxin dose of 375 ng per culture dish . The viability decreased after 16 h incubation and reached zero percent at 48 h incubation after exposure to C2T, while complete rounding of the cells occurred at 4 h incubation . These changes in viability of CMK cells were inversely parallel to the detachment of the cells .

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Fig . 5 . Immunofluorescence labeling of CMK cells exposed to two components of C2T . The cells in Slide-Chamber were incubated at 37 ° C for 5 min with : (a), 2 µg trypsinized component II ; (b), 2 µg untrypsinized component II and (c), a mixture of 1 pg component I and 2 µg trypsinized component II . The cells were then reacted with : (a) and (b), IgG for component II and (c), IgG for component I, and stained as described in 'Materials and methods' . Magnification of all photographs is the same . The bar represents 5 pm .

Immunofluorescence labeling of CMK cells

To examine the binding of two components of C2T, the cells were incubated with the toxin components and stained by immunofluorescence . When the cells were exposed to component II, either trypsinized or untrypsinized, a particulate form of fluorescence labeling was observed on the cell surface (Fig . 5 (a) and (b) ) . However, the size and the distribution of the fluorescence labeling was different depending on the pretreatment of component II ; larger and more irregular for the labeling of trypsinized component II . When the cells were exposed to component I alone, no fluorescence labeling was observed (data not shown) . A fluorescence labeling of component I could be seen after staining with the specific antibody to component I of cells incubated with component I in the presence of trypsinized component II, but not in the presence of untrypsinized component II (Fig . 5 (c) ) . The size and distribution of fluorescence labeling of component I was very similar to that of trypsinized component II, both of which appeared to aggregate on the cell surface . In no case was fluorescence labeling observed when either the toxin component or the specific IgG for each component was omitted for the reaction .

Discussion C2T has various biological activities, which are elicited by a combination of untrypsinized component I and trypsinized component II but not by the combination of untrypsinized components I and II ; the components individually have little activity .` The present studies demonstrated that the mixture of untrypsinized component I and trypsinized component II induced high rounding activity in tissue-cultured cells, whereas the components alone and the mixture of untrypsinized components I and II caused little activity . These results agree well with the previous findings described above . The rounding activity induced by combination of untrypsinized component I and trypsinized component II was 400 times higher than that of untrypsinized



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components I and II . However, the mixture of untrypsinized components I and II was not completely inactive . In addition, the dose response curve represented as the rounding activity vs log dose of the toxin represented a straight line for the mixture of untrypsinized component I and trypsinized component II, whereas that for the mixture of untrypsinized components I and II did not . This seems to be due to the activation of component II with trypsin-like protease(s) of the cells, which resulted in higher rounding activity with the higher toxin doses . The activity of the mixture of untrypsinized components I and I I is also due to the assay for C2T using tissue-cultured cells, which could detect a wide range of concentration of the toxin starting from ng levels . The trypsinized component II alone, though at relatively high dose, also caused some rounding activity . Previous studies have shown that the biological activity of C2T increases proportionally to the increasing amount of component II when mixed with a constant amount of component 1 .7 This indicates that the activity of C2T is dependent mostly on the concentration of component II but not of component I . Therefore, if a trace amount of component I, which could not be detectable by usual methods to check the purity of the protein, is present as a contaminant in the preparation of component II, rounding of the cultured cells could be induced by trypsinizing component II, as the assay method used in the present study was highly sensitive to C2T . In fact, the rounding activity of trypsinized component II was neutralized with anti-component I IgG (data not shown) . The rounding activity induced with untrypsinized component II is also due to the same reason . Botulinum C2T, in addition to lethality, has novel biological activities, including enterotoxic and vascular permeability activities . s•' The response of the target cells for these activities is initiated by the binding of two components of the toxin to the cell surface . Previous studies have shown that the binding of component II to the cell membrane introduces a binding site for component I, which appears to be internalized into the cells ." The present study also demonstrated that immunofluorescence labeling of component I on the cell surface membrane was observed only in the presence of trypsinized component II but not of untrypsinized component II, although the labeling of component I I alone, either trypsinized or untrypsinized, was observed . These results are consistent with those reported previously with the isolated cells and the brush borders of mouse intestine ." However, the distribution of components I and II bound to the membrane of the tissue-cultured cells was quite different from the binding to the isolated intestinal cells and the brush borders . The fluorescence labeling of the tissue-cultured cell membrane was particular, whereas that of the intestinal cells and the brush borders was evenly distributed on the surface, although the fluorescence labeling on the intestinal cells was confined to the brush border of the cells ." This difference between the binding of the two components of C2T to the isolated intestinal cells and to the tissue-cultured cells is possibly due to the fluidity of the cell membrane, which could be lost in the isolated intestinal cells and the brush borders . The similarity in distribution pattern of the fluorescence labeling between the trypsinized component II and the component I in the presence of trypsinized component II suggests either that component I binds to a site modified by trypsinized component II or that component I binds to the trypsinized component II bound to the cell membrane . The difference in the distribution pattern of the fluorescence labeling of untrypsinized and trypsinized component IIs suggests that untrypsinized component II binds to a site different from that for trypsinized component II . C2T elaborated by C. botulinum types C and D is cytopathogenic for various mammalian cell lines .' A common and characteristic change in the cell morphology of these cell lines is rounding . The present study also demonstrated that the cells intoxicated with CT were not 'dead' for a certain period after exposure to the toxin

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when examined by the classical viability tests based on vital staining with trypan blue . This is consistent with the finding that macromolecular syntheses in the tissue-cultured cells were not damaged with C2T, even after the cells were completely rounded . Previously, we have reported that component I of C2T ADP-ribosylates the cytoplasmic actin and that the intracellular ADP-ribosylation of the actin is correlated with the rounding . 12 This enzymatic modification of intracellular actin with C2T possibly causes an irreversible change in the function of the microfilaments of the cells, resulting in rounding of the cells without an inhibition in macromolecular syntheses . Thus, we suggest as the mode of action of C2T the following : (1) trypsinized component II binds to the cell membrane, (2) and introduces a binding site for component I ; (3) component I enters the cells, (4) ADP-ribosylates the cytoskeletal actin, and (5) this causes cytopathogenic effect ." Materials and methods Cell culture. CMK cells obtained from National Institute of Health, Tokyo, were grown in Eagle's minimal essential medium (Nissui Pharmaceutical Co ., Tokyo) containing 0 .03% glutamine and 0 .1% sodium bicarbonate (MEM) supplemented with 10% fetal calf serum (MEM-FCS) . Cultures were incubated at 37 ° C in a humid atmosphere containing 5% CO 2 and 95% air and used for test within 2 to 4 days . Assay for rounding activity. Cells obtained as the monolayer in Corning tissue culture flasks (# 25100) were detached by trypsinization at 37°C for 10 min with 0 .25% trypsin in PBS and were suspended in MEM-FCS . Tissue culture plates of 98-wells (Corning, #25860) were seeded with 4x 10 4 cells per well . After incubation at 37 ° C for 24 h, the cells were washed with 150 mm NaCl, 10 mm K-Na phosphate buffer, pH 7 .3 (PBS), and 100 µl of C2T diluted with MEM was added to each well . The plates were incubated for the periods indicated . For counting the rounded cells, the cells were stained by adding 50 µl of 0 .05% methylene blue in 0 .1 M Na 2 CO 3 NaHC0 3buffer, pH 10 .0, into each well and by incubating at room temperature for 10 min . The rounded and unrounded cells in randomly selected four fields of 0 .08 mm 2 under micrometer per well were counted with an inverted phase-contrast microscope (Nikon, type TMS, Tokyo) . The percentage of the rounding was calculated from the average of the four countings . Assays for viability and detachment. To determine the percentages of detached and viable cells, plastic dishes of 35 mm in diameter (Corning, #25000) were seeded with 1 ml of cell suspension containing 4 .0x10 5 cells . After incubation at 37°C for 24 h, the cells were washed with PBS and incubated with 375 ng of C2T in 1 ml of MEM for the periods indicated . The cells detached from the dishes were removed and counted in a BOrker-Turk chamber, while the attached cells were removed by trypsinization at 37°C for 10 min with 0 .25% trypsin in PBS, suspended in 1 ml of PBS, and counted in the chamber . The viability of the cells was determined by removing the whole cells with 0 .25% trypsin in PBS as described above and by counting the cells stained and unstained with 0 .25% trypan blue at room temperature . The percentages of the detachment and the viability were calculated from the average of the data of four dishes . Macromolecular synthesis. The effects of C2T on protein, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses were examined by using [U- 14 C]leucine (355 Ci/mmol, Amersham Japan, Tokyo), [ 3 H)uridine (28 .4 Ci/mmol, Amersham Japan) and [methyl5 H]thymidine (41 Ci/mmol, Amersham Japan), respectively . For protein and DNA syntheses, monolayers of CMK cells were established in a tissue culture plate of 98 wells as described above . Immediately before the experiment, the monolayer in each well was washed with PBS and incubated at 37°C in 100 ftl of MEM without leucine but with 0 .1% bovine serum albumin, 120 ng C2T and 0 .2 µCi of [U- 14 C]leucine or 0 .3 µCi [methyl3 H)thymidine . The incubation was stopped by cooling the plate on ice and washing the cells with PBS . The cells were solubilized in 50 µl KOH for 30 min at room temperature . After the addition of 50 µl of 0 .15% BSA as carrier protein, 75 µl of the solubilized cells was transferred into test tube and mixed with 500µl of ice-cold 15% trichloroacetic acid (TCA) . After standing in ice-bath for 30 min, the TCA-precipitates were collected by filtration on glass fiber filters



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and washed with 600µl of ice-cold 15% TCA . The precipitates on filter were solubilized in 300 yl NCS (Amersham Japan) in vials . The radioactivity was determined by using ACSI I scintillation cocktail (Amersham Japan) . For RNA synthesis, cells were treated as described above, except that the monolayer of CMK cells in each well was incubated with 100 yl MEM containing 120 ng C2T, 0 .1 µCi of [ 3 H]Uridine, and 0 .1% BSA . After incubation at 37°C, the cells were washed with PBS, solubilized in 50 µl 0 .1 % sodium dodecyl sulfate and treated as described above . Preparation of C2T . Components I and II of C2T were purified from the culture of C . botulinum type C strain 92-13 as described previously .' C2T in the present study was defined as containing untrypsinized component I and trypsinized component II in the ratio of 1 : 2 on a protein basis . Trypsinized component II was prepared as described previously ." Preparation of specific immunoglobulins for components l and ll . Anti-component I and -component II sera were prepared as described previously .' Specific antibodies for components I and II were purified by affinity chromatography as described previously .' The immunoglobulins were dialyzed against PBS and stored at -20°C until used . Immunofluorescence staining. CMK cells in MEM-FCS were seeded in a Slide-Chamber (#4808, Lab-Tek Products, Naperville, III .) with 4 .0x10 ° cells per well and incubated for 24 h . The cells were washed with PBS containing 0 .1% bovine serum albumin (PBS-BSA) on ice, and incubated with the toxin components in PBS-BSA at 37°C for 5 min . The cells were washed again with PBS-BSA and the immunoglobulin specific for the toxin component used was added . The final concentrations of the immunoglobulin for components I and II were 40 and 20 µg/ml, respectively . After incubation for 30 min on ice, the cells were washed with PBS-BSA and reacted with dichlorotriazinylaminofluorescein-labeled antibody to rabbit immunoglobulin G (Kirlegaard & Perry Laboratories, Inc ., Gaithersburg, Md .) for 30 min in an ice bath . Finally, unreacted fluorescein-labeled IgG was removed by washing with PBS-BSA and the cells were observed and photographed in an incident fluorescence microscope (Model EPI, Nikon, Tokyo) . Protein determination. Protein concentrations were determined by the method of Lowry et a/., with bovine serum albumin as the standard ."

References 1 . Mason JH, Robinson EM . The antigenic components of the toxins of Cl. botulinum types C and D . Onderstepoort J Vet Sci Anim Ind 1935 ; 5 : 65-75 . 2 . Bulatova TI, Matveev KI, Samsonova VS . Biological characteristics of CI. botulinum type C strains isolated from minks in the U .S .S .R . In : Ingram M, Roberts TA, eds. Botulism 1966 . London : Chapman and Hall, 1967 ; 391-9 . 3 . Eklund MW, Poysky FT . Activation of a toxic component of Clostridium botulinum types C and D by trypsin . Appl Microbiol 1972 ; 24 :108-13 . 4 . Jansen BC . The toxic antigenic factors produced by Clostridium botulinum types C and D . Onderstepoort J Vet Res 1971 ; 38 : 93-8 . 5 . Ohishi I, Iwasaki M, Sakaguchi G . Purification and characterization of two components of botulinum C 2 toxin . Infect Immun 1980; 30 : 668-73 . 6 . Ohishi I . Response of mouse intestinal loop to botulinum C 2 toxin : enterotoxic activity induced by cooperation of nonlinked protein components . Infect Immun 1983 ; 40: 691-5 . 7 . Ohishi I, Iwasaki M, Sakaguchi G . Vascular permeability activity of botulinum C 2 toxin elicited by cooperation of two protein components . Infect Immun 1980 ; 31 : 890-5. 8. Simpson LL . A comparison of the pharmacological properties of Clostridium botulinum type C, and type C2 toxins . J Pharmacol Exp Ther 1984 ; 230 : 691-701 . 9 . Ohishi I, Miyake M, Ogura K, Nakamura S . Cytopathic effect of botulinum C 2 toxin on tissue-culture cells . FEMS Microbiol Lett 1984 ; 23 : 281-4 . 10 . Lowry OH, Rosebrough NJ, Farr AL, Randall RJ . Protein measurements with the Folin phenol reagent . J Biol Chem 1951 ; 193 : 265-75 . 11 . Ohishi I, Miyake M . Binding of the two components of C 2 toxin to epithelial cells and brush borders of mouse intestine. Infect Immun 1985; 48: 769-75 . 12 . Ohishi I, Tsuyama S . ADP-ribosylation of nonmuscle actin with component I of C2 toxin . Biochem Biophys Res Commun 1986 ; 136 : 802-6 . 13 . Ohishi I . NAD-glycohydrolase activity of botulinum C 2 toxin : a possible role of component I in the mode of action of the toxin . J Biochem 1986; 100 : 407-13 .