Volume 119, Issue 1, Pages 139-150 (July 2000) Clostridium difficile toxin A causes early damage to mitochondria in cultured cells  D. He, S.J. Hagen, C. Pothoulakis, M. Chen, N.D. Medina, M. Warny, J.T. LaMont  Gastroenterology  Volume 119, Issue 1, Pages 139-150 (July 2000) DOI: 10.1053/gast.2000.8526 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Confocal microscopy of toxin A binding to nonpermeabilized CHO cell monolayers. Confluent CHO cell monolayers growing on cover slides (105 cells) were incubated with 5 μg/mL of purified toxin A at 4°C to inhibit internalization. After incubation, cells were fixed in 4% paraformaldehyde and exposed to a goat polyclonal anti–toxin A IgG (1:100), followed by incubation with FITC-labeled antigoat IgG (1:80), as described in Materials and Methods. (A) Note the presence of staining on the cell surface at 2-5 minutes and more intense staining at 15 minutes. Omission of anti–toxin A IgG (Ctrl 1) or exposure of CHO cells to buffer alone (Ctrl 2) resulted in complete disappearance of fluorescence staining. (B) Statistical analysis of the relative fluorescent intensity of 6-10 cells randomly chosen from 4-5 different high-power fields (100×) of the images shown in A shows significantly increased fluorescence intensity 5, 10, and 15 minutes after toxin A exposure, compared with fluorescent intensity of control monolayers. *P < 0.05. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Confocal microscopy of toxin A binding to nonpermeabilized CHO cell monolayers. Confluent CHO cell monolayers growing on cover slides (105 cells) were incubated with 5 μg/mL of purified toxin A at 4°C to inhibit internalization. After incubation, cells were fixed in 4% paraformaldehyde and exposed to a goat polyclonal anti–toxin A IgG (1:100), followed by incubation with FITC-labeled antigoat IgG (1:80), as described in Materials and Methods. (A) Note the presence of staining on the cell surface at 2-5 minutes and more intense staining at 15 minutes. Omission of anti–toxin A IgG (Ctrl 1) or exposure of CHO cells to buffer alone (Ctrl 2) resulted in complete disappearance of fluorescence staining. (B) Statistical analysis of the relative fluorescent intensity of 6-10 cells randomly chosen from 4-5 different high-power fields (100×) of the images shown in A shows significantly increased fluorescence intensity 5, 10, and 15 minutes after toxin A exposure, compared with fluorescent intensity of control monolayers. *P < 0.05. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Colocalization of toxin A and GRP 75 in CHO cells. CHO cell monolayers growing in cover slides (105 cells) were exposed to 5 μg/mL toxin A for 5 and 10 minutes. Cells were then washed in PBS, fixed, and permeabilized with saponin as described in Materials and Methods. Cells were stained with a polyclonal goat anti–toxin A antibody and a monoclonal antibody directed against the mitochondrial protein GRP 75. Cells were then washed and incubated with (B and E) Texas Red–conjugated antigoat IgG and (A and D) FITC-labeled antimouse IgG and examined by confocal microscopy. Toxin A (red) can be visualized within the cells after 10 and 15 minutes, and GRP 75–positive signal is present in all cells. (C and F) Merged images show colocalization (arrows) of toxin A with the mitochondrial protein GRP 75 in some cells (yellow fluorescence). Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Time-course effects of toxin A on ATP concentration and cell rounding. Confluent CHO cell monolayers were exposed to 5 μg/mL toxin A (■) or buffer (●) at 37°C. At the indicated time points, medium was aspirated and cells were processed for measurements of cellular ATP concentration using the luciferase/luciferin reaction, as described in Materials and Methods. Cell rounding (▵, expressed as percentage of 200 cells) was estimated in separate CHO cell monolayers exposed to toxin A under the same conditions. Results are expressed as mean ± SEM of 4 dishes per group and are representative of 3 separate experiments. Toxin A caused a dramatic decrease in ATP concentration within 15 minutes of exposure before the onset of cell rounding at 15-20 minutes. (B) Time-course effect of toxin A on Rho A glucosylation. Confluent CHO cell monolayers were exposed to 5 μg/mL toxin A, and the extent of Rho A glucosylation was determined by the C3-mediated ADP ribosylation assay described in Materials and Methods. Data are representative of 3 independent experiments. Toxin A–induced Rho A glucosylation was evident after 30 minutes of toxin exposure. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Time-course effects of toxin A on ATP concentration and cell rounding. Confluent CHO cell monolayers were exposed to 5 μg/mL toxin A (■) or buffer (●) at 37°C. At the indicated time points, medium was aspirated and cells were processed for measurements of cellular ATP concentration using the luciferase/luciferin reaction, as described in Materials and Methods. Cell rounding (▵, expressed as percentage of 200 cells) was estimated in separate CHO cell monolayers exposed to toxin A under the same conditions. Results are expressed as mean ± SEM of 4 dishes per group and are representative of 3 separate experiments. Toxin A caused a dramatic decrease in ATP concentration within 15 minutes of exposure before the onset of cell rounding at 15-20 minutes. (B) Time-course effect of toxin A on Rho A glucosylation. Confluent CHO cell monolayers were exposed to 5 μg/mL toxin A, and the extent of Rho A glucosylation was determined by the C3-mediated ADP ribosylation assay described in Materials and Methods. Data are representative of 3 independent experiments. Toxin A–induced Rho A glucosylation was evident after 30 minutes of toxin exposure. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of toxin A on mitochondrial membrane potential. Confluent CHO cell monolayers were exposed to 5 μg/mL toxin A at 37°C. At the indicated time points, cells were trypsinized and incubated with fluorescent rhodamine 123, a marker of electrochemical potential of mitochondrial membranes. Rhodamine 123 fluorescence levels were measured in the cells by fluorescence-activated cell sorter analysis. Data are representative of 3 independent experiments. Rhodamine 123 fluorescence was reduced 15 minutes after toxin A exposure and further reduced during the 2-hour incubation period. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of toxin A on ROIs. CHO cells were pretreated with DHR 123, a specific marker for ROIs, and exposed to either (A) buffer alone or (B–D) buffer-containing toxin A for various time points. Cells were then fixed in 4% paraformaldehyde and then examined by confocal microscopy. Increased fluorescence signal is present 5 minutes after toxin A exposure (B) compared with control, buffer-exposed monolayers (A). (C) Ten and (D) 15 minutes after toxin A exposure, the signal remains high. (E) Analysis of the relative fluorescent intensity of 6-10 cells randomly chosen from 4-5 different high-power fields (100×) of the images in A shows a significant increase in oxygen radicals 5 minutes after toxin A exposure, compared with control. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of toxin A on ROIs. CHO cells were pretreated with DHR 123, a specific marker for ROIs, and exposed to either (A) buffer alone or (B–D) buffer-containing toxin A for various time points. Cells were then fixed in 4% paraformaldehyde and then examined by confocal microscopy. Increased fluorescence signal is present 5 minutes after toxin A exposure (B) compared with control, buffer-exposed monolayers (A). (C) Ten and (D) 15 minutes after toxin A exposure, the signal remains high. (E) Analysis of the relative fluorescent intensity of 6-10 cells randomly chosen from 4-5 different high-power fields (100×) of the images in A shows a significant increase in oxygen radicals 5 minutes after toxin A exposure, compared with control. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of antioxidants on toxin A–induced generation of ROIs. CHO cell monolayers loaded with DHR 123 were preincubated (30 minutes at 37°C) with either medium alone or medium containing 200 μmol/L of the antioxidants BHA or BHT. Cells were then exposed to toxin A and, at the indicated time intervals and fluorescence levels, were measured by fluorescence-activated cell sorter analysis. Data are representative of 3 independent experiments. Toxin A exposure increased the relative DHR 123 fluorescence intensity after 10, 15, and 30 minutes; this increase was inhibited by BHA and BHT at all time points. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of toxin A on mitochondrial cytochrome c levels. Isolated CHO cell mitochondria or cytoplasmic preparations were exposed to either buffer or 5 μg/mL toxin A. At the indicated time points, proteins from isolated mitochondria or cytosolic preparations were separated in 12% SDS-PAGE, transferred to membranes, and blotted with an antibody directed against cytochrome c. Cytochrome c was markedly reduced in cell mitochondria 30 minutes after toxin A exposure and absent after 60 and 120 minutes. In whole cells exposed to toxin, cytochrome c is absent in cytoplasm from control preparations but present 30 minutes after toxin A exposure. Data are representative of 3 independent experiments. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Electron microscopy of toxin A–exposed CHO cells. Confluent CHO cell monolayers exposed to either (A) buffer or (B) buffer containing 5 μg/mL toxin A for 15 minutes were examined by electron microscopy. The apical surface of CHO cells contained many mitochondria (M; A and B). In control cells, the mitochondrial matrix was electron dense, and each mitochondrion possessed many electron-lucent cristae (arrows, A). In toxin A–exposed cells, the mitochondria showed significant swelling, the mitochondrial matrix was less dense, and cristae were not readily apparent. N, nucleus. Original magnification 25,847×; bar = 1 μm. (C) Statistical analysis of mitochondrial diameter of electron microscopy images measured with an ocular micrometer was performed as described in Materials and Methods. The diameter of the mitochondria was significantly enlarged in toxin A–exposed (15 minutes) vs. buffer-exposed cells. *P < 0.00001 vs. buffer. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Electron microscopy of toxin A–exposed CHO cells. Confluent CHO cell monolayers exposed to either (A) buffer or (B) buffer containing 5 μg/mL toxin A for 15 minutes were examined by electron microscopy. The apical surface of CHO cells contained many mitochondria (M; A and B). In control cells, the mitochondrial matrix was electron dense, and each mitochondrion possessed many electron-lucent cristae (arrows, A). In toxin A–exposed cells, the mitochondria showed significant swelling, the mitochondrial matrix was less dense, and cristae were not readily apparent. N, nucleus. Original magnification 25,847×; bar = 1 μm. (C) Statistical analysis of mitochondrial diameter of electron microscopy images measured with an ocular micrometer was performed as described in Materials and Methods. The diameter of the mitochondria was significantly enlarged in toxin A–exposed (15 minutes) vs. buffer-exposed cells. *P < 0.00001 vs. buffer. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Electron microscopy of toxin A–exposed CHO cells. Confluent CHO cell monolayers exposed to either (A) buffer or (B) buffer containing 5 μg/mL toxin A for 15 minutes were examined by electron microscopy. The apical surface of CHO cells contained many mitochondria (M; A and B). In control cells, the mitochondrial matrix was electron dense, and each mitochondrion possessed many electron-lucent cristae (arrows, A). In toxin A–exposed cells, the mitochondria showed significant swelling, the mitochondrial matrix was less dense, and cristae were not readily apparent. N, nucleus. Original magnification 25,847×; bar = 1 μm. (C) Statistical analysis of mitochondrial diameter of electron microscopy images measured with an ocular micrometer was performed as described in Materials and Methods. The diameter of the mitochondria was significantly enlarged in toxin A–exposed (15 minutes) vs. buffer-exposed cells. *P < 0.00001 vs. buffer. Gastroenterology 2000 119, 139-150DOI: (10.1053/gast.2000.8526) Copyright © 2000 American Gastroenterological Association Terms and Conditions