Figure 1.
The actin-ADP-ribosylating toxins CDT and C2 toxin induce formation of cellular processes.
(A) Subconfluent Caco-2 cells were treated with 20 ng/ml CDTa and 40 ng/ml CDTb. After 1 h the formation of cellular processes starts. Length and number of processes increases over time. In each panel the incubation time (h) is indicated. Scale bar represents 20 µm. (B) Quantification of formation of protrusions. The lengths of all processes of cells were summated every 15 min after addition of the toxins and normalized by the respective section of the cell perimeter. Cells were incubated with the indicated toxin concentrations. 20 ng/ml CDTa +40 ng/ml CDTb and 5 ng/ml CDTa +10 ng/ml CDTb induced the strongest formation of protrusions. Data are given +/−SEM from n≥3 movies and at least 3 cells per movie. (C) Scanning electron microscopy of Caco-2 (left and middle panel) cells treated with 20 ng/ml CDTa and 40 ng/ml CDTb or HT-29 cells (right panel) treated with 250 ng/ml C2I and 500 ng/ml C2II for 2 h. Controls are from Caco-2 cells. Control cells show microvilli at the cell surface. CDT or C2-treated cells show a strong formation of protrusions. Arrow: corkscrew shaped protrusion with leaf-like extension. Scale bar in upper row represents 10 µm. Scale bar in lower row represents 2 µm.
Figure 2.
Toxin-induced disruption of the actin cytoskeleton and formation of microtubule-based protrusions.
(A) Indirect immunofluorescence of α-tubulin (green) and actin-staining by TRITC-conjugated phalloidin (red) in Caco-2 cells. The nucleus was stained by DAPI (blue). CDT causes increasing disruption of the actin cytoskeleton and a concomitant increased formation of microtubule-based protrusions over time. Cells were treated with 20 ng/ml CDTa and 40 ng/ml CDTb for the indicated times. White dotted line in the right panels indicates cell border. The line was delineated according to a phase contrast picture. White square in lower panel is magnified below. Scale bar represents 20 µm. (B) In vitro 32P-ADP-ribosylation of actin by the enzyme component C2I after intracellular ADP-ribosylation by CDT. Caco-2 cells were treated with 20 ng/ml CDTa and 40 ng/ml CDTb for the indicated times. Cells were lysed and unmodified actin was subsequently modified by C2I in the presence of [32P]NAD. The 32P-ADP-ribosylated actin was analyzed by SDS-PAGE and phosphorimaging. C2I-catalyzed 32P-ADP-ribosylation of actin in control cells was taken as 100%. Data in columns are given from 3 independent experiments (+/−SEM). Below, the autoradiogram of an ADP-ribosylation from a representative experiment is shown.
Figure 3.
Influence of CDT on EB1 and CLIP-170 at microtubule plus-ends.
(A) Indirect immunofluorescence of EB1 (red) and α-tubulin (green) in Caco-2 cells. Control cells show short comet-like EB1 associations at the tip of microtubules. After CDT treatment, EB1 comets are elongated and also present at the tip of microtubule-based protrusions. Cells were treated with 20 ng/ml CDTa and 40 ng/ml CDTb and fixed after 2 h. Areas in white squares show magnifications. Scale bar represents 20 µm. (B) Indirect immunofluorescence of CLIP-170 (green) and EB1 (red) in Caco-2 cells. The nucleus was stained by DAPI (blue). EB1 and CLIP-170 comets are elongated after CDT treatment. Cells were treated with 20 ng/ml CDTa and 40 ng/ml CDTb and fixed after 2 h. Areas in white squares show magnifications. Scale bar represents 20 µm. (C) Quantification of the EB1 and CLIP-170 comet lengths. The figure shows a quantification of the EB1 and CLIP-170 comet length from pictures stained as in Figure 3B. After CDT treatment, both +TIPs have an increased microtubule tip association. +TIPs were measured by Metamorph software. Data are given +/−SEM (*** = p<0.001, n = 100). CDT concentrations were as above.
Figure 4.
Influence of CDT on microtubule growth.
Caco-2 cells transfected with EB3-GFP, were monitored in fluorescence time-lapse microscopy. Cells were recorded 0, 1 and 2 h after treatment with 20 ng/ml CDTa and 40 ng/ml CDTb. (A) Control images of EB3-GFP transfected cells show microtubules losing their EB3-comets as they approach the cell cortex. Asterisk: EB3-GFP comet approaching the cell cortex (white dotted line); cross: same comet during loss of EB3 decoration. (B) Images of CDT-treated (2 h) EB3-GFP transfected cells. Microtubules approaching the cell cortex do not lose EB3-association. Polymerizing microtubule forms initial protrusion and other microtubules follow the same track. Asterisk: EB3-GFP comet forming a protrusion. Arrow: a second EB3-GFP comet following the first into the protrusion. Scale bar in A and B represents 5 µm. Note different time scales in A and B. (C) Quantification of microtubule polymerization rate. After CDT treatment the polymerization rate of microtubules decreases. Growing microtubules (EB3-GFP comets) were tracked over 10 sec at 0 (control), 1 and 2 h after CDT-treatment. Data from 100 comets (5 experiments and 7 different cells) are given +/−SEM (*** = p<0.001). (D) Quantification of microtubule time in growth. After CDT treatment microtubules stay longer in their growth phase. The life time of 30 EB3-GFP comets from appearance to fading was measured (data are given +/−SEM (*** = p<0.001) from ≥5 cells and ≥4 independent experiments).
Figure 5.
Influence of CDT on CLASP2 and ACF7 localization.
(A) Indirect immunofluorescence of CLASP2 (red) and α-tubulin (green) in Caco-2 cells. The nucleus was stained by DAPI (blue). CLASP2 is translocated from the tip of microtubules that reached the cell cortex to filamentous structures in the cell interior. Cells were treated with 20 ng/ml CDTa and 40 ng/ml CDTb and fixed after 2 h. Scale bar represents 20 µm. (B) Indirect immunofluorescence of ACF7 (red) and α-tubulin (green) in Caco-2 cells. The nucleus was stained by DAPI (blue). ACF7 is translocated from the cell cortex to the microtubule lattice in the cell interior. Cells were treated with 20 ng/ml CDTa and 40 ng/ml CDTb and fixed after 2 h. White squares represent magnifications. Scale bar represents 20 µm.
Figure 6.
Influence of the Rho-inactivating toxin B and C3 toxin on CDT-induced protrusion formation.
(A) Series of DIC time-lapse images of cells treated with 300 ng/ml toxin B at time point 0 h. After 3 h, 20 ng/ml CDTa and 40 ng/ml CDTb were added. Incubation was continued for 6 h. CDT induced the formation of microtubule-based protrusions after toxin B intoxication. Scale bar represents 20 µm. (B) Series of DIC time-lapse images of Caco-2 cells treated with 300 ng/ml C3 fusion toxin at time point 0 h. After 3 h, 20 ng/ml CDTa and 40 ng/ml CDTb were added. The incubation was continued for 6 h. CDT induced the formation of microtubule-based protrusions after C3 intoxication. Scale bar represents 20 µm.
Figure 7.
CDT treatment increases the adherence of C. difficile.
(A) Indirect immunofluorescence of C. difficile surface proteins (green) and α-tubulin (red) on Caco-2 cells with adhered bacteria. The nucleus was stained by DAPI (blue). Cells were treated without and with 20 ng/ml CDTa and 40 ng/ml CDTb. After 1 h, 25 µl of an inoculum of C. difficile VPI 10463 (over night culture, OD 1.2, 2.5×108 bacteria/ml) was added to a 24-well dish of confluent Caco-2 cells on a coverslip. After 90 min cells were washed and fixed. CDT intoxication increased the adherence of C. difficile VPI 10463. Scale bar represents 20 µm. (B) CDT also increased the adherence of C. difficile VPI 10463 under anaerobic conditions. 100 µl of a C. difficile over night culture (OD 1.2, 2.5×108 bacteria/ml) was added to a 3 cm dish of confluent Caco-2 cells treated without or with 20 ng/ml CDTa and 40 ng/ml CDTb. The cells were incubated under anaerobic conditions for 4 h. After incubation the cells were washed, scraped off and plated. After 2 days CFUs were counted. Adherence after CDT treatment was quantified as percent of adherence on untreated cells. (C) Scanning electron microscopy of Caco-2 cells. Cells were treated as in Figure 7A. Scale bar represents 5 µm. After CDT treatment Clostridia were caught and wrapped in protrusions (arrows) (D) Time-lapse images of Caco-2 cells incubated together with the indicated strains of C. difficile for the indicated times. Cells were incubated for 5 h under anaerobic conditions (100% N2) and subsequently incubated under aerobic conditions (6.5% CO2 and 9% O2) for further 3 h. CDTb was neutralized by addition of anti-Iota B antibody (1∶50) at time point 0 h (right panel). CDT-producing Clostridia induced the formation of protrusions. A CDT-neutralizing antibody inhibited the formation of protrusions by CDT-producing Clostridia. Scale bar represents 20 µm.