Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria
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.