<i>Clostridium difficile</i> toxin B diminishes the internalization of <i>C</i>. <i>burnetii</i> by HeLa cells.

<p>(A) HeLa cells were infected with <i>C</i>. <i>burnetii</i> for 4 h at 37°C in the presence of 0.05% DMSO (control, panels a-e) or with different concentrations of <i>Clostridium difficile</i> toxin B (panels f-t). Cells were fixed and processed for indirect immunofluorescence to determine <i>C</i>. <i>burnetii</i> internalization and F-actin distribution as described in Materials and Methods. Cells were analyzed by confocal microscopy. Micrographs of representative cells are shown. Cells were incubated sequentially with an antibody against <i>C</i>. <i>burnetii</i> and an appropriate secondary antibody conjugated to Cy5 (white pseudo color) under non-permeabilizing conditions. Under this condition, extracellular bacteria were stained in white pseudo color (panels c, h, m, and r). Then, cells were re-incubated with the same anti-<i>C</i>. <i>burnetii</i> antibody and an appropriate secondary antibody conjugated to Cy3 (red pseudo color) under permeabilizing conditions. Under this condition total bacteria were stained in red pseudo color (panels b, g, l, and q). In the merged images (panels d, i, n and s) and the insets of the merged images (panels e, j, o, and t), extracellular <i>C</i>. <i>burnetii</i> is shown in white and red pseudo colors (arrows), while intracellular <i>C</i>. <i>burnetii</i> is shown in red pseudo color (yellow arrowheads). F-actin was labeled with phalloidin-FITC (green, panels a, f, k, and p). Bars scale: 5 μm. (B) Quantification of <i>C</i>. <i>burnetii</i> internalized in control and toxin-treated HeLa cells. (C) Quantification of total <i>C</i>. <i>burnetii</i> associated to control and treated HeLa cells. Between 100 and 120 cells and 1200 and 1600 bacteria were counted in each experiment. Results are expressed as means ± SE of three independent experiments. *p < 0.05, **p < 0.01 compared to the DMSO treatment (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05).