<i>C</i>. <i>burnetti</i> internalization is inhibited by overexpression of the dominant negative mutants of Rho GTPases.

<p>(A) HeLa cells were transfected with pEGFP (panels a-e), pEGFP-RhoA N19 (panels f-j), pEGFP-Cdc42 N17 (panels k-o), or pEGFP-Rac1 N17 (panels p-t). Cells were infected for 4 h at 37°C with <i>C</i>. <i>burnetii</i> and subsequently fixed and processed for immunofluorescence to determine the levels of <i>C</i>. <i>burnetii</i> internalization as described in Materials and Methods. Cells were analyzed by confocal microscopy. Representative micrographs are presented. As indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145211#pone.0145211.g001" target="_blank">Fig 1</a>, extracellular and total bacteria were stained in white pseudo color (panels c, h, m, and r) and red pseudo color (panels b, g, l, and q), respectively. 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). Bars scale: 5 μm. (B) Quantification of <i>C</i>. <i>burnetii</i> internalized by transfected HeLa cells. (C) Quantification of total <i>C</i>. <i>burnetii</i> associated to HeLa cells. Between 40 and 60 cells and between 400 and 600 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 EGFP control (one-way ANOVA and Dunnett's <i>post hoc</i> test). ns: non-significant differences between groups (p > 0.05).