FIG 2.

Surface localization of CpnT within macrophages. (A) Fluorescence microscopy of infected RAW 264.7 macrophages with WT, WT+ CpnT, and WT+ _AxxxECpnT. All bacterial strains carried L5::gfp for fluorescence detection. Macrophages were infected at an MOI of 2 and fixed at 24 h postinfection. Cells were permeabilized with 0.2% Triton X-100 and immuno-labeled with anti-HA antibody (CpnT-HA). Additional dyes were used to label the nuclei (Hoechst) and the actin filaments (Phalloidin). The scale bar represents 10 μm. Images are a representation of five biological replicates. (B) The fluorescence quantification of the anti-HA signal per infected RAW macrophage was calculated using automated image analysis software (CellProfiler). Analysis was performed on one representative experiment out of three independent experiments, with a total number of 40 to 46 infected macrophages. One-way ANOVA and multiple comparisons using Dunnet’s statistical test were performed for statistical significance. ns, P > 0.05; ****, P < 0.0001. (C) Confocal microscopy of macrophages infected with WT+ CpnT strain. Infection conditions and labeling procedure were identical to those for panel A. The scale bar represents 10 μm.