DipA is exposed to the host cytosol during macrophage infection.

<p>(A) Representative confocal micrographs of BMMs infected for 10 h with SchuS4Δ<i>dipA</i>(p<i>dipA-HA</i>), SchuS4(p<i>iglI-HA</i>), or SchuS4(p<i>iglA-HA</i>). Using conditions that permeabilize host plasma membranes but not bacterial membranes (described in Materials and Methods), samples were processed for immunofluorescence labelling of HA-tagged proteins (green) and bacterial LPS (red), and counterstained with DAPI to label DNA (blue). Magnified insets show single channel images of the boxed area. Scale bars, 10 or 2 µm. (B) Quantification of CCF2/AM cleavage in J774A.1 cells that were either uninfected or infected with SchuS4, or SchuS4 expressing C-terminal TEM1 fusions with IglI, IglA or DipA. After 16 h, infected macrophages were loaded with CCF2/AM and analyzed by live cell microscopy for blue fluorescence emission. At least 100 cells were scored per experiment. Data are means ± SD from a representative experiment performed in triplicate out of three independent repeats. Asterisks indicate statistically significant differences compared to uninfected, SchuS4-infected, and SchuS4 expressing IglA-TEM1-infected controls (* <i>P < 0.05</i>, 1-way ANOVA, Tukey’s post-test). (C) Representative fluorescence micrographs of J774A.1 cells that were either uninfected or infected for 16 h with SchuS4, SchuS4 expressing IglI-TEM1, SchuS4 expressing IglA-TEM1, and SchuS4 expressing DipA-TEM1. Cells emitting blue fluorescence indicate delivery of TEM1 β-lactamase fusions to the cytosol and CCF2/AM cleavage. Intact CCF2/AM, indicating the absence of TEM1 β-lactamase activity in the cytosol, results in green fluorescence emission. Scale bar, 50 µm.