FIGURE 7.

Putative localization of various BAs in the C. difficile cell. Left: Transmission electron micrographs of C. difficile challenged with the four different bile acids (CA, cholic acid, DCA, deoxycholic acid, CDCA, chenodeoxycholic acid, LCA, lithocholic acid) and of untreated cells, all in exponential growth phase. Morphological changes are highlighted as white arrows. Scale bars, top 1 μm and bottom 200 nm. Middle: Scanning electron micrographs of C. difficile challenged with four different bile acids (CA, DCA, CDCA, and LCA) and of untreated cells, scale bar = 1 μm. Right top and center: Immunofluorescence (IF) of C. difficile challenged with BAs and stained with mouse anti-CA antibody (green), mouse anti-CDCA antibody (green), rabbit anti-DCA antibody (green), rabbit anti-LCA antibody (green) as well as DAPI (blue) and rabbit anti-C. difficile antibody (purple). Due to the same antibody host (rabbit), LCA samples were not counterstained with anti-C. difficile but with Nile red (red) and DCA challenged cells were only co-stained with DAPI. Scale bars = 5 μm. Right bottom: IF of TEM sections of C. difficile challenged with BAs and stained with anti-CA (green) and counterstained with anti-C. difficile (purple) and anti-LCA (green) counterstained with Nile red (red). An IF staining of TEM sections of DCA, CDCA and untreated samples was not possible. Scale bars = 5 μm.