FIG. 5.

The conserved residue R149 contributes to the ability of ExoT to disrupt the actin cytoskeletons of infected cells. HeLa cells were infected with isogenic strains of P. aeruginosa at an MOI of 25 for 6 h. (A and B) No bacteria; (C and D) PA103pscJ::Tn5; (E and F) PA103ΔU+pUCP20; (G and H) PA103ΔUΔT+pUCP20; (I and J) PA103ΔUΔT+pExoS; (K and L) PA103ΔUΔT+pExoT; (M and N) PA103ΔUΔT+pExoT(R149G); (O and P) PA103ΔUΔT+pExoT(R149K). Infected cells were fixed, permeabilized, and stained with Texas Red-labeled phalloidin to visualize actin (red). In addition, the fixed and permeabilized cells were labeled with rabbit anti-ExoU antiserum and a secondary goat anti-rabbit–fluorescein isothiocyanate antibody to visualize P. aeruginosa (green). Note that although the bacterial strains shown do not express ExoU, the anti-ExoU antiserum contains additional antibodies, likely to lipopolysaccharide, which result in sufficient labeling of the bacteria. Labeled cells were observed by fluorescence microscopy, and the images shown were captured at a ×1,000 magnification with a CCD camera and SPOT imaging software. (A, C, E, G, I, K, M, and O) Actin staining only; (B, D, F, H, J, L, N, and P) merged image of both actin (red) and bacterial (green) staining.