Figure 6.
Translocation of TEM1∷RalF into J774 macrophages by L. pneumophila.
(A) J774 macrophages were infected with Lp01 (top row) or dotA (bottom row) mutant transformed with plasmids expressing FLAG-TEM-1 fusions for a total of four hours. Translocation efficiency is given as percentage of blue cells. Data is representative of three experiments that produced similar results. Bar, 100 µm. (B) Same experiment as above by using DsRed labeled Lp01 and dotA mutant bearing a plasmid expressing FT∷RalF350–374. These high magnification images were merged from the β-lactamase color channel (Blue and Green), DsRed mono channel (Red) and phase contrast control. The upper blue cell contains Lp01 expressing FT∷RalF350–374 and the bottom green cell contains the dotA mutant expressing the same fusion protein. Bar, 5 µm. (C) Number of bacteria per well. At the end of experiment cells were lysed in 0.5% Tween 20 after incubation for 90 min with 50µg/ml gentamicin. Colony forming units were determined by serial dilution and plating on BYCE plates. (D) Western blot using anti Flag, showing all β-lactamase fusions were expressed and their expression were similar between Lp01 (WT) and DotA mutant (Δ). Numbers on right indicate protein standard in kDa.