(A-E) Representative live cell microscopy images of D. discoideum infected with PflaA-GFP-producing L. pneumophila. D. discoideum Ax3 producing cytosolic mCherry (pDM1042) was infected (MOI 5) with (A, B) wild-type L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp), (C, D) JR32 (Ptac-mCerulean-PflaA-gfp) or (E) ∆flaA (Ptac-mCerulean-PflaA-gfp) and embedded in agarose for 48 h. PflaA-GFP-positive L. pneumophila (A) escapes the LCV (0 sec), followed by other bacteria, which are contained in the cytosol and localize to the plasma membrane, where a PflaA-GFP-positive L. pneumophila first leaves the ruptured host cell (185 sec), or (B) triggers host cell death (loss of cytosolic mCherry) upon exit of the amoeba. PflaA-GFP-positive L. pneumophila (C) escape the LCV after 75 sec and spread within the cytosol for several minutes, and (D) are the first to exit the host cell, leading to the loss of cytosolic mCherry and host cell death. (E) L. pneumophila ∆flaA lyses the LCV and host cell less vigorously. Scale bars: 5 µm. 