(A-D) A. castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of PflaA (Ptac-mCherry-PflaA-gfp) and mCherry constitutively, (B) GFP under the control of PralF (PralF-gfp) and stained with DAPI, (C) GFP under the control of PsidC (PsidC-gfp) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post-infection, analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP-positive) emerge at the colony cluster periphery only after bacterial replication has ceased (red fluorescent Timer). 