(A) Time lapse microscopy of A. castellanii infected with PflaA-GFP-producing L. pneumophila. A. castellanii amoebae were infected (MOI 5) with L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp) and embedded in 0.5% agarose/PYG medium. Approximately 46 h post infection, GFP-positive (motile) bacteria emerged in the mCherry-positive intracellular cluster, which within 1.5 h covered the bacterial cluster and spread from the bursting amoeba. At 55 h p.i., primarily mCherry-positive/GFP-negative (non-motile) L. pneumophila remained clustered. Scale bar: 10 µm. 