Fig. 5.

The Icm/Dot T4SS and the effector LegG1 promote A. castellanii migration through L. pneumophila biofilms. Biofilms of L. pneumophila JR32, ∆icmT, ∆ppgA, ∆legG1 or ∆ppgA‐∆legG1 mutant strains harbouring pNT28 (constitutive production of GFP) were grown in AYE medium at 25°C, A. castellanii was added to preformed biofilms after 6 days and amoeba migration was followed by confocal microscopy.

A. Single amoeba migrating through biofilms were tracked and individually shown as a line in migration plots.

B. The velocities and (C) Euclidean distances of migrating amoebae tracked in (A) were quantified. The data (B, C) are means and standard deviations of the mean derived from all tracked amoebae (n.s., not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; one‐way ANOVA with Tukey post‐test; JR32 vs. mutant strains). Single amoebae were tracked in three biologically independent experiments with a total of n = 30 amoebae for each bacterial strain.