(C, D) Histograms of the (C) number and (D) spreading distance of L. pneumophila released from infected amoebae. Spreading was quantified using the Imaris software ("surface" tool). One mask covered the heavily infected amobae (center: x, y, z = 0), another mask covered each single bacterium outside the infected host cell. The total number of released bacteria were for JR32 (n = 1240), ∆flaA (n = 33) and ∆lqsA (n = 214). The distance of each bacterium to the center was quantified by vector calculations. The red line marks the approximate cell diameter (5 µm). Data shown are means and SEM of four infected amoebae from one biological replicate (two-tailed Student's t test; * P < 0.05, ** P < 0.01, *** P < 0.001). 