Effect of L. pneumophila lqs genes on host cell migration.

D. discoideum strain Ax3 producing GFP (pSW102) was infected (MOI 10, 1 h) with (A) L. pneumophila wild-type, ΔicmT, ΔlqsS, ΔlqsT, ΔlqsS-lqsT, ΔlqsR or ΔlqsA mutant strains harboring pSW001 (DsRed), or with (D) the strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA). An under-agarose assay was used to monitor the migration towards folate (1 mM) for another 4 h. The white lines represent the edge of the sample wells. (B, E) Graphs of the data from (A, D) plotted as per cent GFP fluorescence intensity versus migration distance. (C) Murine RAWs 264.7 macrophages were infected (MOI 10, 1 h) with L. pneumophila wild-type, ΔicmT, ΔlqsS, ΔlqsT, ΔlqsS-lqsT, ΔlqsR or ΔlqsA mutant strains. Cells were stained with Cell Tracker Green BODIPY and let migrate towards CCL5 (100 ng/ml) in an under-agarose assay for another 4 h. Graphs show the per cent fluorescence intensity versus migration distance. (F) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L. pneumophila wild-type, ΔicmT or ΔlqsA mutant strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (G) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of the triplicate samples are shown (pNT28 vs. pNT36: ***p