FIG 4.

Intracellular replication of wild-type and T2SS mutant L. pneumophila strains in amoebae. (A and B) Monolayers of A. castellanii amoebae were infected with GFP-expressing wild-type strain 130b (WT), lspF mutant NU275 (lspF), or the complemented lspF mutant NU275(pMF1) (lspF/lspF+) at an MOI of 20. After centrifugation and a 1-h incubation to facilitate uptake, gentamicin was added to kill remaining extracellular bacteria. The infected amoebae were then lysed at 12, 16, or 20 h postinoculation, and intracellular CFU were enumerated via plating to obtain the fold change in growth relative to the level at time zero (T_x/T₀). The data are presented as means with standard deviations from triplicate infected wells and are representative of four (A) or two (B) independent experiments, and significant differences between results for the strains were obtained at each time point (Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (C and D) Monolayers of A. castellanii amoebae were infected as described above with GFP-expressing wild-type strain 130b (WT), lspF mutant NU275 (lspF), or proA mutant AA200 expressing plasmid-encoded GFP (proA). The GFP fluorescence from L. pneumophila was monitored kinetically every 30 min for the next 20 h, and the fluorescence values obtained were normalized to the GFP signal at t = 0 following gentamicin treatment. The data are presented as means with standard errors from three (n = 3) independent experiments, and the lines with asterisks indicate the time intervals in which significant differences were observed between wild-type- and mutant-infected amoebae (Student's t test; P < 0.05).