FIG 1.

Effect of MOI on T2SS promotion of L. pneumophila infection of amoebae. (A) A. castellanii amoebae were inoculated with wild-type strain 130b (WT) expressing plasmid-encoded GFP at increasing MOIs, and the tissue culture plates were centrifuged to promote contact of L. pneumophila with the amoebae. Following 1 h of incubation, the amoebal monolayers were washed to remove extracellular bacteria and treated with gentamicin for an additional hour to completely kill any noninternalized bacteria. The GFP intensities in the infected wells were immediately determined using a fluorescent microplate reader and plotted against the MOI (left), or the plates were incubated for an additional 10 h, at which time the infected amoebae were lifted and analyzed by flow cytometry for GFP intensity as a measure of productive infection (right). RFU, relative fluorescent units. (B) A. castellanii amoebae were infected with either the wild type or the lspF mutant strain NU275 (lspF) at the indicated MOI, as described above. At 0, 24, 48, and 72 h p.i., net bacterial growth (number of CFU at the denoted time/number of CFU at t = 0) was determined by plating aliquots of the supernatant on BCYE agar. Data in log units are presented as means with standard deviations from quadruplicate infected wells and are representative of two independent experiments. (C) A replotting of the data in panel B showing how wild-type and lspF mutant net growth is impacted by increasing the MOI from 0.1 to 20. In panels B and C, the asterisks indicate significant differences between results for the strains (Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).