FIG 4.

Vfr controls synthesis of TFP and ExlBA, and both are required for cytotoxicity. (A) ExlA-dependent cytotoxicity on A549 epithelial cells of the wild type (WT), various mutants, and the complemented strains. Cytotoxicity was measured after 4.5 h of incubation with the bacteria added at an MOI of 10. The experiments were performed in triplicate, and the bars indicate the standard deviations. The P value (P < 0.01) was determined using a Mann-Whitney U test and is indicated by ** when the difference between the WT strain and each mutant is statistically supported. (B) Twitching ability of the indicated strains after 48 h at 37°C. The twitching area was visualized following staining of the plates with Coomassie blue. (C) Analysis of ExlA secreted by the different strains after a 2-h infection of A549 cell monolayers. Proteins from cell culture medium were TCA precipitated and used in an immunoblot analysis with anti-ExlA and anti-FliC antibody probes. FliC was used as a loading control.