Figure 5.

LasR mutation abolishes AprA/LasB C5a-cleaving activity and enhances neutrophil recruitment. (a) Relative expression of aprA (black columns) and lasB (white columns) determined by RT-PCR and normalized using the housekeeping gene rpsL. Data are displayed as the percentage of the expression of each gene with respect to PA14. Asterisks indicate significant differences in the expression of both genes between strains *P < 0.05 (2-tailed t test). (b) Western blot analysis of C5a cleavage. Purified recombinant human C5a (20 ng) was incubated with LB (control) or the cell-free supernatants from the bacterial stationary cultures in LB. Proteins were separated and subjected to a Western blot with a mouse monoclonal antibody that recognizes C5a. (c) Human BALF was incubated with the cell-free supernatants from the bacterial growth cultures in artificial sputum medium and the amount of C5a was quantified by sandwich ELISA. (d) Human neutrophil transmigration assay with media, purified C5a (100 nM) or C5a treated with cell-free supernatants from the bacterial stationary cultures in LB. Data are the percentage of the neutrophils recovered from the basolateral chamber with respect to total number of neutrophils added to the upper chamber. Results are the mean values obtained from three independent experiments. Error bars represent SEMs. *P < 0.05 (2-tailed t test).