The Pseudomonas aeruginosa Type III Secretion System Has an Exotoxin S/T/Y Independent Pathogenic Role during Acute Lung Infection
Figure 5
Cell death and IL-1β maturation upon infection of the MF4/4 macrophage cell line with different P. aeruginosa T3SS mutants.
MF4/4 cells were pre-stimulated with 100 ng/ml LPS for 4 h and infected with WT P. aeruginosa or ΔSTY or ΔSTY/ΔPopB mutant strains at a MOI of 100. Non infected cells prestimulated with 100 ng/ml LPS for 4 h were used as a control. A. LDH release in the culture medium was determined by spectrophotometry as described in materials and methods and is expressed as optical density (absorbance) at 490 nm. Results are the mean +/− SD of triplicates and are representative for four independent experiments. B. Caspase-3 activity in cell extracts was measured in a fluorometric assay with Ac-DEVD-AMC and is expressed as change in fluorescence over time (Δf/min). Results are the mean +/− SD of triplicates and are representative of three independent experiments. C. Culture supernatants (SN) were collected and analyzed for the presence of mature IL-1β by immunoprecipitation of IL-1β followed by SDS-PAGE and western blotting. The corresponding total cell lysates were analyzed for the presence of proIL-1β by western blotting. Results are representative of three independent experiments.