Figure 3. P. aeruginosa T3SS needle tip complex protein PcrV associates with increased PMVEC permeability during early phase infection.

A. Isolated rat PMVECs were grown on transwells to confluence and permeability measured as a function of FITC-dextran flux across the monolayer. PMVECs were inoculated with medium alone, saline solution (vehicle control), or with either P. aeruginosa mutant at a 40:1 MOI. Flux assays were initiated at 4-hours post-inoculation by the addition of FITC-dextran and flux monitored for 1-hour. Average flux rate data at 5-hours post-inoculation from the SS vehicle control transwells were used to normalized the flux rate from PA103 (ΔU/ΔT) or PA103 (ΔPcrV) (n=3 for each condition). Inoculation with PA103 (ΔU/ΔT) caused significant increases in PMVEC permeability compared to inoculation with PA103 (ΔPcrV). Error bars are the standard error of the mean. The asterisk indicates a significant difference by unpaired t-test (P < 0.05). B. Mock cell culture infection experiments were performed where ~2.5 x 10⁷ CFU/mL of each P. aeruginosa mutant strain was inoculated into DMEM culture medium (no PMVECs) and bacterial growth followed over time by direct plating. Error bars are the standard error of the mean of technical replicates from at least 3 independent experiments. There were no observable differences in growth rates between the two P. aeruginosa mutant strains. C. The ability of each of the P. aeruginosa mutant strains to adhere to PMVECs was assessed. Isolated rat PMVECs were grown in culture dishes to confluence and inoculated with either P. aeruginosa mutant at a 40:1 MOI. At one hour post-inoculation, monolayers were washed copiously to remove planktonic bacteria, PMVECs permeabilized, and total associated bacteria determined by direct plating. Error bars are the standard error of the mean of technical replicates from at least 3 independent experiments. There were no observable differences in adherence to PMVECs between the two P. aeruginosa mutant strains.