FIG. 3.

ExoT inhibits internalization of P. aeruginosa by J774.1 macrophage-like cells. J774.1 cells were infected with isogenic mutants of P. aeruginosa strain PA103, shown at an MOI of 40 to 60. For each infected well, the number of internalized bacteria was divided by the value for total LDH activity released upon lysis of the cells in that well to normalize for J774.1 cell detachment from the culture plate during infection (internalized CFU/LDH). Averages ± the standard errors of the means for five to six experimental points are shown. (A) For strains containing no episomal vectors, the internalized CFU/LDH value for each well was normalized to the average internalized CFU/LDH value for PA103pscJ::Tn5 in experiments performed with J774.1 cells of the same passage. The normalized CFU/LDH values were averaged to calculate the percentages of invasion, and the standard errors of the means are shown. The actual average number of internalized CFU per well ± the standard error of the mean for PA103pscJ::Tn5 in the representative experimental results shown was 3.87 × 10⁶ ± 4.44 × 10⁴, and the average LDH units per well was 9.05 × 10⁴ ± 3.91 × 10³, yielding an average internalized CFU/LDH of 43.1 ± 1.35. (B) For strains containing episomal vectors, the internalized CFU/LDH for each well was normalized to the average internalized CFU/LDH for PA103ΔUΔT+pUCP20. The actual average number of internalized CFU per well ± the standard error of the mean for PA103ΔUΔT+pUCP20 in the representative experimental results shown was 1.26 × 10⁶ ± 6.91 × 10⁴, and the average value for LDH units per well was 4.96 × 10⁴ ± 2.33 × 10³, yielding an average internalized CFU/LDH and standard error of the mean of 25.54 ± 1.65.