Figure 5. Ail and YadA drive translocation by IP32953 into neutrophils in lung single cell suspensions.

Naive BALB/c mouse lungs were harvested, homogenized and infected at an MOI of 1:1 for 1h at 37°C with ETEM expressing IP32953 strains WT, Δail ΔyadA, ΔailΔyadA or ΔyopB. Lung cells were then incubated with gentamicin, CCF4-AM and the indicated cell surface markers. (A) The percentage of ETEM⁺ cells from lung cell suspensions infected with WT was set to 1 and the mean relative percentage of ETEM⁺ cells from lung suspensions infected with various adhesins mutants was normalized to WT. ΔyopB infected cells were used as a gating control for ETEM. The absolute percentage of ETEM⁺ cells infected with WT ranged from 1.33–3.65%. The mean and SEM is from 41.33–3.65%. The mean and SEM is from 4–6 independent 6 independent experiments per strain. Statistical significance was determined by One-Way ANOVA with Tukey’s post-test. No statistical significance was observed. (B-C) White bars represent the percentage of total (B) Ly6G⁺ or (C) TCRβ⁺ cells in the lung cell suspensions and black bars represent percentage of ETEM⁺ cells that are (B) Ly6G⁺ or (C) TCRβ⁺. Data is from 41.33–3.65%. The mean and SEM is from 4–6 independent 6 independent experiments per strain. Statistical significance determined by One-Way ANOVA with (A) Dunnett’s or (B–C) Bonferroni’s post-test. (ns=not significant, *p<0.05, **p<0.01, ***p<0.001)