Figure 7
Inactivation of the flagellin gene in E. gallinarum MRx0518 abolishes its immunostimulatory profile. (A) Changes in gene expression in HT29-MTX after 24 h co-culture with MRx0518 and MRx0518 fliC::pORI19 culture supernatants (MRx0518SN and fliCSN) (MOI 100:1 equivalent) and 1 µg/ml purified MRx0518 recombinant flagellin (FliCMRx0518), measured by qPCR (fold change compared to the YCFA or the empty vector control, as appropriate). Statistical comparisons were performed with GraphPad Prism on ΔCT values using two-way ANOVA followed by Tukey’s multiple comparison tests. Statistically significant differences in comparison to the YCFA or empty vector control group as appropriate are shown on the graph as *(p < 0.05), **(p < 0.01) and ****(p < 0.0001). (B) IL-8 concentrations (pg/ml) detected by ELISA assay in HT29-MTX cell-free supernatant after 24 h co-culture with MRx0518 (MRx0518SN), MRx0518 fliC::pORI19 (fliCSN) and DSM100110 (DSM100110SN) culture supernatants (MOI 100:1 equivalent), and 1 µg/ml purified MRx0518 recombinant flagellin (FliCMRx0518). YCFA was included as a negative control. Statistical comparisons were performed with GraphPad Prism using an ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences with the relevant control are shown on the graphs as ***(p < 0.001) and ****(p < 0.0001).
