Figure 9
Mechanisms underlying FlaR contribution to S. pneumoniae virulence. (a) Phagocytosis assay. Mice peritoneal cells were plated onto 96 well culture plates (2 × 105 cells/well). Adherent cells were incubated with either WU2 WT, WU2ΔflaRErm or WU2ΔflaRflaR/Erm/Kan complemented strains (2.5 × 104 CFU/well) for 40 min. Aliquots of the supernatant were plated onto blood agar plates for enumeration. The number of phagocytosed bacteria is presented as the difference between initial bacterial CFU/well and the residual live bacteria CFU/well. Presented here is the summary of 2 biological independent experiments each performed in triplicates (One-way ANOVA, p = 0.1190, non-significant). (b) FlaR mediates bacterial adhesion to host cells. The WU2 WT, WU2ΔflaR Erm and complemented WU2ΔflaR flaR/Erm/Kan plasmid and WU2ΔflaR flaR/Erm/Kan chromosome strains (MOI 10:1) were added to A549 cells, cultured in 96 well plates for 1 h at 37 °C. Excess bacteria were then removed, cells released with 0.25% trypsin-EDTA and plated onto blood agar plates for enumeration. The WU2ΔflaR Erm mutant and, to a lesser extent, WU2ΔflaR flaR/Erm/Kan chromosome strain exhibited significant inhibition of adhesion, relative to the WU2 WT and to the WU2ΔflaR flaR/Erm/Kan plasmid. The experiment was repeated three times on different occasions. Results presented here is of a representative experiment performed in quadruplicates (one way ANOVA with Dunnett’s post hoc multiple comparisons test, n = 4 in each group, *p = 0.0103; ***p < 0.0004; ****p = 0.0001).
