Capacity of streptococcal pathogens to activate TLR2 and NF-κB. A
S. uberis, but not other streptococcal species failed to activate NF-κB in the HEK293 reconstitution system of TLR2-signaling. HEK293 cells were transfected with constructs expressing the bovine TLR2 receptor (200 ng) and the Renilla luciferase expressing reporter gene being driven by NF-κB through its ELAM promoter. Subsequently, the cells were challenged for 24 h with different dose (abscissa) of heat-killed streptococcal strains or E. coli
1303. The luciferase activity was measured from cell lysates and normalized against their protein concentration. Values are expressed as fold increase above the level of the unstimulated control (ordinate). Each transfection was run in duplicate and assayed from triplicate challenges. (*p < 0.05; ***p < 0.001, regarding the difference to the unstimulated control). B
S. uberis bacteria, dead or alive failed to activate NF-κB in pbMEC. pbMEC were transfected with the ELAM driven reporter gene construct (100 ng) and either stimulated with 30 µg/mL of the heat-killed bacteria (as indicated; left hand panel) or incubated for 1 h with 107 live bacteria/mL. Bacteria were subsequently killed with 100 µg/mL gentamicin and the cultures incubated for another 23 h. Thereafter, the Renilla activity was measured from cell lysates and expressed as multiple of the respective unstimulated control (ordinate; tabulated values below the graph, mean ± SEM, n = 2 independent experiments, each assayed in triplicate). Only the NF-κB inductions in response to the E. coli challenges were statistically significant (p < 0.001).