Figure 2.

Generation and characterization of recombinant FlaA- and rFlaA:Betv1-mutants replacing either the TLR-activating sequence stretch in the D1 domain or truncating the C-terminal D0 domain. To investigate the mechanism of rFlaA:Betv1-mediated macrophage activation, four mutant proteins were generated (A). For the generation of rFlaA^*D1 and rFlaA^*D1:Betv1 eight amino acids of the flagellin D1 structural domain (QRMRQLAV) reported to contribute to recognition of flagellin by TLR5 were replaced with the sequence DTVKVKAT (A). For rFlaA^ΔDC0 and rFlaA^ΔDC0:Betv1 the flagellin DC0 domain was deleted (A). The ability of wild-type and protein mutants to activate human TLR5 was tested using HEK-Blue™ hTLR5 reporter cells (B). Data are the mean results of three independent experiments ± SD. Statistical comparisons were performed between the wild-type proteins (rFlaA or rFlaA:Betv1) and respective mutants treated with equimolar stimulation doses and statistical significance was shown as ***: p-value < 0.001.