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Figure 2 | Veterinary Research

Figure 2

From: Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella

Figure 2

Site-directed mutagenesis of H7 flagellin to reduce TLR5 signalling. (A) Predicted TLR5-binding regions of H7 flagellin (FliC). Top panel shows the structure of FliC (Salmonella Typhimurium) from PDB entry 1UCU (R-type straight flagellar filament) and is coloured in UCSF Chimera according to structural domains as indicated. TLR5 binding residues have been mapped within the D1b regions (dark grey). The bottom panel shows the FliC-H7 D1b regions which are homologous to those from S. Typhimurium and the 4 residues that have been mutated in this study are shown in red. Q89 and R90 in the D1b amino terminal regions have the same position in S. Typhimurium, whereas the L500 and I504 in FliC-H7 are equivalent to I411 and L415 (leucine and isoleucine are interchanged) respectively in S. Typhimurium phase 1 FliC (13). (B) Coomassie-stained SDS-PAGE of WT-H7 and variants. Flagella were purified from E. coli O157 TUV93-0 ΔfliC and total protein concentration adjusted following a BCA assay to 2.5 μg per lane. The left margin shows the approximate molecular size (kDa). (C) Motility of E. coli O157 expressing altered flagellins. Motility was assessed following inoculation of E. coli O157 (TUV93-0) ΔfliC containing WT fliC H7 clone (pEW7) and site-directed mutants as indicated. Motility was assessed after overnight incubation.

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