Flagellin is a wide-spread bacterial virulence factor sensed by the membrane-bound Toll-like receptor 5 (TLR5) and by the intracellular NAIP5/NLRC4 inflammasome receptor. TLR5 recognizes a conserved region within the D1 domain of flagellin, crucial for the interaction between subunits in the flagellum and for bacterial motility. While it is known that a deletion of the D0 domain of flagellin, which lines the interior of flagella, also completely abrogates activation of TLR5, its functional role remains unknown. Using a protein fusion strategy, we propose a role for the D0 domain in the stabilization of an active dimeric signaling complex of flagellin-TLR5 at a 2:2 stoichiometric ratio. Alanine-scanning mutagenesis of flagellin revealed a previously unidentified region of flagellin, the C-terminal D0 domain, to play a crucial role in TLR5 activation. Interestingly, we show that TLR5 recognizes the same hydrophobic motif of the D0 domain of flagellin as the intracellular NAIP5/NLRC4 inflammasome receptor. Further, we show that residues within the D0 domain play a previously unrecognized role in the evasion of TLR5 recognition by Helicobacter pylori. These findings demonstrate that TLR5 is able to simultaneously sense several spatially separated sites of flagellin that are essential for its functionality, hindering bacterial evasion of immune recognition. Our findings significantly contribute to the understanding of the mechanism of TLR5 activation, which plays an important role in host defense against several pathogens, but also in several diseases, such as Crohn’s disease, cystic fibrosis and rheumatoid arthritis.
Toll-like receptors encounter a diversity of degradation products in endosomes. TLR7 and TLR8 have been shown to be activated by RNA degradation products. Here we show that although TLR9 requires single-stranded DNA longer than 20 nucleotides for a robust response, TLR9 activation is augmented by CpG-containing oligodeoxyribonucleotides (sODNs) as short as 2 nucleotides, which, by themselves, do not induce activation in cell cultures, as well as in mice. sODNs also activate human TLR9 in combination with ODNs containing a single CpG motif that by themselves do not activate human TLR9. The specific sequence motif of sODN and colocalization of ODN and sODN suggest that the mechanism of activation involves binding of both ODN and sODN to TLR9. sODNs augment TLR9 activation by mammalian genomic DNA indicating the role of short DNA degradation products in the endosomes in response to infection or in autoimmune disease, particularly at limiting concentrations of ODNs.
Toll-like receptor 5 (TLR5) is a receptor of the innate immune system that recognizes flagellin from certain bacterial species and triggers an inflammatory response. The Salmonella dublin flagellin in complex with zebrafish TLR5 has been crystallized previously. In the present study, we extrapolate the structure of this complex using structure-guided mutagenesis to determine the recognition modes of human and mouse TLR5 receptors and demonstrate species-specific differences in flagellin recognition. In general, the recognition mode of the mouse receptor can be said to be more robust in comparison to that of the human receptor. All-atom molecular dynamics simulation showed differences between the two receptors within the primary binding region. Using a functional motility assay, we show that although the highly conserved area of the flagellin analyzed in this study encompasses key structural requirements for flagella formation, a direct correlation between immune recognition and structure on the level of amino acid residues is not observed.
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