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Abstract

Toll-like receptor 5 (TLR5) is a Pattern Recognition Receptor which targets and responds to conserved sites on bacterial flagellin, the protein subunit of bacterial flagella, which confers motility. TLR5-flagellin binding is widely used as a proxy for immune activity. However, we recently identified a class of flagellins termed “silent”. They contain the TLR5 epitope facilitating binding at the canonical nD1-domain binding site, but elicit a weak immune response.
To better characterise the relationship between TLR5-epitope binding and signalling, we determined the relative binding and activity profiles of 116 flagellins, with the TLR5 epitope, against a truncated human TLR5 construct, containing only TLR5-epitope binding site. This revealed a continuum in binding strengths with only a weak correlation to the immune response, decoupling binding and activity. We then explored the binding kinetics of the silent R. hominis FlaB and agonist S. typhimurium FliC flagellins to the truncated TLR5. This revealed temporal differences in their binding profiles, and diverging roles during binding for the ‘PIM’ residues, considered essential for TLR5 binding. Structural analysis was performed to uncover how differences in known binding residues influences their contrasting binding profiles.
Our findings reveal the complexity of TLR5-flagellin interactions, and indicate how silent innate immune evasion may be achieved. This could lead to new avenues for developing TLR5-targetted treatments.