The N‐terminal D1 domain of Treponema pallidum flagellin binding to TLR5 is required but not sufficient in activation of TLR5

Abstract

Syphilis is a chronic bacterial infection caused by Treponema pallidum (T pallidum) and the pathogenesis that T pallidum infection induces immunopathological damages in skin and other tissues remains unclear. We have previously reported that recombinant flagellins of T pallidum can elicit IL‐6 and IL‐8 transcriptions via TLR5 pathway. To identify the domains which induced the pro‐inflammatory activity and the importance of the interactions between TLR5 and domains, homology‐based modelling and comparative structural analyses revealed that Tpflagellins can combine with TLR5 directly. Deletion mutations showed that the ND1 domain binding to TLR5 is required but not sufficient in TLR5 activation. Moreover, site‐directed mutagenesis analysis indicated that the arginine residue (Tpflagellins R89) of the ND1 domain and its adjacent residues (Tpflagellins L93 and E113) constitute a hot spot that elicits IL‐6, IL‐8 transcriptions and TLR5 activation, and affects the binding of Tpflagellins to TLR5. Taken together, these results give insight into the pathogenesis of T pallidum and may contribute to the future design of Tpflagellins‐based therapeutics and syphilis vaccine.

Escape of TLR5 Recognition by Leptospira spp.: A Rationale for Atypical Endoflagella

“…We also found in FlaB the consensus in the D0 domain involved in the flagellin/TLR5 complex stabilization ( Figures 6A,C ). We compared the leptospiral FlaB sequences in these 3 consensus binding TLR5 regions with other spirochetes, Borrelia burgdorferi and Treponema spp., the latter known to signal via TLR5 when FlaB are expressed as recombinant proteins ( 42 ) and also with bacteria known to dodge the TLR5 response such as Helicobacter pylori ( 43 ) and Bartonella bacilliformis ( 10 ), presenting variations in those consensus sequences of their flagellins ( Supplementary Figure 4A ). In addition, we also found this FlaB region to be 100% conserved in a panel of major species of Leptospira circulating all over the world, including potential human pathogens, such as L. borgpeterseni, L. kirschneri , L. noguchii , L. weilii , L. santarosai , as well as L. licerasiae , belonging to another clade of species of lower virulence ( 2 ) ( Supplementary Figure 4B ).…”

“…However, our attempts to express recombinant FlaB monomers have failed. We cannot exclude a caveat in our cloning strategy but this failure was quite surprising considering that T. denticola and T. pallidum FlaB were expressed as stable recombinant proteins that were able to signal through TLR5 in THP1 monocytes or in human keratocytes, respectively (42). One hypothesis could be that the FlaBs that encompass a different shape than FliC would need to be stabilized by polymerization into the complex filament structure.…”

The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification

“…Many studies have proved that TLR5 recognises flagellins via the extracellular domain, which leads to the expression of a variety of genes. However, due to variation in the sequences and domains of flagellins, flagellins from diverse bacterial species use different TLR5 recognition mechanisms ( 34 ). For activation, the receptor must undergo a ligand-induced conformational change ( 27 ).…”

Research progress on Toll-like receptor signal transduction and its roles in antimicrobial immune responses

“…The shortest flagellin consists of the D0 and D1 domains in Bacillus subtilis ( B. subtilis , W23), lacks the hypervariable domains, and still triggers efficient TLR5 recognition and the subsequent signal transduction (Song et al 2017 ). R89, L93, and E113 residues in the D1 domain of flagellin are critical to interact with the TLR5 LRR9 loop for inducing IL-6 and IL-8 transcription and TLR5 activation in B. subtilis and Treponema pallidum , whereas R89T and E114D flagellin mutants of Campylobacter jejuni ( C. jejuni ) and H. pylori failed to bind with TLR5, thus evading immune recognition by TLR5 (Song et al 2017 ; Xu et al 2019 ). Thus, the D0 and D1 domains are the minimal regions required for TLR5 activation; even the CagL protein of type IV secretory system from the highly virulent H. pylori strain contains a D1-like motif that can activate TLR5 and downstream signal transduction in vitro (Pachathundikandi et al 2019 ).…”