Abstract:
The gut microbiome, a complex community of trillions of archaea, fungi, viruses, and bacteria in the gastrointestinal tract, has a well-established role in host health and homeostasis. The balance of microbial species plays a crucial role in disease and pathogenicity, making understanding host- microbe interactions of great therapeutic interest. Toll-like receptor 5 (TLR5), is an innate immune receptor, which specifically responds to bacterial flagellin. TLR5 overactivity is linked to pro- inflammatory diseases like IBD, while disrupted activity increases susceptibility to pathogens. This dissertation explores how flagellins from the microbiome modulate TLR5 responses, providing insights essential for developing targeted therapeutics.
Through a screen of interactions between TLR5 and flagellins from common commensal bacteria, we identified "silent" flagellins, primarily from the Lachnospiraceae family, which bind to TLR5 but elicit a weak immune response, suggesting a widespread mechanism of immune avoidance. TLR5 signalling was modulated by interactions at the flagellin D1 and D0 domains, with the latter containing an allosteric binding site thought to induce conformational change in TLR5. A novel kinetic analysis revealed distinct binding profiles between the D1 domain binding TLR5N14 and the stimulatory S. typhimurium FliC (StFliC) and silent R. hominis FlaB (RhFlaB) flagellins, which are predicted to impact their ability to trigger an immune response. A novel structure of RhFlaB revealed distinct surface characteristics, absent from StFliC, which were likely causal to its unique kinetic binding profile.
I further explored the role of conformational change in TLR5 signalling, and produced two TLR5- TIR dimers capable of partially activating the TLR5 signalling pathway, though less effectively than StFliC, which warrant further investigation. I also produced and purified high concentrations of functional recombinant full-length TLR5 and TLR5 ectodomain constructs for structural analyses. Preliminary negative staining and Cryo-EM identified TLR5 monomers in the expected conformation, but high-resolution density maps have not yet been resolved.
My work reveals how microbes modulate TLR5 immune responses and the structural mechanisms affecting binding kinetics, and lays a solid foundation for further research. These findings underscore the crucial role of microbial interactions in host health and homeostasis, and highlight the significant scientific and medical benefits of continued research in this field.
Silent interactions