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Abstract:
Roseburia hominis is an abundant constituent of the human gut microbiome and a member of the Lachnospiraceae bacterial family. Its ability to produce short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate has been associated with the modulation of gut microbial ecology and host energy homeostasis. We have recently shown that R. hominis uniquely produces “silent” flagellins that can bind to host toll-like receptor 5 (TLR5) without initiating a pro-inflammatory response. This suggests that these organisms can actively modulate their interaction with the host immune system, challenging our current understanding of flagellin-TLR5 interactions. We now seek to understand the genetic basis for silent and stimulatory flagellins and the mechanisms for their interaction with the host. To achieve this we systematically identified the requirements for successful DNA transfer to R. hominis, resulting in the first genetic system for this non-model organism. We identified four restriction-modification defence systems in R. hominis and characterised the methyltransferases and their subunits responsible for protecting its own DNA. Next, we developed an in vitro methylation strategy that was applied to a series of E. coli- Lachnospiraceae shuttle vectors, enabling DNA transfer and uptake at high efficiencies. We then constructed knock-out vectors to sequentially remove each flagellin gene from the chromosome and characterised mutant impact on R. hominis in vitro growth kinetics, substrate utilisation, and motility.
