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Zusammenfassung:
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. Significantly, we have recently shown that R. hominis 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. Little is known about this flagellin type, or about the purpose of the four separate flagellins encoded in its genome; without a genetic system, it is unclear how much we can fully elucidate about role flagellin expression and production plays for the bacterium, or its host. In response, we developed a genetic system for R. hominis to unravel the specific molecular mechanisms driving these flagellin-host interactions. We showed that R. hominis has four restriction-modification systems and characterised the methyltransferases and their subunits responsible for differentiating self and non-self DNA. Next, we constructed a series of E. coli-Lachnospiraceae shuttle vectors and developed a strategy for both in vivo plasmid methylation according to R. hominis-specific nucleotide motifs and DNA transfer. We will then construct knock-out vectors to sequentially remove each flagellin gene from the chromosome and will characterise mutant impact on R. hominis in vitro growth kinetics, substrate utilisation, and motility.
