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Abstract:
Methanobrevibacter smithii is the dominant archaeon in human gut and promotes bacterial fermentation by consuming hydrogen, a major fermentation waste product. The aggregation of M. smithii with fermentative bacteria enhances the availability of hydrogen, yet the underlying mechanisms governing this aggregation process remain elusive. Bioinformatic analysis based on homology to bacterial adhesins annotated a high presence of adhesin-like proteins (ALPs) in the Methanobrevibacter pan-genome, potentially involved in cell-cell aggregation. However, to date, no experimental characterization of these ALPs has been conducted for M. smithii. Our study aims to address this knowledge gap by experimentally identifying the M. smithii protein adhesins responsible for mediating cell-cell interactions, thereby validating the adhesive function of annotated ALPs using phage display and binding assays. To find the adhesive protein with phage display, An M13 phage library has been constructed using M. smithii genome fragments, and multiple rounds of affinity screening with various bacterial baits have been conducted to discover the adhesive proteins. Genes enriched due to their binding to the baits were identified using Illumina sequencing. Up to date, the preliminary results suggested several annotated ALPs being enriched during the selection against Christensenella spp. Additionally, we are conducting binding assays of M. smithii against its bacterial counterparts, followed by cross-linking proteomics to detect the proteins involved in the binding process. In the future, we will further characterize the expression of ALPs and other genes in order to determine the conditions that mediate ALPs production and aggregation, utilizing multi-omics to access the regulatory profile of ALPs in varying coculture combinations within a bioreactor system. The outcomes of this research will enhance our comprehension of how the dominant methanogen, M. smithii, directly mediates metabolic cooperation with bacterial fermenters within the human gut. The insights gained from this study may have broader implications for understanding gut microbiota interactions and their impact on host health.
