2023 AIChE Annual Meeting

Deciphering the Adhesion Mechanism of Segmented Filamentous Bacteria

Segmented Filamentous Bacteria (SFB) are gut microbes which are known to stimulate the host immune system by anchoring to intestinal epithelial cells. SFB have been shown to recruit vital Th17 and Th22 populations to protect the host from pathogenic and opportunistic bacteria. Except for the interaction through flagellum, the mechanism of adhesion of SFB is currently unknown. With more knowledge about the way in which SFB anchors to epithelial cells and induces immunity, this mechanism could be utilized for the efficient delivery of biologic drugs directly into the cytoplasm of patients’ cells.

Bioinformatic analysis of predicted SFB adhesion proteins revealed P12260, a surface protein of the bacteria with a predicted carbohydrate binding domain. It is plausible that P12260 could bind to glycans in the intestinal mucin, the extracellular matrix, or on epithelial cell surfaces. In order to investigate the properties of this protein, His-tagged P12260 was recombinantly expressed in E. coli and purified using Nickel-Immobilized Metal Affinity chromatography. Prior work in our lab had used two-dimensional carbohydrate microarrays to probe carbohydrate binding, but results were inconclusive.

In order to immobilize sugars, monosaccharides D-glucose, L-fucose, L-mannose, L-rhamnose, and N-acetyl-D-glucosamine were bound by their hydroxyl groups to epoxy-activated agarose resin. FITC-tagged Concanavalin A, glucose binding lectin, and FITC-tagged Lotus tetragonolobus Lectin, known to bind fucose, were used as controls. All three proteins were loaded on individual columns made of agarose-immobilized specific sugar. After column washing and protein elution, we were able to determine each protein’s binding affinity to each sugar. Western blot was used to determine the amount of protein in the elution. This method can be used to identify the principal monosaccharide residue which is involved in binding, but the full glycan structure is still unknown. In future research, epoxy-activated agarose resin can be used to immobilize P12260. This resin can be used in pull down experiments with gut epithelial cell lysate in order to identify the in vivo binding partner.