Effect of the nanoscale S-layer protein armoring on tolerance, adhesion, and colonization potential of Bifidobacterium adolescentis Bf 15703

A proteinaceous surface layer (known as an S-layer) of numerous lactic acid bacteria has been shown to confer tissue adherence, specifically to epithelial cells, and protection against environmental stress. To investigate the potential of recombinant S-layer proteins to assemble on Bifidobacterium, we co-incubated S-layer proteins with Bifidobacterium adolescentis Bf 15703. This process resulted in a significant increase in fluorescence intensity at 533 nm compared to the control group, alongside a notable shift in zeta potential from −45.36 ± 4.05 mV to −24.31 ± 2.35 mV, confirming successful protein assembly. Morphological characterization of the armored bacteria supported these findings. In vitro digestion assays demonstrated significantly enhanced gastrointestinal tolerance in the modified bacterial cells. Furthermore, adhesion to HT-29 cells (a human colorectal adenocarcinoma cell line) was increased by approximately fivefold, with an adhesion rate of 1.05%. In vivo studies revealed a significantly prolonged retention time, as supported by ex vivo optical imaging and cryosection analysis. qPCR analysis confirmed sustained colonization for > 27 days. These results demonstrate that heterologously expressed S-layer proteins can successfully assemble on B. adolescentis Bf 15703, thereby enhancing its gastrointestinal resilience, adhesion, and long-term colonization capabilities in vivo.