Extracellular vesicles produced by Bifidobacterium longum export mucin-binding proteins.

Extracellular proteins are important factors in host-microbe interactions; however, the specific factors that enable bifidobacterial adhesion and survival in the gastrointestinal (GI) tract are not fully characterized. Here, we discovered that Bifidobacterium longum NCC2705 cultured in bacteria-free supernatants of human fecal fermentation broth released a myriad of particles into the extracellular environment. The aim of this study was to characterize the physiological properties of these extracellular particles. The particles were approximately 50–80 nm in diameter with high protein and dsDNA content, suggesting that they were extracellular vesicles (EVs). A proteomics analysis showed that the EVs primarily consisted of cytoplasmic proteins with crucial functions in essential cellular processes. We identified several mucin-binding proteins using biomolecular interaction analysis, including phosphoketolase, GroEL, EF-Tu, phosphoglycerate kinase, transaldolase (Tal), and Hsp20. The recombinant GroEL and Tal proteins showed high binding affinities to mucin. Further, the immobilization of these proteins on microbeads affected the permanence of microbeads in the murine GI tract. These results suggest that bifidobacterial exposure conditions that mimic the intestine stimulate B. longum EV production. The resulting EVs exported several cytoplasmic proteins that may have promoted B. longum adhesion. This study improved our understanding of the Bifidobacterium colonization strategy in the intestinal microbiome. ImportanceBifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. Morphological observations revealed that extracellular appendages of bifidobacteria in complex microbial communities are important for understanding its adaptations to the GI tract environment. We identified dynamic extracellular vesicle (EV) production by Bifidobacterium longum in bacteria-free fecal fermentation broth that was strongly suggestive of differing bifidobacterial extracellular appendages in the GI tract. In addition, export of the adhesive moonlighting proteins mediated by EVs may promote bifidobacterial colonization. This study provides new insight into the roles of EVs on bifidobacterial colonization processes as they adapt to the GI environment.