Ribosome-engineered Lacticaseibacillus rhamnosus GG with enhanced adhesion and immune activation via surface moonlighting proteins.

Commercial interest in using probiotics to enhance human health and animal production is growing. Accordingly, there is great demand for probiotic lactic acid bacteria exhibiting enhanced robustness and functionality. The present study focused on ribosome engineering (RE), a simple, economical, and safe non-genetically modified organism microbial breeding technology. Ribosome-engineered mutants of Lacticaseibacillus rhamnosus GG (LGG), one of the most widely used probiotic strains, were characterized. We found that an LGG strain with the K56N mutation within the rpsL gene (MTK56N: K56N) expressed a variety of proteins, primarily moonlighting proteins, on the cell surface, likely due to increased translational efficiency and/or activated protein transport via ABC transporters. The K56N mutation is suggested to be a major contributing factor to the enhanced expression of moonlighting proteins, although contributions from additional mutations cannot be ruled out. The K56N surface proteins, including DnaK, GroEL, PyK, and GAPDH, mediated strong bacterial adhesion to intestinal cells and exerted immunostimulatory effects in macrophages by enhancing tumor necrosis factor (TNF)-α production. Interestingly, although LGG-wild-type extracellular vesicles (EVs) suppressed lipopolysaccharide-induced TNF-α production, this effect was not observed with K56N-EV. These findings highlight the potential use of K56N as a probiotic strain with immune-activating properties and the feasibility of using RE to develop probiotic strains for future industrial applications. IMPORTANCE: Ribosome engineering, a microbial breeding strategy that induces spontaneous mutations via treatment with antibiotics, has led to novel functionalities in Lacticaseibacillus rhamnosus GG. A study of streptomycin-resistant rpsL mutants identified the K56N mutation as a key driver of enhanced protein display on the bacterial surface. This mutation promoted strong adhesion to intestinal cells and immunostimulatory effects in macrophages. The mutation also triggered transcriptomic shifts and morphologic changes, highlighting the broad range of bacterial modifications.