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

Area of Science:

• Microbial genetics and breeding
• Probiotics and functional foods
• Immunology and host-microbe interactions

Background:

• Commercial demand for robust probiotic bacteria is increasing.
• Lacticaseibacillus rhamnosus GG (LGG) is a widely used probiotic strain.
• Ribosome engineering (RE) is a safe, non-GMO method for microbial strain improvement.

Purpose of the Study:

• To characterize ribosome-engineered mutants of LGG for enhanced probiotic functionality.
• To investigate the role of the K56N mutation in the rpsL gene on LGG surface protein expression and function.
• To evaluate the potential of engineered strains for industrial applications.

Main Methods:

• Ribosome engineering of Lacticaseibacillus rhamnosus GG using antibiotic treatment to induce mutations.
• Characterization of K56N mutant (MT_K56N) for surface protein expression, including moonlighting proteins.
• Assessment of bacterial adhesion to intestinal cells and immunostimulatory effects in macrophages.
• Analysis of extracellular vesicles (EVs) from wild-type and K56N strains.

Main Results:

• The K56N mutation in the rpsL gene led to increased expression of surface proteins, including moonlighting proteins like DnaK, GroEL, PyK, and GAPDH.
• K56N surface proteins enhanced bacterial adhesion to intestinal cells.
• K56N strain exhibited immunostimulatory effects, increasing TNF-α production in macrophages.
• Extracellular vesicles from the K56N strain did not suppress LPS-induced TNF-α production, unlike wild-type EVs.

Conclusions:

• The K56N mutation is a key factor in enhancing LGG's probiotic properties, including adhesion and immune activation.
• Ribosome engineering is a viable strategy for developing improved probiotic strains with specific functionalities.
• The MT_K56N strain holds potential for use as an immune-activating probiotic in industrial applications.