The Extracellular Wall-Bound β- N -Acetylglucosaminidase from Lactobacillus casei Is Involved in the Metabolism of the Human Milk Oligosaccharide Lacto- N -Triose

Abstract

ABSTRACT Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto- N -triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto- N -biose and galacto- N -biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA , encoding a β- N -acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto- N -triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of β- N -acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto- N -triose into N -acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3′- N -acetylglucosaminyl-mannose and 3′- N -acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3′- N -acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3′- N -acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA -knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3′- N -acetylglucosaminyl-mannose. Here we describe the physiological role of a β- N -acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N -acetylglucosaminide-rich gut niche.