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

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.

FIG 1

(A) Schematic presentation of the 11.2-kbp Lactobacillus casei BL23 DNA fragment containing the β-N-acetylglucosaminidase precursor and a mannose-6P isomerase in the surroundings of the galacto-N-biose (gnb) operon (18). The first (gnbR) and last (gnbA) genes of this operon are represented. (B) Schematic presentation of proposed pathways for lacto-N-triose and 3′-N-acetylglucosaminyl-mannose metabolism in L. casei BL23. PTS, phosphoenolpyruvate:sugar phosphotransferase system; GlcNAc, N-acetylglucosamine; Gal, galactose; Man, mannose; LacG, phospho-β-galactosidase; NagA, N-acetylglucosamine-6P deacetylase; NagB, glucosamine-6P deaminase.

FIG 2

Coomassie brilliant blue-stained 10% SDS-polyacrylamide gel showing the His-tagged proteins β-N-acetylglucosaminidase BnaG and mannose-6P isomerase ManA. Lane P, protein standards. The numbers on the right are molecular masses.

FIG 3

(A) Growth curves of Lactobacillus casei wild-type strain BL23 on MRS basal medium without a carbon source (black) or with lacto-N-triose (red), 3′-N-acetylglucosaminyl-mannose (blue), 3′-N-acetylgalactosaminyl-galactose (dark cyan), or lactose (green). (B) Growth curves of L. casei mutant strain BL380 (bnaG) on MRS basal medium without a carbon source (black) or with lacto-N-triose (red), 3′-N-acetylglucosaminyl-mannose (blue), or lactose (green). The data presented are mean values based on at least three replicates. Error bars indicate standard deviations.

FIG 4

Growth curves of Lactobacillus casei wild-type strain BL23 (A) and L. casei mutant strain BL381 (manA) (B) on MRS basal medium without a carbon source (black) or with 3′-N-acetylglucosaminyl-mannose (blue) or mannose (pink). Data are presented as mean values based on at least three replicates. Error bars indicate standard deviations.