An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides

Abstract

Lactating mothers secrete milk sialyloligosaccharides (MSOs) that function as anti-adhesives once provided to the neonate. Particular infant-associated commensals, such as Bifidobacterium longum subsp. infantis, consume neutral milk oligosaccharides, although their ability to utilize acidic oligosaccharides has not been assessed. Temporal glycoprofiling of acidic HMO consumed during fermentation demonstrated a single composition, with several isomers, corresponding to sialylated lacto-N-tetraose. To utilize MSO, B. longum subsp. infantis deploys a sialidase that cleaves α2-6 and α2-3 linkages. NanH2, encoded within the HMO catabolic cluster is up-regulated during HMO fermentation and is active on sialylated lacto-N-tetraose. These results demonstrate that commensal microorganisms do utilize MSO, a substrate that may be enriched in the distal gastrointestinal tract.

Figures

FIGURE 1.

Temporal glycoprofile of abundant MSO consumption by B. longum subsp. infantis ATCC15697. MSO compositions are represented by an m/z value signifying a characteristic oligosaccharide composition. Only 999 (SLNT) is consumed appreciably (≥50%) at the initiation of stationary phase.

FIGURE 2.

B. longum subsp. infantis ATCC15697 sialic acid utilization cluster. The analogous loci in the closely related B. longum subsp. longum NCC2705 is included for comparative purposes. Dark gray arrows depict genes conserved in B. longum subsp. infantis ATCC15697 (BI ATCC15697) and B. longum subsp. longum NCC2705 (BL NCC2705). White arrows denote genes unique to ATCC15697, including sialic acid catabolism, with light gray arrows marking those genes specific to NCC2705. Loci are preceded by BL and Blon_ for NCC2705 and ATCC15697, respectively.

FIGURE 3.

Domain structure of the two ATCC15697 sialidase enzymes. Catalytic residues appear above the protein, with domain boundaries demarked below. SCOP Superfamily sialidase domains (SSF50939) are depicted encompassing several bacterial neuraminidase repeats (BNR) (i.e. Asp-boxes).

FIGURE 4.

Phylogenetic relationship of sialidases encoded by gut bacteria. Branch lengths are in the same units (number of amino acid substitutions per site) as those of the evolutionary distances used to construct the tree. The organism and loci are listed in which these sialidases are found.

FIGURE 5.

ATCC15697 sialidase gene expression during carbohydrate fermentation. Gene expression is calculated relative to levels while growing on lactose as a sole carbon source. Bars represent averages from three independent experiments ± standard error. *, p ≤ 0.05.

FIGURE 6.

B. longum subsp. infantis ATCC15697 sialidase substrate specificity assay. NanH1 (A) and NanH2 (B) were assayed for activity on a library of sialosides with either α2,3- or α2,6-sialyl linkages depicted in white and black, respectively. The error bars in the graphs represent the standard errors of experimental values obtained from duplicated samples in an individual experiment. Significant differences were determined with a Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Digestion of MSO species with 3 recombinant sialidases for 30 min. SLNT and DFSLNH were purified from a pool of HMO and DSLNT was obtained from a commercial source. HPLC fraction before digestion of purified HMO (i.e. SLNT and DFSLNH) (a); digestion of purified HMO with an α2–3-neuraminidase (b); digestion of purified HMO with NanH2 (c); digestion of purified HMO with NanH1 (d); commercially obtained DSLNT prior to digestion (e); DSLNT digestion with an α2–3-neuraminidase (f); DSLNT digestion with NanH2 (g); DSLNT digestion with NanH1 (h).