We previously reported that the oligosaccharide chains of hog gastric mucin were degraded by unidentified subpopulations numbering 1% of normal human fecal bacteria. Here we report on the enzyme-producing properties of five strains of mucin oligosaccharide chain-degrading bacteria isolated from feces of four healthy subjects. Four were isolated from the greatest fecal dilutions yielding mucin side chain-degrading activity in culture, and thus were the numerically dominant side chain-degrading bacteria in their respective hosts. Three were Ruminococcus strains and two were Bifidobacterium strains. Two Ruminococcus torques strains, IX-70 and VIII-239, produced blood group A-and H-degrading a-glycosidase activities, sialidase, and the requisite jl-glycosidases; these strains released >90% of the anthrone-reacting hexoses from hog gastric mucin during growth in culture. The Bifidobacterium strains lacked A-degrading activity but were otherwise similar, these released 60-80% of the anthrone-reacting hexoses but not the A antigenic structures from hog gastric mucin. Only Ruminococcus AB strain VI-268 produced blood group B-degrading a-galactosidase activity, but this strain lacked ,5-N-acetyl exosaminidases to complete degradation of B antigenic chains. When this strain was co-cultured with a strain that produced jl-N-acetylhexosaminidases, release of hexoses from blood group B salivary glycoprotein increased from 50 to >90%, and bacterial growth was enhanced. The glycosidases required for side chain degradation were produced by these strains in the absence of mucin substrate, and a substantial fraction of each activity in stationary phase cultures was extracellular. In contrast, none of 16 other fecal Bacteroides, Escherichia coli, Streptococcus faecalis, and Bifidobacterium strains produced ABH blood group-degrading enzymes; other glycosidases produced by these strains were predominantly cell bound except for extracellular j-N-acetylhexosaminidases produced by the five S. faecalis strains.We conclude that certain Bifidobacterium and Ruminococcus strains are numerically dominant populations degrading mucin oligosaccharides in the human colon due to their constitutive production of the requisite extracellular glycosidases including blood group antigen-specific a-glycosidases. These properties characterize them as a functionally distinct subpopulation of normal human enteric microflora comprised of An abstract of portions of this work was published in Gastroenterology 1983; 84:1191.
Oligosaccharide side chains of human colonic mucins contain 0-acetylated sialic acids and glycosulfate esters. Although these substituents are considered to protect the chains agiist degradation by bacterial glycosidases, sialate O-acetylesterase, N-acetylneuraminate lyase, and glycosulfatase activities have been found in fecal extracts. To better define the source of these activities, we measured extracellular and cell-bound sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities produced by 23 isolates of human fecal bacteria grown anaerobically in a hog gastric mucin culture medium; these represented dominant populations of fecal anaerobes, facultative anaerobes, and the subset of mucin oligosaccharide-degrading bacteria. Every strain produced sialidase and high levels of arylesterase, and all but five facultative anaerobes produced sialate O-acetylesterase. Sialic acids containing 2 mol or more of O-acetyl ester per mol of sialic acid were cleaved from mucin glycoproteins more slowly by sialidases of mucin oligosaccharide-degrading stains than were sialic acids containing 1 or 0 mol, and only N-acetyland mono-O-acetylated sialic acids were recovered from enzyme digests of a mucin containing di-O-acetylated sialic acids. No detectable N-acetylneuraminate lyase activity was produced by any strain, but low activity was induced by increasing the glycoprotein-bound sialic acid concentration in the culture medium of six Escherichia coli strains. Using lactitol-6-sulfate as a substrate, we found weak glycosulfatase activity in the partially purified, concentrated enzyme mixture in the culture supernatants of four mucin oligosaccharide-degrading strains but in none of the unconcentrated culture fractions. We conclude that the presence of two or more O-acetyl groups on sialic acids inhibits enteric bacterial sialidases but that production of sialate O-acetylesterases by several populations of enteric bacteria lessens the likelihood that mucin oligosaccharide chains terminating in 0-acetylated sialic acids are protected from degradation. Sialate O-acetylesterases have a role * Corresponding author.
A subset of normal human faecal bacteria composed o f niucin oligosaccharide-dcgrading (MOD) strains appears to be the principal sotircc of intraluminal. extracellular glycosidases degrading the oligosaccharides of gut mucin glycoproteins and epithelial membrane glycocon.jupates. Here we report studics characterising the saccharides released from hog gastric mucin during incubation with the extracellular enzymes in their cell-free culture supernatcs. We have also tested the ability ofthe saccharide products to support growth of larger populations of faecal bacteria. Gel exclusion chromatography of the residual inticin following bacterial growth in mucin-containing medium confirmed that MOD strains extensively degraded the carhohydrate moieties (60-96 per cent) whereas strains of bacteroides and bifidobacteria of larger faecal populations degraded the carbohydrate moieties less extensively (median loss, 29 per ccnt; range 8-42 per cent). Saccharides released from hog gastric niucin by MOD strain enzymes were identified by biochemical and NMR spectroscopic methods as galactose. fucose. N-acetylglucosamine. N-acetylgalactosamine and the disaccharides GalB3tialNAc and Galfi3GlcNAc. Larger oligosaccharides were not recovered. Carbon-13 NMR spectra of the residual mucin were consistent with complete removal of some but not all oligosaccliaride chains. The monosaccharides but not thc disaccharides were also recovered following incubation of mucin with the sterile cell-free supernate of a heavily inoculated faecal culture. Saccharides rcleased from hog gastric mucin by partially purified culture supernate enzyincs from Rutninococcus toryuc~s strain IX-70 were 70-98 per cent utilised by larger populations of faecal bacteria during growth in culture. Bacteria growing from 1 x 10-'Og faecal inocula from five subjects showed minimal growth in medi;i containing intact hog gastric much but grew well if the niucin-containing medium was preincubated with a sterile enzyme preparation from R. toryuu.s IX-70. We conclude that glycosidases produced by MOD strains can scne ; I nutritionally supportive role for larger populations of cnteric bacteria. especially during fasting.
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