A bacterial sulfoglycosidase highlights mucin O-glycan breakdown in the gut ecosystem.

Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family.

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on ß-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of ß-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-ß-D-Frup (diheterolevulosan II) and ß-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-ß-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with ß-D-Fruf and ß-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).

Three Candidate Probiotic Strains Impact Gut Microbiota and Induce Anergy in Mice with Cow's Milk Allergy.

Cow's milk allergy is a worldwide public health issue, especially since there is no effective treatment, apart from milk and dairy product avoidance. The aim of this study was to assess the beneficial role of three probiotic strains previously selected for their prophylactic properties in a mouse model of ß-lactoglobulin allergy. Administration of Lactobacillus rhamnosus LA305, L. salivarius LA307, or Bifidobacterium longum subsp. infantis LA308 for 3 weeks post-sensitization and challenge modified the composition of the gut microbiota, with an increase in the Prevotella NK3B31 group and a decrease in Marvinbryantia, belonging to the Lachnospiraceae family. Although no impact on markers of sensitization was detected, modifications of foxp3, tgfß, and il10 ileal gene expression, as well as plasma metabolomic alterations in the tryptophan pathway, were observed. Moreover, ex vivo studies showed that all probiotic strains induced significant decreases in cytokine production by ß-lactoglobulin-stimulated splenocytes. Taken together, these results suggest that the three probiotic strains tested lead to alterations in immune responses, i.e., induction of a tolerogenic anergy and anti-inflammatory responses. This anergy could be linked to cecal microbiota modifications, although no impact on fecal short-chain fatty acid (SCFA) concentrations was detected. Anergy could also be linked to a direct impact of probiotic strains on dendritic cells, since costimulatory molecule expression was decreased following coincubation of these strains with bone marrow-derived dendritic cells (BMDCs). To conclude, all three candidate probiotic strains induced strain-specific gut microbiota and metabolic changes, which could potentially be beneficial for general health, as well as anergy, which could contribute to oral tolerance acquisition.IMPORTANCE We showed previously that three probiotic strains, i.e., Lactobacillus rhamnosus LA305, L. salivarius LA307, and Bifidobacterium longum subsp. infantis LA308, exerted different preventive effects in a mouse model of cow's milk allergy. In this study, we evaluated their potential benefits in a curative mouse model of cow's milk allergy. When administered for 3 weeks after the sensitization process and a first allergic reaction, none of the strains modified the levels of sensitization and allergic markers. However, all three strains affected gut bacterium communities and modified immune and inflammatory responses, leading to a tolerogenic profile. Interestingly, all three strains exerted a direct effect on dendritic cells, which are known to play a major role in food sensitization through their potentially tolerogenic properties and anergic responses. Taken together, these data indicate a potentially beneficial role of the probiotic strains tested in this model of cow's milk allergy with regard to tolerance acquisition.

Identification and structural basis of an enzyme that degrades oligosaccharides in caramel.

Cooking with fire produces foods containing carbohydrates that are not naturally occurring, such as α-d-fructofuranoside found in caramel. Each of the hundreds of compounds produced by caramelization reactions is considered to possess its own characteristics. Various studies from the viewpoints of biology and biochemistry have been conducted to elucidate some of the scientific characteristics. Here, we review the composition of caramelized sugars and then describe the enzymatic studies that have been conducted and the physiological functions of the caramelized sugar components that have been elucidated. In particular, we recently identified a glycoside hydrolase (GH), GH172 difructose dianhydride I synthase/hydrolase (αFFase1), from oral and intestinal bacteria, which is implicated in the degradation of oligosaccharides in caramel. The structural basis of αFFase1 and its ligands provided many insights. This discovery opened the door to several research fields, including the structural and phylogenetic relationship between the GH172 family enzymes and viral capsid proteins and the degradation of cell membrane glycans of acid-fast bacteria by some αFFase1 homologs. This review article is an extended version of the Japanese article, Identification and Structural Basis of an Enzyme Degrading Oligosaccharides in Caramel, published in SEIBUTSU BUTSURI Vol. 62, p. 184-186 (2022).

Identification and characterization of endo-α-, exo-α-, and exo-ß-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-ß-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and ß-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.