Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

Abstract

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL. IMPORTANCE Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown

“…4). For Bacteroides species belonging to the HGM, the GH16 enzymes that have been characterised so far include specificities towards porphyran, agarose, β-1,3-glucan, and mixed linkage glucan [24][25][26][27] . The genetic loci encoding the GH16 enzymes upregulated on mucin also contain genes encoding SusCD pairs for the Bacteroides spp.…”

Linear and branched β-Glucans degrading enzymes from versatile Bacteroides uniformis JCM 13288T and their roles in cooperation with gut bacteria

“…Assuming the importance of three GHs (GH3, GH16 and GH158) associated with PUL36 and one GH (GH30_3) of PUL21 for degrading laminarin, these enzymes were cloned and heterologous expressed in E. coli . While we were working on these enzymes, the study of Dejean et al (2020) 52 appeared in which they characterized Bu GH3, Bu GH16, and Bu GH158. Therefore in the study, we mainly focused to work with Bu GH158, which is not identical to one that characterized (Supplementary Fig.…”

“…S7) and Bu GH158 ( Figure 4(b -d)) indicated that both enzymes can produce oligosaccharides, which can be utilized by other bacteria present in their vicinity. 52 Especially, such cooperativity is rarely seen with Gram-positive bacteria. In order to check this hypothesis, Gram-positive bacteria were spotted near to B. uniformis JCM 13288 T on agar plate and subsequently, CFUs were calculated.…”

“…Interestingly, B. fluxus lacks GH16 and others lack GH158 but showed growth on laminarin. 52 The pustulan-PUL is also pervasive among Bacteroides and other genera regardless of presence of α-mannan utilization locus. 37 This PUL is most likely to be involved in utilization of dietary edible mushroom and utilization of cell wall glycans of gut commensal fungal species such as Saccharomyces cerevisiae.…”

“…Capability of adopting long branched chain glucan by Bu GH16 was also convincingly demonstrated by Dejean et al (2020). 52 The Bu GH158 has for the first time been used for generation of β- 1, 3-OSs after optimization of the type of curdlan. Pustulan-PUL in the B. uniformis JCM 13288 T encodes single Bu GH30_3 that cleaves β- 1, 6-linked polysaccharides (pustulan and lentinan) in to β- 1, 6-OSs with the help of SGBP (65% homologous to B. thetaiotaomicron VPI-5482) and then imports it into the periplasmic via SusCD complex ( Figure 7 ).…”