Mucin-driven ecological interactions in an in vitro synthetic community of human gut microbes

Abstract

Specific human gut microbes inhabit the outer mucus layer of the gastrointestinal tract. Certain residents of this niche can degrade the large and complex mucin glycoproteins that constitute this layer and utilise the degradation products for their metabolism. In turn, this microbial mucin degradation drives specific microbiological ecological interactions in the human gut mucus layer. However, the exact nature of these interactions remains unknown. In this study, we designed and studied an in vitro mucin-degrading synthetic community that included mucin O-glycan degraders and cross-feeding microorganisms by monitoring community composition and dynamics through a combination of 16S rRNA gene amplicon sequencing and qPCR, mucin glycan degradation with PGC-LC-MS/MS, production of mucin-degrading enzymes and other proteins through metaproteomics, and metabolite production with HPLC. We demonstrated that specialist and generalist mucin O-glycan degraders stably co-exist and found evidence for cross-feeding relationships. Cross-feeding on the products of mucin degradation by other gut microbes resulted in butyrate production, hydrogenotrophic acetogenesis, sulfate reduction and methanogenesis. Metaproteomics analysis revealed that mucin glycan degraders Akkermansia muciniphila, Bacteroides spp. and Ruminococcus torques together contributed 92% of the total mucin O-glycan degrading enzyme pool of this community. Furthermore, comparative proteomics showed that in response to cultivation in a community compared to monoculture, mucin glycan degraders increased carbohydrate-active enzymes whereas we also found indications for niche differentiation. These results confirm the complexity of mucin-driven microbiological ecological interactions and the intricate role of carbohydrate-active enzymes in the human gut mucus layer.

Keywords: Akkermansia; CAZymes; gut microbiota; mucin; synthetic community.

Graphical Abstract

Fig. 1

Relative abundance of MDSC species over time based on a combination 16S rRNA gene amplicon sequencing and qPCR. Relative abundances of B. caccae, B. fragilis, B. thetaiotaomicron, and P. vulgatus were determined by species-specific qPCR. Data is normalised for 16S rRNA gene copy number per species.

Fig. 2

Number of fucosidases, galactosidases, hexosaminidases, sialidases, and sulfatases detected in MDSC during the stable phase, coloured by the organism that produced them.

Fig. 3

Detection of different enzyme categories per species. A circle around the species code signifies that a gene coding for this enzyme type was present in the genome (that was used as a database for the analysis of proteomics data) of this species. A filled circle indicates that this enzyme was detected in our metaproteomic analysis. Am = A. muciniphila; Bc = B. caccae; Bf = B. fragilis; Bt = B. thetaiotaomicron; Pv = P. vulgatus; Ac = A. caccae; Fd = F. duncaniae; Ah = A. hallii; Ar = A. rectalis; Ri = R. intestinalis; Bh = B. hydrogenotrophica; Dp = D. piger; Ms = M. smithii.

Fig. 4

Glycosyl hydrolases (GHs) per GH family detected in the synthetic community.

Fig. 5

Mucin monosaccharide metabolism by MDSC. A) Fucose, B) sialic acid, and GlcNAc, C) galactose. A circle around the species code signifies that this enzyme type was present in the genome (that was used as a database for the analysis of proteomics data) of this species. A filled circle indicates that this enzyme was identified in our proteomics analysis. Am = A. muciniphila; Bc = B. caccae; Bf = B. fragilis; Bt = B. thetaiotaomicron; Pv = P. vulgatus; Ac = A. caccae; Fd = F. duncaniae; Ah = A. hallii; Ar = A. rectalis; Ri = R. intestinalis; Bh = B. hydrogenotrophica; Dp = D. piger; Ms = M. smithii.

Fig. 6

Hydrogen scavenging pathways in MDSC: Acetogenesis by B. hydrogenotrophica (Bh), sulfate reduction by D. piger (Dp), and methanogenesis by M. smithii (Ms). A circle around the species code signifies that this enzyme type was present in the genome (that was used as a database for the analysis of proteomics data) of this species. A filled circle indicates that this enzyme was identified in our proteomics analysis.

Fig. 7

Differential detection of mucin-targeting glycosyl hydrolases by mucin glycan degraders in community compared to monoculture.

Fig. 8

Overview of mucin degradation within MDSC. Mucin glycan degraders apply carbohydrate-active enzymes and sulfatases to release monosaccharides and sulfate groups from mucin (A). Next, saccharides can be utilised by the mucin glycan degraders themselves or cross-feeding organisms (B), which results in the production of SCFAs. Hydrogen that is produced during the fermentation of mucin is mainly consumed through acetogenesis and sulfate reduction (C).

Fig. 9

Experimental overview. The inoculum was prepared from fresh precultures and used to inoculate the triplicate fermentors (F1-F3) at t = 0 h. From t = 0 h–6 h, the fermentors were operated in batch mode. From t = 6 h onwards, fermentors were operated in continuous mode and sampled every 24 h for community composition (16S rRNA gene amplicon sequencing and qPCR) and metabolite production (HPLC). During the stable phase (t = 72 h–120 h), additional samples were taken to assess protein production (metaproteomics) and mucin O-glycan degradation (PGC-LC–MS/MS).