Proteomic analyses of Bacteroides ovatus and Bifidobacterium longum in xylan bidirectional culture shows sugar cross-feeding interactions.

The intestinal microbiome is a community of anaerobic microorganisms whose activities significantly impact human health. Its composition can be modulated by consuming foods rich in dietary fiber, such as xylan, a complex polysaccharide that can be considered an emerging prebiotic. In this work, we evaluated how certain gut bacteria acted as primary degraders, fermenting dietary fibers, and releasing metabolites that other bacteria can further use. Different bacterial strains of Lactobacillus, Bifidobacterium, and Bacteroides were evaluated for their ability to consume xylan and interact with one another. Results from unidirectional assays gave indications of possible cross-feeding between bacteria using xylan as a carbon source. Bidirectional assays showed that Bifidobacterium longum PT4 increased its growth in the presence of Bacteroides ovatus HM222. Proteomic analyses indicated that B. ovatus HM222 synthesizes enzymes facilitating xylan degradation, such as ß-xylanase, arabinosidase, L-arabinose isomerase, and xylosidase. Interestingly, the relative abundance of these proteins remains largely unaffected in the presence of Bifidobacterium longum PT4. In the presence of B. ovatus, B. longum PT4 increased the production of enzymes such as α-L-arabinosidase, L-arabinose isomerase, xylulose kinase, xylose isomerase, and sugar transporters. These results show an example of positive interaction between bacteria mediated by xylan consumption. Bacteroides degraded this substrate to release xylooligosaccharides, or monosaccharides (xylose, arabinose), which might support the growth of secondary degraders such as B. longum.

Screening Microbial Interactions During Inulin Utilization Reveals Strong Competition and Proteomic Changes in Lacticaseibacillus paracasei M38.

Competition for resources is a common microbial interaction in the gut microbiome. Inulin is a well-studied prebiotic dietary fiber that profoundly shapes gut microbiome composition. Several community members and some probiotics, such as Lacticaseibacillus paracasei, deploy multiple molecular strategies to access fructans. In this work, we screened bacterial interactions during inulin utilization in representative gut microbes. Unidirectional and bidirectional assays were used to evaluate the effects of microbial interactions and global proteomic changes on inulin utilization. Unidirectional assays showed the total or partial consumption of inulin by many gut microbes. Partial consumption was associated with cross-feeding of fructose or short oligosaccharides. However, bidirectional assays showed strong competition from L. paracasei M38 against other gut microbes, reducing the growth and quantity of proteins found in the latter. L. paracasei dominated and outcompeted other inulin utilizers, such as Ligilactobacillus ruminis PT16, Bifidobacterium longum PT4, and Bacteroides fragilis HM714. The importance of strain-specific characteristics of L. paracasei, such as its high fitness for inulin consumption, allows it to be favored for bacterial competence. Proteomic studies indicated an increase in inulin-degrading enzymes in co-cultures, such as ß-fructosidase, 6-phosphofructokinase, the PTS D-fructose system, and ABC transporters. These results reveal that intestinal metabolic interactions are strain-dependent and might result in cross-feeding or competition depending on total or partial consumption of inulin. Partial degradation of inulin by certain bacteria favors coexistence. However, when L. paracasei M38 totally degrades the fiber, this does not happen. The synergy of this prebiotic with L. paracasei M38 could determine the predominance in the host as a potential probiotic.

Metabolic Modeling and Bidirectional Culturing of Two Gut Microbes Reveal Cross-Feeding Interactions and Protective Effects on Intestinal Cells.

The gut microbiota is constituted by thousands of microbial interactions, some of which correspond to the exchange of metabolic by-products or cross-feeding. Inulin and xylan are two major dietary polysaccharides that are fermented by members of the human gut microbiota, resulting in different metabolic profiles. Here, we integrated community modeling and bidirectional culturing assays to study the metabolic interactions between two gut microbes, Phocaeicola dorei and Lachnoclostridium symbiosum, growing in inulin or xylan, and how they provide a protective effect in cultured cells. P. dorei (previously belonging to the Bacteroides genus) was able to consume inulin and xylan, while L. symposium only used certain inulin fractions to produce butyrate as a major end product. Constrained-based flux simulations of refined genome-scale metabolic models of both microbes predicted high lactate and succinate cross-feeding fluxes between P. dorei and L. symbiosum when growing in each fiber. Bidirectional culture assays in both substrates revealed that L. symbiosum growth increased in the presence of P. dorei. Carbohydrate consumption analyses showed a faster carbohydrate consumption in cocultures compared to monocultures. Lactate and succinate concentrations in bidirectional cocultures were lower than in monocultures, pointing to cross-feeding as initially suggested by the model. Butyrate concentrations were similar across all conditions. Finally, supernatants from both bacteria cultured in xylan in bioreactors significantly reduced tumor necrosis factor-α-induced inflammation in HT-29 cells and exerted a protective effect against the TcdB toxin in Caco-2 epithelial cells. Surprisingly, this effect was not observed in inulin cocultures. Overall, these results highlight the predictive value of metabolic models integrated with microbial culture assays for probing microbial interactions in the gut microbiota. They also provide an example of how metabolic exchange could lead to potential beneficial effects in the host. IMPORTANCE Microbial interactions represent the inner connections in the gut microbiome. By integrating mathematical modeling tools and microbial bidirectional culturing, we determined how two gut commensals engage in the exchange of cross-feeding metabolites, lactate and succinate, for increased growth in two fibers. These interactions underpinned butyrate production in cocultures, resulting in a significant reduction in cellular inflammation and protection against microbial toxins when applied to cellular models.

Molecular Insights Into O-Linked Glycan Utilization by Gut Microbes.

O-linked glycosylation is a post-translational modification found mainly in eukaryotic cells, which covalently attaches oligosaccharides to secreted proteins in certain threonine or serine residues. Most of O-glycans have N-acetylgalactosamine (GalNAc) as a common core. Several glycoproteins, such as mucins (MUCs), immunoglobulins, and caseins are examples of O-glycosylated structures. These glycans are further elongated with other monosaccharides and sulfate groups. Some of them could be found in dairy foods, while others are produced endogenously, in both cases interacting with the gut microbiota. Interestingly, certain gut microbes can access, release, and consume O-linked glycans as a carbon source. Among these, Akkermansia muciniphila, Bifidobacterium bifidum, and Bacteroides thetaiotaomicron are prominent O-linked glycan utilizers. Their consumption strategies include specialized α-fucosidases and α-sialidases, in addition to endo-α-N-acetylgalactosaminidases that release galacto-N-biose (GNB) from peptides backbones. O-linked glycan utilization by certain gut microbes represents an important niche that allows them to predominate and modulate host responses such as inflammation. Here, we focus on the distinct molecular mechanisms of consumption of O-linked GalNAc glycans by prominent gut microbes, especially from mucin and casein glycomacropeptide (GMP), highlighting the potential of these structures as emerging prebiotics.