Molecular strategies and interactions between representative bacteria of the human gut microbiota during the utilization of glycomacropeptide
Authors:   , González Morelo Kevin José 
Published: April 2024

The human gut microbiota plays an essential role in metabolizing complex dietary compounds that host enzymes cannot degrade. The prebiotic role of milk glycans, such as free human milk oligosaccharides (HMO), currently included in milk formulas to stimulate a healthy gut microbiota, has recently been validated. In addition, O-glycans are similarly complex oligosaccharides commonly found in intestinal mucins and milk proteins. While large extensions of O-glycans protect the epithelium cells, some gut microbes have been described to utilize them as carbon sources for colonization. O-glycans have been proposed as emerging prebiotics due to their remarkable similarity to host-associated glycans and HMO. Glycromacropeptide (GMP) is an O-glycosylated peptide obtained from whey during cheese manufacture. GMP could be considered a simple model of O-glycans to analyze the molecular mechanisms involved in metabolic interactions between gut microbes. This doctoral work aimed to determine the molecular strategies and microbial interactions while utilizing GMP as a carbon source. For this reason, this work pursues three specific objectives: i) To study co-expression networks in human milk oligosaccharides in Bifidobacterium species, ii) To study the mechanism of GMP O-glycan utilization by bacteria representative of the adult intestinal microbiome, and iii) To determine the metabolic cross-feeding interactions between representative bacteria of the human gut microbiome using GMP as a source of Oglycans. Chapter I critically discusses the different molecular strategies of O-glycan consumption by prominent bacteria of the gut microbiota currently available in the literature. Certain gut microbes can access, release, and consume O-linked glycans as a carbon source. Among these, Bifidobacterium bifidum and Bacteroides thetaiotaomicron are prominent O-linked glycan utilizers. Their consumption strategies include specialized α-fucosidases, α-sialidases, and endo-α-Nxvii 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. Chapter II focuses on the gene regulation of HMO to observe possible differences in gene expression architecture among Bifidobacterium species, as these oligosaccharides are chemically related to O-linked glycans. This chapter highlights in silico results from the co-expression networks obtained through WGCNA. This powerful systems biology methodology allows a better understanding of these oligosaccharides' consumption and gene expression. RNA-seq data obtained from Geo Datasets were obtained for Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum and Bifidobacterium longum subsp. longum. Between 10 and 20 co-expressing modules were obtained for each dataset. HMO-associated genes appeared in the modules with more genes for B. infantis and B. bifidum, in contrast with B. longum. Hub genes were identified in each module and generally participated in conserved essential processes. Certain modules were differentially enriched with LacI-like transcription factors, and others with specific metabolic pathways, such as the biosynthesis of secondary metabolites. The three Bifidobacterium transcriptomes showed distinct regulation patterns for HMO utilization. HMO-associated genes in B. infantis co-expressed in two modules according to their participation in galactose or N-Acetylglucosamine utilization. Instead, B. bifidum showed a less structured coexpression of genes participating in HMO utilization. Finally, this category of genes in B. longum clustered in a small module, indicating a lack of co-expression with main cell processes, and suggesting a recent acquisition. Chapter III highlights complex strategies for utilizing O-glycans in GMP consumption among gut microbes based on cross-feeding and competition. Individual cultures of representative bacteria allowed the identification of the major GMPdegraders. Unidirectional assays identified galacto-N-biose, galactose, Nxviii Acetylgalactosamine, and sialic acid as by-products, providing a perspective on microbial interactions during GMP fermentation. Bidirectional assays demonstrated cross-feeding activity, competition between gut microbes, and promotion of butyrate from the fatty acids derived from GMP. O-glycan-specific enzyme expression was identified for B. infantis ATCC 15697 and B. bifidum JCM 1254 during GMP crossfeeding consumption. This study highlights complex strategies for utilizing o-glycans in GMP consumption among gut microbes based on cross-feeding and competition.