Metabolism of human milk oligosaccharides by infant-associated bifidobacteria
Bifidobacteria are Gram-positive, anaerobic bacteria belonging to the Actinobacteria phylum, and are commensals of the mammalian, avian and occasionally insect gastrointestinal tracts. In humans, bifidobacteria are typically highly abundant in the intestinal microbiota of healthy breastfed infants, in particular a small number of infant-associated species, including Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum and Bifidobacterium kashiwanohense. A key adaptation, facilitating the establishment and dominance of these species in the breastfed infant gut microbiota, is the ability to consume and metabolise specific glycans only found in breastmilk, and indigestible for the infant, known as Human Milk Oligosaccharides (HMOs). Fascinatingly, the adaptation to utilise HMO as a substrate by bifidobacteria is almost exclusively reserved for infant-associated species, and even more fascinatingly, these species have developed strikingly varying strategies for the consumption of different HMO components. Strategies for the metabolism of various individual HMO glycans in different species of bifidobacteria shall be discussed in this thesis, with particular focus on B. breve UCC2003 and the novel isolate B. kashiwanohense APCKJ1. Chapter II of this thesis is comprised of work elucidating the mechanisms and components of LNT, LNnT and LNB utilisation pathways in Bifidobacterium breve UCC2003. Using a combination of experimental approaches, the enzymatic machinery involved in the metabolism of LNT, LNnT and LNB is identified and characterised. Furthermore, the distribution across the genus, of homologs for the key genes involved in the utilisation of these substrates, is analysed. Chapter III focuses on identifying the regulatory network responsible for the transcriptional control of the genes involved in LN(n)T and LNB metabolism, as described in Chapter II. Three transcriptional regulators and corresponding operator and associated (inducible) promoter sequences are characterised, the latter governing transcription of the genetic elements involved in LN(n)T/LNB metabolism. Furthermore, identification of the transcriptional effectors reveals the presence of a series of positive-feedback loops, inducing expression in the presence of breakdown products of key HMO-derived metabolites. In Chapter IV, Bifidobacterium isolates are obtained by screening a number of faecal samples from breastfed infants, using HMO components fucosyllactose and sialyllactose as selective carbohydrates, and compared with isolates obtained using lactose or GOS. A range of bifidobacterial species were obtained, varying between the selective carbohydrate used, and supporting the notion of selective HMO consumption. Analysis of the glycosyl hydrolase profiles of representative strains of the species obtained reveals interesting correlations with their preferential carbohydrate-based selection. In Chapter V, a novel B. kashiwanohense isolate, APCKJ1, which was isolated in the work of Chapter IV, is demonstrated to consume fucosyllactose, and the mechanisms of its utilisation of both fucosyllactose and L-fucose is examined. Using a combination of approaches, the main cellular machinery involved in the uptake and degradation of fucosyllactose is characterised, and heterologous expression of these genes in a B. breve UCC2003 host reveals not only the mechanisms of the utilisation of fucosyllactose, but a potentially functional pathway for the catabolism of fucose, in both strains. The work presented in this thesis represents novel information on the metabolism of HMO glycans in bifidobacteria, particularly in the species B. breve and B. kashiwanohense, as well as key insights into the strategies of HMO utilisation by infant-associated bifidobacteria in general, as an adaptation to the GIT of breastfed infants.
Microbiology, Probiotics, Prebiotics, Bifidobacteria, Infant Health, Human milk oligosaccharides, Carbohydrate metabolism, Molecular biology
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