The influence of milk oligosaccharides on host-commensal interactions in the GI tract

This thesis sets out to explore the influence of milk oligosaccharides on the colonisation of the intestinal microflora and their potential downstream impact upon components of the gastrointestinal tract. Milk oligosaccharides are known to act as prebiotics for the early microflora, but it was not yet known if these molecules possessed alternative bioactivities relating to intestinal adhesion. Various oligosaccharides present in human and bovine milk were screened for their ability to influence the adhesion of an infant-associated commensal, Bifidobacterium longum subsp. infantis ATCC 15697, to in vitro intestinal models (Chapter II). Screening of individual oligosaccharides identified that exposure to 6'sialyllactose, but not 3'sialyllactose, could promote adhesion to the HT-29 cell line. Interestingly, exposure to a combination of 3'- and 6'-sialyllactose resulted in a dramatic increase in adhesion to the HT-29 cell line. 3'- and 6'-sialyllactose, alone or in combination, increased adhesion to the Caco-2 model, though to a reduced extent. Parameters such as oligosaccharide concentration, duration of oligosaccharide exposure, enzymatic treatments of the bacteria, and a screen of other bacterial strains were used to explore the activity of 6'sialyllactose to promote adhesion to the HT-29 cell line in greater detail. The capability of 6'sialyllactose to induce an adhesive phenotype was unique to Bifidobacterium longum subsp. infantis ATCC 15697, required a threshold oligosaccharide concentration above 0.5mg/ml, and likely involves a surface protein or combination of proteins. Following the discovery of the novel bioactivity of 6'sialyllactose, the genetic basis for the induced adhesive-phenotype was investigated. Oligosaccharide exposures were replicated as in the previous in vitro adhesion studies. Genetic expression was assessed through the use of whole genome DNA microarray analysis. Exposure to the combination of 3'- and 6'-sialyllactose resulted in both, the highest number of differentially transcribed genes and the greatest magnitude of transcription. Levels of gene transcription correlated with the trend of increased adhesion to the HT-29 cell line. The study identified several genes related to either stress-response or colonising factors (dnaK, groEL, sortase, dps-ferritin), which were further validated through the use of qPCR. Several of the genes have been confirmed as colonising factors in alternative strains of bacteria. This study demonstrates a connection between exposure to a combination of predominant sialylated milk oligosaccharides and an adaptive colonising response of B. longum subsp. infantis ATCC 15697. Milk oligosaccharides have demonstrated activity as both prebiotic and anti-infective molecules. Although human milk contains the highest concentration and greatest diversity of oligosaccharides, bovine milk and dairy whey streams contain several of the common acidic oligosaccharides and represent a scalable source of bioactive oligosaccharides. Bovine milk oligosaccharides (BMO) isolated from dairy whey streams and 6'sialyllactose were tested against an established commercial prebiotic, oligofructose, to assess their safety profile and impact on the microbial communities in the murine gut (Chapter III). Findings indicated that none of the treatments significantly affected markers of probiotic activity via short chain fatty acid production or altered IgA or cytokine profiles. Oligofructose demonstrated mixed activity to alter bacterial family proportions, while BMO and 6'sialyllactose were associated with significantly reduced proportions of bacterial families containing notable pathogens. The findings of chapter III demonstrate the overall safety of oligosaccharide supplementation and their ability to modulate the murine intestinal microbiome, with potential applications in infant formulations and functional foods. With the ability of milk oligosaccharides to promote the growth and adhesion of bifidobacteria, which are in turn decorated with numerous glycans on their cell walls, it is likely that altering the numbers of bifidobacteria will influence many lectin-mediated interactions in the gastrointestinal tract. Accordingly, a panel of commensal bacteria were screened for their ability to interact with galectin-3, a galactose-binding glycoprotein, which is notably expressed in the epithelial cells of the respiratory and gastrointestinal tract and can influence both metastasis and pathogenic bacterial colonisation (Chapter IV). Surface plasmon resonance was employed for the screening assay, identifying two strains of HMO-consuming commensal bacteria, B. longum subsp. infantis, which interacted with galectin-3 to a greater extent than the pathogenic positive control. The interaction was further validated and characterized through the use of agglutination and solid-phase binding experiments. The galectin-3-bacteria interaction is mediated through a carbohydrate moiety; however, the entire galectin-3 protein is required for optimal binding. The results demonstrate for the first time a novel interaction between galectin-3 and commensal bacteria. As the two strains can be influenced by HMOs via increased growth or colonisation, the findings present a potentially novel means to modulate the activity of galectin-3 in the GI tract. Overall these studies build a strong case for the use of milk oligosaccharides to modulate the intestinal microflora, with bovine milk and dairy whey streams presenting an abundant and yet untapped resource. Future research will aid in further elucidating the intricacies of the oligosaccharide-microflora-GI tract interaction, thereby identifying novel therapeutic targets and means by which to maintain or restore host health.2016-11-1