Sugar-pathogen interactions: breast milk as the first line of defence against infections
thesisposted on 28.03.2022, 00:45 by Wai Yuen Cheah
Bacterial adhesion to host cell surfaces is the first step leading to bacterial infection and involves adhesins on the bacterial cell surface binding to host cell receptors. Milk glycans, carried on glycoproteins and glycolipids, and as free oligosaccharides, are thought to offer protection to infants by binding to pathogenic bacteria with subsequent clearing of the harmful microorganisms from the gastrointestinal tract. The quantitative interaction between milk glycoproteins and fluorescently-labelled human gastrointestinal-colonising bacteria were studied using glass slide glycan microarray technology and a new 96 well microtitre plate based assay. The latter assay more closely resembled the complex binding of total milk proteins to the human gastrointestinal bacteria, Escherichia coli, Salmonella typhimurium, Campylobacter jejuni and Lactobacillus rhamnosus, and demonstrated that the bacteria bound differentially to the milk proteins and that the adhesion was glycan-dependent. Reduced binding was observed for all four gastrointestinal bacterial strains when sialic acid was removed from the milk glycoproteins, suggesting that bacterial binding was at least partially sialic acid specific. The binding to total human milk proteins by the oral streptococci species that are encountered at the initial stage of digestion in the mouth was shown to also involve sialic acid in Streptococcus gordonii adhesion but Streptococcus mutans appeared to recognise different binding epitopes. Bovine and human milk protein fractions were compared for their capacity to bind to human gastrointestinal-colonising bacterial strains. Although there was more protein in bovine milk, volume equivalents of each type of milk protein bound the bacteria differentially with the pathogenic S. typhimurium and the commensal bacterium L. rhamnosus having a higher binding affinity to total human milk proteins as compared to total bovine milk proteins. All the human gastrointestinal bacteria bound more strongly to the whey proteins than to caseins, with involvement of a number of different whey glycoproteins (e.g. immunoglobulins, lactoferrin, β-lactoglobulin) in bacterial adhesion. Again, sialic acid was shown to be involved in the bacterial binding to both bovine and human milk glycoprotein fractions. As further indication of the importance of glycans as receptors for bacterial adhesion to gastrointestinal cells and for the role of milk glycoproteins in competitively inhibiting this binding, N-linked glycans released from both human and bovine milk whey proteins were shown to significantly inhibit all three pathogens, C. jejuni, E. coli and S. typhimurium, from binding to human gastrointestinal epithelial cells. Interestingly, N-linked glycans released from human milk whey proteins were more effective at inhibiting the binding of C. jejuni and L. rhamnosus to the intestinal cells than bovine milk whey protein N-linked glycans, whereas milk glycans from both mammalian species inhibited E. coli and S. typhimurium adhesion to approximately the same extent. The human milk N-linked glycans were also significantly more effective in inhibiting invasion of C. jejuni into the cells than the bovine milk N-linked glycans. This study showed predominately sialylated, core fucosylated structures in the released N- and O-linked glycans of both human and bovine milk proteins. There was a significant difference in the N-glycosylation specific structures between the secreted milks and human cell surface glycoproteins with a high abundance of sialylated structures, well described bacterial lectin receptors, seen across all three sources. Both the human milk glycoproteins and the human epithelial cells carried Lewis epitopes, known receptors for bacterial adhesion. In summary, we have shown that the bacterial interactions with cell surface (epithelial cells) and secreted glycoproteins in both human and milk are glycan-dependent. The data in this study repeatedly emphasises the anti-adhesive and anti-invasive properties of human (and to a lesser extent bovine) milk protein glycans to prevent enteric bacterial infections in the gastrointestinal tract of breastfed infants. It also provides further insights into the possibility of using N-linked human milk glycan structures to mimic this innate immune protection of breastfed infants and improve the formulation of breast milk substitutes.