Targeting the glycome of the milk fat globule membrane for anti-infective properties
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This thesis sets out to explore the potential bioactivities of bovine milk fat globule membrane (MFGM) to enhance our understanding of the roles bovine milk glycoconjugates may play in humans and to contribute towards further discovery and development of functional ingredients which may promote human health. Glycosylated MFGM proteins and lipids in particular, have been accredited with a number of bioactivities, but the full extent of their potential health promoting properties have yet to be realised. Given the reported anti-infective properties associated with bovine MFGM glycoproteins, a defatted bovine MFGM glycoprotein fraction, rich in proteins and glycoproteins (dMFGM), was generated. The ability of this fraction to inhibit Escherichia coli O157:H7 association with human colonic adenocarcinoma, HT-29 cells was subsequently investigated. The dMFGM fraction was observed to reduce the association of several E. coli O157:H7 strains with the HT-29 cells. This activity was strain-specific and concentration dependent. The parameters of the experimental assays were varied to identify the potential mechanisms by which the dMFGM fraction exerted its activity. The anti-infective activity was shown to occur as a result of the dMFGM interacting with the E. coli rather than the HT-29 cells. This study identified the potential use of defatted bovine MFGM to prevent the onset of E. coli infections in humans. Since the glycan portion of milk glycoconjugates may be an important factor in their health-promoting activities, the changes occurring in buttermilk, a source of MFGM, were profiled throughout the course of lactation. Buttermilk samples were generated at 13 time points during lactation for three multiparous animals and their glycosylation patterns were profiled through the use of lectin microarrays and lectin blotting. These techniques provided a platform for high-throughput analysis of the buttermilk sample glycosylation. The data suggested differences in glycosylation, including N-glycosylation, sialylation and fucosylation, between early and late time points. In addition, differences were evident between individual animals at various time points throughout the lactation cycle. The findings of this study may be invaluable in order to target the isolation of glycosylated ingredients for functional foods from particular lactation time points to maximise the abundance of particular glycan structures. In relation to further investigation of the anti-infective activities described above, defining the glycosylation patterns of MFGM and/or buttermilk is of importance however defining the surface glycosylation of pathogenic bacteria is also of great importance. Similarly, the host’s glycan presentation is important for pathogen binding and thus identification of the glycan binding preferences of pathogenic species is necessary to better understand how to reduce the threat of infection. The cell surface glycosylation and carbohydrate binding profiles of 5 enterohaemorrhagic E. coli (EHEC) O157:H7 strains and 2 enteropathogenic E. coli (EPEC) strains were characterized and compared using lectin and neoglycoconjugate microarrays in order to investigate if their surface glycosylation patterns and binding preferences were markedly different. The lectin microarray profiles suggested the presence of a variety of cell surface glycan structures with different apparent abundance or accessibility for each strain screened. An apparent difference between the surface glycosylation of the EHEC and EPEC strains was evident. Furthermore, the neoglycoconjugate microarray profiling identified the bacterial strains could bind to a relatively high number of oligosaccharide structures and interestingly, the binding preferences of EHEC and EPEC were markedly different. These results demonstrate that EHEC and EPEC surface glycosylation profiles and binding preferences differ. This may partially explain the different colonisation properties observed in chapter 2. Furthermore, knowledge of these glycan profiles may be invaluable in the design of anti-infectives targeted at specific bacterial strains. In addition to its anti-infective activity, bovine buttermilk and its components have been reported to possess immune-modulating properties. The transcriptional responses of colonic epithelial cells to buttermilk generated from colostrum and from mature milk were compared since the glycosylation status is known to change through the milk maturation process. Genomic microarrays were employed to identify any processes influenced by exposure to buttermilk. A number of processes were shown to be affected by both treatments including apoptosis, reproduction, immune system processes, barrier function, metabolism and stimulus response. Subsequently, immune system processes were chosen for further analysis as these were the most significantly modulated by the colostrum and mature buttermilk treatments. Polymerase chain reaction (PCR) validation indicated that the buttermilk treatments influenced the expression of a number of immune-associated genes including chemokine, cytokine and anti-viral genes. Interestingly, an apparent anti-inflammatory effect was observed indicating the potential of bovine buttermilk in preventing inflammatory diseases. Overall these studies indicate the potential of bovine MFGM fractions to promote human health by reducing the threat of infection and modulating immune response and inflammation. Moreover, the selection of bovine MFGM from certain lactation stages may be advantageous in order to increase the level of bioactivity by using the most appropriate glycosylation profile in the dairy fraction. Future research, including in vivo trials, are necessary to help identify the impact such fractions may have on the maintenance and preservation of human health.