Glycan structural determinants and their role in microbial interaction

Microbial infection is initiated only after adherence to the host cell surface. In many cases the microbial interactions with the host surface are mediated between the glycans on the host cell surface and the carbohydrate binding proteins of the pathogen. Mucosa! epithelial surfaces, such as coat the oral cavity, are potential sites for colonisation by oral microorganisms. Saliva constantly bathes the buccal epithelial cell (BEC) surface of the mouth and in this study we have used the oral cavity as a model system to demonstrate the innate immune protective role played by the glycan receptors on the proteins of saliva against the adhesion of the oral pathogen C. a/bicans to the BEC. Thereby, this work could help design glycan inhibitors similar to the host's evolved innate immune system to naturally evade pathogens and thus address the problem of increased microbial resistance to antibiotics. In the first phase of this work (Chapter 2, Publication I), a flow cytometry based adhesion assay was developed to quantify the interaction between buccal cells and the commensal oral pathogen Candida albicans. The structures of the N-and 0-linked oligosaccharides on the glycoproteins of saliva and BEC membranes were analysed using capillary carbon negative ion LC-ESI MS/MS. A total of 190 glycan structures were characterised and found to be qualitatively similar between saliva and epithelial buccal cell membrane proteins, but differed quantitatively in their relative amounts. The similarity of the terminal glycan epitope structures on saliva and BEC membrane glycoproteins, and the fact that whole saliva and released glycans from salivary proteins inhibited the interaction of C. albicans with BEC, confirmed the protective role of the glycans on salivary glycoproteins against pathogen infection of the oral surface mucosa. Further investigation of the glycan determinants identified on the terminal N-and 0-glycan structures of BEC and saliva supported the proposed function of blood group antigens as an evolutionary selection against pathogen infection. The detailed mass spectrometric glycan characterisation and relative quantification of BEC membrane glycans (Chapter 3) was carried out on 19 individuals of various A, 8, AB and O blood group types. The N-glycans of BEC were similar in all secretor individuals and did not display the A and B determinants; while non-secretors did not possess the 0/H antigens. Jn contrast, the 0-glycans on the membrane proteins of BEC from secretory individuals expressed the A, B and H antigens, while the non-secretors lacked any of these structures. The Lewis x/a and Lewis y/b blood group antigens were observed on secretor individuals N- and O-glycans; in non-secretor individuals, as expected, only Lewis x/a antigens were present. Multivariate statistical analysis showed that C. albicans demonstrated a significantly (p < 0.05) higher preference to adhere to BEC of blood group O individuals. The diagnostic and signature fragment ions produced by negative ion MS/MS fragmentation, together with the elution selectivity of PGC retention, were identified and applied to differentiate the N- and 0-glycan isomer structures of the complex salivary glycans (Chapter 4, Publication 2). This labour-intensive approach led to the construction of a PGC-LC-ESI-IT-MS2 tandem mass spectral repository on the online UniCarb-DB database which was further expanded by an online MS2 fragment spectral library of 30 common glycan substructures that typically occur at the non-reducing terminus of glycoconjugates, fragmented in the positive and negative ion mode (Chapter 5, Publication 3). The substructure spectra were used to identify and confirm terminal glycan determinants from the multistage (MS3) mass spectra of the salivary N- and O-glycans. These mass spectrometric insights will enable the easier identification and confirmation of glycan determinants on oligosaccharides released from glycoproteins in future analyses. The work presented here applies negative ion PGC-LC-ESI-MS/MS analysis for the detailed characterisation of the N- and O-glycans on epithelial cell surface and secreted fluid proteins and demonstrates the role played by terminal glycan structural determinants as receptors for pathogen binding.