Bacterial biofilms are surface associated colonies that are of considerable concern and interest to industry, medicine and research. They are resistant to antibiotics, their host’s defences and are able to survive under harsh conditions. Biofilm formation in many bacterial strains are dependent on the production of a polysaccharide intercellular adhesion (PIA), a beta-(1-->6)-N-acetylglucosamine polymer. Vaccines derived from biologically isolated PIA have shown efficacy against clinically isolated strains of E. coli and pathogenic strains of S. aureus in animal models. Accordingly, chemically synthesized neoglycoconjugates based on PIA glycosides will be developed to serve as lead compounds for the development of new antibiotics as well as vaccines against biofilm dependent infections. Described in this thesis is a comprehensive study of the synthesis of PIA oligosaccharides and their deacetylated equivalents, the strategy for installing a stable linker on the free reducing oligosaccharide terminus and finally the conjugation to a model carrier protein for the development of potential neoglycoprotein vaccines.

Bacterial biofilms are surface associated colonies that are of considerable concern and interest to industry, medicine and research. They are resistant to antibiotics, their host’s defences and are able to survive under harsh conditions. Biofilm formation in many bacterial strains are dependent on the production of a polysaccharide intercellular adhesion (PIA), a beta-(1-->6)-N-acetylglucosamine polymer. Vaccines derived from biologically isolated PIA have shown efficacy against clinically isolated strains of E. coli and pathogenic strains of S. aureus in animal models. Accordingly, chemically synthesized neoglycoconjugates based on PIA glycosides will be developed to serve as lead compounds for the development of new antibiotics as well as vaccines against biofilm dependent infections. Described in this thesis is a comprehensive study of the synthesis of PIA oligosaccharides and their deacetylated equivalents, the strategy for installing a stable linker on the free reducing oligosaccharide terminus and finally the conjugation to a model carrier protein for the development of potential neoglycoprotein vaccines.

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)