Investigating the interactions of microbial pathogens with membrane glycoproteins

Binding of pathogen to host often involves glycans on host epithelial surfaces, a complex process that poses challenges for in vitro studies. In this thesis, methods were developed to study the binding of bacteria to human epithelial membrane glycoproteins by Pseudomonas aeruginosa, a common respiratory pathogen of individuals with cystic fibrosis (CF). A plate assay was developed and revealed differences between the adhesion of clinical strains isolated from four CF patient sputum samples, and a laboratory prototype of P. aeruginosa, to epithelial mucins. Isothermal titration calorimetry was also trialled for measurement of this interaction, but the generation of high background heat compromised the measurement of receptor-binding. Thus the plate assay was used to investigate the role of the glycan component of the glycoproteins in pathogen binding, by assessing bacterial binding before and after treatment of membranes from human epithelial lung cells with glycosidases. All strains increased adhesion following removal of sialic acid, but galactosidase treatment had a varied effect, demonstrating differences between the P. aeruginosa strains in their affinity to different glycan substructures. The findings and techniques developed by this thesis will be useful in future studies that seek to understand more about glycan involvement in pathogen to host adhesion.