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• W122156902 abstract "Phase variation is the reversible, high frequency on/off switching of gene expression and is a common feature of surface expressed virulence determinants in pathogenic bacteria. Phase variation of surface associated genes permits the bacterium to evade host immune responses by the alteration of surface epitopes that elicit an immune response. Phase variation is also used to create a phenotypically diverse population of bacteria, some of which will be better suited to survival in the rapidly changing host micro-environment. The study of phase variable modifications in Haemophilus influenzae and pathogenic Neisseria will expand the understanding of how these bacteria can invade and persist in their human hosts. Critically, these studies are hampered by a lack of efficient and sensitive methods for the detection and analysis of such modifications. This thesis describes the development and use of protocols for the analysis of either glycosylation or methylation. Three distinct biological problems were addressed during the course of this study, each focused on a specific phase variable modification in H. influenzae and the pathogenic Neisseria. Firstly, a strategy to selectively label glycoproteins in H. influenzae for the purpose of identifying novel, unreported glycoproteins was developed. The strategy was a technical success with only the known H. influenzae glycoproteins, HMW1 and HMW2, detected via the modified Leloir pathway. Despite the presence of multiple glycoproteins in other pathogenic bacteria, no other galactose and glucose dependent glycoproteins were found to be expressed in this strain of H. influenzae. Interestingly, analysis of the Leloir pathway enzyme, PgmB, showed that it is not required for HMW glycosylation and processing, despite what had been previously reported by others. Furthermore, it was conclusively shown that the OpsX enzyme is not phenotypically linked to the HMW glycoproteins. Secondly, this thesis describes steps towards the development of an efficient and sensitive method for the identification of methylated adenosines, specifically ModA2 methylated adenosines. Such a method should make it possible to identify the binding sites of the remaining ModA methyltransferases. Five independent approaches were developed throughout the course of the study, specifically: 3H-labelling, immunoblot detection, biotin labelling, electrophoretic mobility shift assay and restriction inhibition. Finally, the mechanisms governing modA diversity were investigated using a bioinformatic approach. Recombination was identified as having a major role in modA diversity early in the evolution of H. influenzae and the pathogenic Neisseria. Successful mod alleles have been widely disseminated and their DNA recognition sequences strictly maintained. More recently, there is evidence that entire DNA recognition domains have been exchanged between mod genes in different genetic backgrounds, enabling rapid evolution of regulatory responses to host immune responses. It is predicted that the number of modA groups will increase as further modA and genome sequencing is undertaken, with an additional two alleles (modA19 and modA20) identified in this study alone. The role of phase variable modifications in H. influenzae and the pathogenic Neisseria is complex and the true extent of the role that methylation and glycosylation plays in the pathogenicity of these organisms is a fascinating web of molecular interactions that have yet to be completely unravelled." @default.
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• W122156902 date "2011-02-01" @default.
• W122156902 modified "2023-09-27" @default.
• W122156902 title "Phase Variable Modifications in Haemophilus influenzae and the Pathogenic Neisseria" @default.
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