Molecular mechanism of alginate polymerisation and modifications in Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements for degree of Doctor of Philosophy in Microbiology at Massey University, New Zealand

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Massey University
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Pseudomonas aeruginosa is an ubiquitous opportunistic human pathogen in immunocompromised patients. It is of particular relevance to cystic fibrosis (CF) patients where it frequently causes chronic bronchopulmonary infection and is the leading cause of morbidity and mortality. The decline in lung function is caused by the emergence of a mucoid variant showing excessive production of the exopolysaccharide, alginate. The alginate-containing biofilm matrix of this mucoid variant protects P. aeruginosa from the immune system and antibiotics. Here the alginate biosynthesis/modification/secretion multiprotein complex was investigated with regard to protein-protein interactions constituting the proposed multiprotein complex and the molecular mechanisms underlying alginate polymerisation and modifications. This study sheds light on the structure and function of various alginates from a material property and biological function perspectives. The binary interactions of AlgK-AlgE, AlgX-Alg44, AlgK-Alg44 and Alg8-Alg44 were identified proposing a new model for this multiprotein complex organisation. Proteinprotein interactions were found to be independent of c-di-GMP binding to PilZAlg44 domain. C-di-GMP-mediated activation of alginate polymerisation was found to be different from activation mechanism proposed for cellulose synthesis. This study showed that alginate polymerisation and modifications were linked. It was shown that the molecular mass of alginate was reduced by epimerisation, while it was increased by acetylation. It was determined that previously characterized proteins AlgG (epimerase) and AlgX (acetyltransferase) have mutual auxiliary and enhancing roles. Biofilm architecture analysis showed that acetyl groups lowered viscoelasticity of alginates and promoted cell aggregation, while nonacetylated polymannuronate alginate promoted stigmergy. Experimental evidence was provided that Alg44 boosted acetylation while the periplasmic domain of this protein was critical for protein stability and regulation of alginate modifications. Full-length Alg44 was purified and it was found to be a dimer in solution. Overall, this study sheds new light on the arrangement of the proposed alginate biosynthesis/modification/secretion multiprotein complex. Furthermore, the activation mechanism and the interplay between polymerisation and modification of alginate were elucidated and new functions and interactive role of alginate-polymerising and– modifying subunits were further understood.
Pseudomonas aeruginosa, Molecular aspects, Alginates, Polymerization