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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Date
2015
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Massey University
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Abstract
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.
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Keywords
Pseudomonas aeruginosa, Molecular aspects, Alginates, Polymerization