Surviving and thriving in the cystic fibrosis lung: contribution of an arginine specific aminopeptidase to sustained infection by Pseudomonas aeruginosaTools Spencer, Daniella Lucy (2019) Surviving and thriving in the cystic fibrosis lung: contribution of an arginine specific aminopeptidase to sustained infection by Pseudomonas aeruginosa. PhD thesis, University of Nottingham.
AbstractPseudomonas aeruginosa is a prominent pathogen within the Cystic Fibrosis (CF) community which can survive the challenges posed by the immune system and antibiotic treatment in patient lungs. P. aeruginosa is such a success in this environment for numerous reasons that including (i) a multifactorial virulence strategy, (ii) capacity to form biofilms, and (iii) the ability to thrive under low oxygen conditions. P. aeruginosa possesses an Arginine specific Autotransporter Aminopeptidase (AaaA). The arginine that AaaA releases from the N-terminal of proteins/peptides is used by P. aeruginosa as a nitrogen and energy source, which is hypothesized to confer a fitness advantage in oxygen limited environments. To further understand the relevance of AaaA to chronic P. aeruginosa CF airway infections, this work investigated whether aaaA: (i) is controlled by regulators relevant to the CF environment; (ii) is present and active in CF isolates; (iii) can be purified for the purpose of inhibitor development, and (iv) plays a role in biofilm formation. A panel of CF isolates showed the presence of a highly conserved aaaA gene with some SNPs outside the active site. The levels of AaaA activity in these strains differed, but did not correlate with any particular phenotypic trait. Comparison of aaaA promoter sequences from GenBank revealed a 169 bp promoter deletion in some P. aeruginosa strains including the Liverpool epidemic strain (LES). AaaA activity responded positively to a reduced oxygen concentration in the environment, and in line with this, activation of aaaA was found to be under the potential control of several transcription factors including ArgR, NarX/NarL plus the alternative sigma factor RpoN and AaaA itself. A qRT-PCR assay was developed for further testing of aaaA expression under low oxygen tensions. A recombinant protein was created to facilitate further studies including crystallisation and inhibitor screening. Finally, aaaA was found to be involved in biofilm development depending on the strain background and environmental conditions. This data supports the idea of a subtle, but important, role for AaaA in the chronic persistence of CF lung P. aeruginosa infections.
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