Exploring the effects of redox within the bacterial biofilm lifecycle of Pseudomonas aeruginosa – the importance of sensory domains

Abstract

Bacteria form sessile antimicrobial-tolerant communities called biofilms, threatening health, infrastructure, and the environment. Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen, responsible for causing many chronic infections, particularly in the lungs of Cystic Fibrosis patients. Environmental stimuli regulate biofilm dispersal to benefit the survival of the bacterial cells. Redox changes regulate biofilm dispersal, and nitric oxide has a role in this process. Several proteins respond to redox changes to disperse the biofilm, and these often contain the sensory PAS domains. The complete pathway of this redox-stimulated biofilm dispersal is currently not fully comprehended.
Within this thesis the concept of bacterial biofilms and the involvement of redox in their lifecycle is explored. With a focus on PAS domains, several proteins which are redox-responding and regulate biofilm lifecycle are studied, namely BdlA, PipA, PA2072, and RbdA. Firstly, analysis was completed to further a bioinformatical study to predict the functions of lesser researched proteins in P. aeruginosa. Comparing the PAS domains phylogenetically and sequentially to a reference set of structurally characterised PAS domains generated testable hypotheses and highlighted PAS domains for which the ligand/cofactor has not been yet discovered in the P. aeruginosa genome; some of which were followed up with structure prediction analysis. BdlA, a biofilm dispersal protein with two PAS domains, was identified to have a disulphide bond which could be responsible for the reaction to redox changes. This disulphide was mutated, and the structure solved, indicating movement of residues surrounding this bond. PipA, a phage-inducing phosphodiesterase which can disperse the biofilm, has two PAS domains, the structures of which were each individually solved. The PAS1 domain is shown not to have a cofactor/ligand bound but to have a large cavity of interest. The PAS2 domain non-covalently binds an FAD cofactor, shown via UV-visible spectroscopy to be able to chemically reduce in solution and photoreduce as a cause of X-rays in crystallo, but the structure of the flavin did not vastly differ between the oxidised and reduced states seen at synchrotrons or XFEL sources. However, there was some evidence of minor change occurring in X-ray pump-probe analysis which. Finally, PA2072 and RbdA are compared and contrasted for their similar domain composition and opposite functionalities, including comparison of PAS and periplasmic domains with structural prediction highlighting key differences and a potential mechanism is unveiled. The structure of the EAL domain of the RbdA protein in a catalytically primed dimer form is shown. Overall, this thesis contributes to further understanding of PAS domains, and adds further insight into how redox induced bacterial biofilm dispersal is conducted.

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Identifiers

ORCID for Charlotte Rose Cordery: orcid.org/0000-0003-2321-8144

ORCID for Ivo Tews: orcid.org/0000-0002-4704-1139

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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