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Papangeli, Maria (2022) SadB is a Pseudomonas aeruginosa global regulator. PhD thesis, University of Nottingham.

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Abstract

Bacteria can adhere to almost any surface and form resistant biofilm communities. These pose a major clinical burden as they can form on medical devices such as catheters and are hard to prevent. Catheter- associated urinary tract infections (CAUTIs) are the most common nosocomial infection as they represent ~40% of all nosocomial infections.

Hook et al., previously developed a series of polymers that prevent biofilm formation. Following a screen of 20,000 polymers and copolymers and subsequent scale-up, the hit acrylate polymer, EGdPEA has been clinically approved for the coating of urinary tract catheters and presents superior antibiofilm properties compared with silver-embedded silicone catheters. The bacterial biofilm resistance mechanism and surface sensing pathways involved are under investigation. In P. aeruginosa surface sensing includes the flagella, type IV pili and the wsp system.

The present study sought to investigate SadB, an essential gene for biofilm formation and CheA, a histidine kinase of the chemotaxis system for their involvement in surface sensing. SadB is involved in the switch from reversible to irreversible surface attachment. Expression of sadB under a constitutive promoter overcomes the resistance of EGdPEA to biofilm formation. CheA is responsible for transmitting environmental signals to the cell and activating Pch phosphodiesterase, which is important in surface sensing. Biofilm and motility assays and monitoring on-surface gene expression were used as a test of potential involvement in surface sensing. In agreement with the literature, SadB was found to be essential for biofilm formation and to affect swarming motility. sadB was also found to be differentially expressed on the anti and pro-biofilm surfaces. In contrast, CheA affected swarming but did not affect biofilm formation on most polymer surfaces and glass. The promoter of cheA also controls the cheI operon with 13 more genes. The expression of the operon was found to be different on different surfaces but CheA is unlikely to be important in surface sensing.

The literature and findings of the present study highlighted the importance of sadB in surface sensing, but the regulatory pathway was still unknown. Whole transcriptome analysis (RNAseq) was employed to investigate the downstream regulation of sadB in an untargeted manner. RT-PCR and phenotypic assays were used to validate the data. SadB was found to be a global regulator controlling up to 50% of the genome. sadB was shown to negatively affect the pqs system and subsequently the rhl system, rhamnolipid and pyocyanin production and the denitrification pathway. The levels of the PQS signal, pyocyanin and rhamnolipids were also shown to be affected by sadB. The denitrification pathway downregulation in the absence of sadB was shown to affect growth under microaerophilic conditions. Furthermore, sadB positively affected biofilm related targets such as Psl and c-di-GMP production.

Advances were made in the present study in discovering the downstream targets of sadB that explain some of the observed phenotypes, however the function of SadB is still unknown. A study from Muriel et al., reported that a sadB homolog in P. fluorescens binds c-di- GMP. The sadB homolog of P. aeruginosa was successfully purified and tested using SPR and thermal shift assay. The results showed no binding of c-di-GMP. Additionally, attempts were made to resolve the crystal structure of SadB. SadB crystals were obtained after screening commercial crystal screens and optimisation of the conditions but the resolution of the diffraction was not sufficient. Finally, SadB was found to localise in the cytoplasm using an anti-SadB antibody and Western blot and a SadB fluorescent fusion and super-resolution microscopy.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Williams, Paul Alexander, Morgan Heeb, Stephan Jean-Frederic, Dubern
Keywords:	Pseudomonas aeruginosa, SadB, Bacteria
Subjects:	Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	71682
Depositing User:	Papangeli, Maria
Date Deposited:	10 Nov 2022 10:58
Last Modified:	31 Jul 2024 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/71682

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