PQS-dependent quorum sensing in pseudomonas aeruginosa is linked to protein export via the twin-arginine translocation (tat) system

Smith, Frances (2022) PQS-dependent quorum sensing in pseudomonas aeruginosa is linked to protein export via the twin-arginine translocation (tat) system. PhD thesis, University of Nottingham.

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Pseudomonas aeruginosa is a leading cause of nosocomial infection due to its inherent resistance to multiple classes of antibiotics and ability to readily form protective antibiotic-tolerant biofilms. P. aeruginosa has a vast number of regulatory systems as well as multiple interlinked hierarchical quorum sensing (QS) systems that allows tight control over its large genome. The PQS-dependent QS system utilises alkyl-quinolones (AQs) as autoinducers, including the Pseudomonas quinolone signal (PQS) and its immediate precursor, 2-heptyl-4-hydroxyquinoline (HHQ). PQS-dependent QS coordinates the production and release of virulence factors, secondary metabolites, rhamnolipids, microvesicles, and extracellular DNA, which are important for biofilm formation. The Twin-arginine transport (Tat) System translocates fully-folded proteins across the inner membrane and inhibition, mutation or deletion of the tat genes resulted in down-regulated PQS biosynthesis. The primary aim of this PhD was to understand why loss of the Tat system causes perturbation of pqs QS. This was studied in two ways, through a) transcriptome analysis and b) mutagenesis of each known Tat substrate to discover whether a single substrate could account for the phenotype.

Comparison of tat mutant transcriptome with wild-type PAO1-DK revealed highly up-regulated genes involved in anthranilate degradation. Anthranilate is degraded and fed into tri-carboxylic acid (TCA) cycle for energy generation but is also used in the first step of AQ biosynthesis. Up-regulated anthranilate degradation as seen in the transcriptome data may account for the perturbation of pqs QS in tat mutants as the two are reciprocally regulated. Expression from promoters of the first two operons in the anthranilate degradation pathway (antABC and catBCA) and their respective regulators (antR and catR) was confirmed to be up-regulated upon deletion or mutation of the tat system using bioluminescent transcriptional reporters.

Perturbed pqs QS and up-regulated anthranilate degradation may be due to failed translocation of one or more of the effectors translocated by the Tat system. Deletion mutants of 34 Tat system substrates were screened for reduced pqsA expression and up-regulated antA expression. Both were observed in a petA mutant, which encodes the Rieske subunit of the cytochrome bc1 complex. cytB and cytC1 mutants similarly exhibited reduced pqsA expression and AQ production. PA14 DpetA mutants grew thin, flat, eDNA-deficient biofilms similar to PA14 DtatABC, and these were partially restored to a mature biofilm phenotype upon complementation. Results suggested that failure to translocate PetA caused the cell to shift from producing secondary metabolites, many of which are controlled by PQS quorum sensing, to primary metabolic processes such as degradation of aromatic amino acids in order to conserve energy.

The Tat pathway is an ideal target for novel antivirulence agents due to its involvement in export of virulence factors and of effectors involved in anaerobic respiration and osmotic stress response. Deletion of the tat system disrupts pqs QS and subsequently biofilm formation, which is often implicated in chronic or acute recurring P. aeruginosa infections. As such, the second aim of this PhD was to develop inhibitors of the Tat system. Analogues of the published small-molecule Tat inhibitor (2E)-3-[(4-methylphenyl)sulfonyl]acrylonitrile (also known as Bay 11- 7082) were screened for their activity against pqsA expression and pyoverdine production, and structure-activity relationships (SAR) were investigated. Replacement of the nitrile group with an amide (CONH2) functional group increased pqsA-inhibitory activity, but addition of an ethoxycarbonyl (COOEt) functional group had the opposite effect. By altering substituents on the phenyl ring, the combination 3-Cl, 4-F increased inhibitory activity for pqsA expression. All compounds exhibited some bacterial growth inhibition, except for those with an N,N-dimethylcarboxamide (CONMe2) functional group which had very little effect on growth. Investigation of eukaryotic cell viability following incubation with Bay 11-7082 and 4-Me-PhSACONH2 showed the latter was less cytotoxic.

This work gives new insight into the effects of tat mutation upon the transcriptome of P. aeruginosa PAO1-DK, and shows that the slower growth, perturbed PQS-dependent quorum sensing, and thin, flat biofilm formation seen in tat mutants is likely due to failed export of the Rieske subunit of the cytochrome bc1 complex, PetA. SAR analysis of analogues of a known Tat inhibitor, Bay 11- 7082, should help advance knowledge for the development of inhibitors of pqsA expression and potentially also the Tat system.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Hardie, Kim
Williams, Paul
Keywords: Pseudomonas aeruginosa, Twin-arginine translocation system, Anthranilate, PQS, Quorum sensing, Protein export, Cytochrome bc1, Inhibitor structure-activity relationship
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: 68640
Depositing User: Smith, Frances
Date Deposited: 29 Jul 2022 04:40
Last Modified: 29 Jul 2022 04:40
URI: https://eprints.nottingham.ac.uk/id/eprint/68640

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