Structural and functional elucidation of four putative PQS-binding proteins in Pseudomonas aeruginosa

*Pseudomonas aeruginosa* is a multi-drug resistant, human opportunistic pathogen. If left untreated, *P. aeruginosa* can cause severe to life-threatening infections in people with burns, cystic fibrosis, and in immunocompromised patients. During chronic infections, *P. aeruginosa* primarily co-ordinates virulence in the host through a cell-to-cell communication mechanism called quorum sensing (QS). There are three key QS systems in *P. aeruginosa* responsible for driving global changes in virulence gene expression: the *las*, *rhl*, and *pqs* systems. Each of the *las*, *rhl*, and *pqs* systems rely on a receptor-autoinducer relationship: these receptor-autoinducer complexes are LasR-OdDHL, RhlR-BHL, and PqsR-PQS, respectively. When the receptors (LasR, RhlR, PqsR) bind with their cognate autoinducer (OdDHL, BHL, PQS, respectively), they act as transcription factors that ultimately stimulate the expression of hundreds of virulence-associated genes. The influence these QS systems have on the expression of virulence determinants has led to decades of scientific research focusing on the characterisation of these regulators. Although LasR and PqsR have been structurally elucidated, the RhlR crystal structure has long eluded characterisation and has been highly sought after due to its obvious potential as a therapeutic target. In a collaborative research effort, I helped to identify ten additional proteins as putative binding partners of the *pqs* autoinducer, PQS. Four of the ten proteins identified were the cyanide synthase (HcnC), a putative protease (PfpI), a phenazine biosynthetic protein (PhzD1), and the QS regulator RhlR. For this PhD project, I aimed to structurally and biochemically characterise these four proteins to, in part, confirm their proposed interaction with PQS. A novel ligand (benzoic acid) was discovered bound in the active site of PhzD1 (crystal structure solved to 1.1 Å). Additionally, the crystal structure for PfpI was resolved at 1.4 Å resolution. The PfpI tertiary and quaternary structures obtained in this study suggested a possible role in electrophile detoxification, a hypothesis which I confirmed *in vitro* using 1D NMR. To complement the novel PfpI structural and biochemical data, I generated and confirmed “clean” *pfpI* deletion mutants for phenotypic and ‘omic analyses. I observed discrepancies in phenotypes between the *pfpI* deletion mutant and the *pfpI* transposon mutants previously reported in the published literature, which I sought to reconcile through subsequent whole genome sequencing (WGS) of these previously published strains. WGS of the *pfpI* transposon mutants revealed a plethora of unexpected mutations elsewhere in the genome, which likely contribute to many of the reported phenotypes. The “clean” deletion mutant that I generated harboured no significant additional mutations. Proteomic profiling of the *pfpI* deletion mutant exhibited altered protein expression in systems involved in Type VI secretion, motility, and metabolism. Overall, the work presented in this dissertation further illustrates the intractability of purifying the QS transcriptional regulator, RhlR. I report benzoic acid to be a novel binding partner for the phenazine biosynthetic protein, PhzD1. Phenotypic analyses of *pfpI* mutants and consequent WGS highlight the need for rigorous strain validation when using transposon mutant libraries. Using the PfpI structural data I obtained during this study, I hypothesised and confirmed a novel detoxification role for PfpI in *P. aeruginosa*. Lastly, proteomic analysis of a *pfpI* deficient mutant revealed global dysregulation of key biological processes.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Grimm, Larson

Advisor dc:contributor.advisor

• Welch, Martin

• Crystal structure
• Cystic fibrosis
• Electrophile detoxification
• Mutants
• Phenotype
• Proteomic profiling
• Pseudomonas aeruginosa
• Quorum sensing
• Whole genome sequencing

Licence

• https://www.repository.cam.ac.uk/bitstreams/af0411aa-db1d-4cb8-975d-1722a26dbf9b/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.101975

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/358204