Evolution and Regulation of Virulence and Antimicrobial Resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterium responsible for various life-threatening infections. There are several factors that make P. aeruginosa a successful pathogen, including its metabolic versatility, its propensity to acquire antimicrobial resistance (AMR) and the ability to produce numerous virulence factors. However, the expression of virulence factors is costly for the cell and, therefore, tightly regulated via quorum sensing (QS). P. aeruginosa has three main interconnected QS systems (las, rhl and pqs), which are in turn modulated by the Stringent Response (SR). This system allows bacteria to sense and respond to nutrient limitations and related stresses by modulating cellular transcription patterns. Notably, it has always been assumed that the stringent response is essential for the activation of QS systems, and therefore, for the expression of virulence factors in P. aeruginosa. Furthermore, the structure of the regulatory network linking QS and the SR remains unknown. Here, I studied the regulation of virulence factors in P. aeruginosa by characterising the mechanism by which avirulent strains become virulent via compensatory mutations. I constructed and characterised an extensive collection of mutants in a mutant that is defective in the SR (due to deletion of the relA and spoT genes) and discovered that QS-dependent virulence factor production can be restored independently of the SR. Whole-genome sequencing of these “bypassing mutants” revealed mutations in several genes, including mexT. Through genome editing, I confirmed that mutations in mexT are responsible for the increased virulence and, surprisingly, resistance to aminoglycoside antibiotics and the “last-resort” antibiotic, colistin. RNA-seq of the mexT mutants revealed that many virulence and AMR-related genes were upregulated, including the H2-T6SS and the mexGHI-opmD efflux pump. Genomics analysis of mutations in mexT in P. aeruginosa genomes showed that those are prevalent amongst clinical and environmental isolates and not restricted to a particular region of the gene. My findings describe a novel mechanism by which P. aeruginosa can restore virulence in an SRindependent manner and show that mexT can be a mutational hotspot for the emergence of antimicrobial resistance.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Figueroa Chávez, Wendy

Advisor dc:contributor.advisor

• Welch, Martin

• Antimicrobial resistance
• Virulence
• Pathogenesis
• Regulatory networks
• Pseudomonas aeruginosa

Licence

• https://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.89161

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/341739