Pseudomonas aeruginosa PQS mediated virulence regulation and interference

Abstract

Pseudomonas aeruginosa is a ubiquitous bacterium that can be found in most mesophilic aquatic and terrestrial habitats. Furthermore, P. aeruginosa (PA) is an important opportunistic pathogen that can infect humans, animals, insects and plants. P. aeruginosa has three hierarchically organised quorum sensing (QS) systems named las, rhl and pqs. The las and rhl are classic QS systems that use an N-acylhomoserine lactone as autoinducers. The las system is the major QS system of the cell and controls the other two and there is partial redundancy with the rhl system regarding the genes and functions controlled by the system. Activated functions are related to motility, virulence and biofilm formation. The pqs system is a QS system based in alkyl-4(1H)-quinolones (AQs) molecules, specifically the 2-heptyl-3,4-dihydroxyquinoline called pseudomonas quinolone factor (PQS). The pqs system is under control of las and rhl and can in turn influence the expression of rhl. The pqs system controls virulence, iron acquisition, biofilm maturation and the oxidative stress response. The PqsR is the main regulator of the pqs system upon activation with PQS. The inhibition of QS and in particular the pqs system is an approach to decrease the virulence of P. aeruginosa in vivo to improve the outcome of antibiotic treatments and decrease the P. aeruginosa associated morbidity. The SENBIOTAR project (sensitising Pseudomonas aeruginosa biofilms to antibiotics and reducing virulence through novel target inhibition, MRC project MR/N501852/1) aims at developing PqsR antagonists that inhibit the pqs system. A series of potential inhibitors were tested for activity in a pqsA-lux transcriptional reporter in Chapter 3. Two compounds, SEN016 and SEN066 were found to actively inhibit expression and SEN016 was used for further in silico optimisation, developing a series of compounds that were tested for IC50 and isothermal titration calorimetry (ITC). The lead compound SEN089 showed a K(D) (dissociation constant) of 2.66 nM and IC50 of 67 nM while compounds SEN022 and SEN066 did not bind to PqsRLBD even if they inhibited pqs. The compounds were tested in a variety of phenotypes and strains. Results from Chapter 4 show that compounds effectively inhibit phenotypes under pqs regulation but not others controlled by different regulatory systems. The data evidenced significant differences on the efficacy of compounds when tested in other strains. Notably, SEN089 remained the overall best compound. Moreover, data provided evidence that P. aeruginosa has at least two autolytic mechanisms, one is activated by pqs whereas the other is repressed by PQS. A novel hypothesis for the role of PQS in the CF lung is also discussed there. In Chapter 3, some compounds were tested in biofilm models to assess the relevance of pqs inhibition. There was a significant increased bioactivity between the compounds and antibiotics tested as well as between the compounds and shearing forces. Several guidelines are provided for optimal confocal imaging aimed at quantitation. Furthermore, another novel hypothesis is presented regarding the role of pqs in the biofilm of the CF lung describing it as a response mechanism rather than an active system. In summary, a series of PqsR antagonists were developed and analysed for binding affinity to PqsR, secondary unspecific activity, pqs inhibition, biofilm impact and an additive effect on antibiotic treatment in different strains of P. aeruginosa. The compounds successfully inhibit pqs and had a significant impact in virulence modulation as well as sensitivity towards tobramycin or ciprofloxacin. This work explores the inhibition of pqs as an effective therapeutic target and suggests multiple novel mechanisms through which pqs regulates the physiology of the cell