Disruption of thePseudomonas aeruginosaTat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochromebc1sub-unit
Authors: Soh Eliza Ye-Chen , Smith Frances , Gimenez Maxime Rémi , Yang Liang , Vejborg Rebecca Munk , Fletcher Matthew , Halliday Nigel , Bleves Sophie , Heeb Stephan , Camara Miguel , Givskov Michael , Hardie Kim R. , Tolker-Nielsen Tim , Ize Bérengère , Williams Paul 
Published: March 2021

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix ofPseudomonas aeruginosabiofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening aP. aeruginosatransposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin.P. aeruginosa tatmutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes inpqsAexpression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to thetatmutants did not returnpqsA,rhlAorphzA1expression or pyocyanin production to wild type levels. However, transformation of thetatmutants with the AQ-independentpqseffectorpqsErestoredphzA1expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. ApqsA::luxfusion was introduced into each of 34 validatedP. aeruginosaTat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochromebc1complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production,rhlAexpression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance ofP. aeruginosabiofilms.

Author Summary

Pseudomonas aeruginosais a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened aP. aeruginosamutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability oftatmutants to translocate the Rieske subunit of the cytochromebc1complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation inP. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.