Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon 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 tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc(1) complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC(1). Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc(1) genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production. Author summary Pseudomonas aeruginosa is 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 a P. aeruginosa mutant 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 of tat mutants to translocate the Rieske subunit of the cytochrome bc(1) complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

Automated summary

The study revealed that perturbation of the twin-arginine translocation (Tat) pathway in P. aeruginosa led to defective biofilm architecture, increased susceptibility to antibiotics, and disrupted pseudomonas quinolone signal (PQS) transmission, resulting in altered production and release of biofilm components.