An autoinducer-independent RhlR quorum-sensing receptor enables analysis of RhlR regulation

Abstract

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa , an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P . aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N -butryl-homoserine lactone (C 4 HSL), and the RhlR:C 4 HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo . RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C 4 HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL ( meta -bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification.

Identification of FDA-approved antivirulence drugs targeting thePseudomonas aeruginosaquorum sensing effector protein PqsE

“…Moreover, pqsE deletion halved P. aeruginosa MIC values for tobramycin in M9-Glu, while this effect was absent in MHB. This indicates that PqsE could positively contribute to P. aeruginosa resistance to tobramycin depending on the growth medium, in agreement with studies showing that PqsE-dependent virulence genes are differentially regulated depending on the growth condition [ 40 , 41 ]. Among the antibiotics tested, pqsE deletion affected only the susceptibility of P. aeruginosa to tobramycin, indicating that PqsE could be required for the expression of factors contributing to resistance to aminoglicosides.…”

“…It was noticed that the effect of nifuroxazide was stronger against rhl- and pqs- than against las -controlled virulence factors [ 87 ]. This observation, together with recent findings highlighting the strong connection between the rhl and pqs QS systems [ 40 , 41 ], supports the hypothesis that the main antivirulence activity of nitrofurans could be dependent on their ability to interfere with PqsE-controlled regulatory pathway(s).…”

“…The pqs QS system controls the expression of multiple virulence factors and biofilm formation, as inferred by the evidence that P. aeruginosa mutants defective in the pqs QS system display attenuated pathogenicity in different plant and animal models of infection [ 35 , 36 , 40 , 41 , 46 , 69 – 75 ]. Although the central role played by the pqs system in P. aeruginosa pathobiology has been extensively studied, a clear characterization of the molecular mechanism by which its individual elements control gene expression is still unclear.…”

“…Indeed, a P. aeruginosa mutant unable to produce C 4 -HSL was as virulent as the wild type isogenic strain, while pqsE and rhlR mutant strains showed complete loss of virulence and attenuated virulence, respectively [ 40 ]. Finally, a recent work showed that a mutated variant of RhlR active in the absence of any ligand could only partially restore pyocyanin production in a pqsE mutant, also depending on the environmental conditions [ 41 ]. This study, together with the previous observation that the pqsE mutant was less virulent than the rhlR mutant in vivo , supports the hypothesis that PqsE could have multiple mechanisms of action besides being involved in AQs synthesis and being required for the production of the RhlR alternative ligand.…”

“…Intriguingly, PqsE controls the transcription of more than 140 genes, including key virulence genes, e.g . genes required for pyocyanin and rhamnolipids production, and genes involved in swarming motility and biofilm formation, via a still poorly understood pathway that is both PqsR- and AQs-independent [ 34 – 41 ]. Accordingly, P. aeruginosa pqsE mutants are impaired in biofilm formation and display reduced virulence in plant and animal models of infection [ 35 , 36 , 40 ].…”