Pseudomonas aeruginosa LasR overexpression leads to a RsaL-independent pyocyanin production inhibition in a low phosphate condition.

Several Pseudomonas aeruginosa virulence-related traits like pyocyanin are regulated by an intricate regulatory network called quorum sensing (QS) that relies on transcriptional regulators that are activated through binding to a self-produced molecule called an autoinducer (AI). QS is composed of three systems, Las, Rhl and Pqs. In the Las system, the regulatory protein LasR interacts with its AI to activate the other two QS systems. In turn, the Rhl and Pqs systems regulate the expression of multiple virulence-related genes, such as the genes of the reiterated operons phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2 involved in pyocyanin production. The Las system also regulates the negative regulator RsaL, which provides negative feedback to the QS-response, including repression of pyocyanin synthesis genes. In this work, we describe that LasR can act as a negative regulator of phzA1 transcription and hence of pyocyanin production and that this regulation is independent of RsaL activity. This work contributes to the understanding of QS-dependent pyocyanin production and demonstrates a previously uncharacterized role of LasR as a repressor.

PqsR-independent quorum-sensing response of Pseudomonas aeruginosa ATCC 9027 outlier-strain reveals new insights on the PqsE effect on RhlR activity.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum-sensing response regulates the expression of several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame-shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE-dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR-dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE-dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains. HIGHLIGHTS: Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR. This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE. Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE. The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction. The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation. Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.

The Rhl Quorum-Sensing System Is at the Top of the Regulatory Hierarchy under Phosphate-Limiting Conditions in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a major nosocomial pathogen that presents high-level resistance to antibiotics. Its ability to cause infections relies on the production of multiple virulence factors. Quorum sensing (QS) regulates the expression of many of these virulence factors through three QS systems: Las, Rhl, and PQS. The Las system positively regulates the other two systems, so it is at the top of a hierarchized regulation. Nevertheless, clinical and environmental strains that lack a functional Las system have been isolated, and, surprisingly, some of them still have the ability to produce virulence factors and infect animal models, so it has been suggested that the hierarchy is flexible under some conditions or with atypical strains. Here, we analyze the PAO1 type strain and its ΔlasR-derived mutant and report, for the first time, a growth condition (phosphate limitation) where LasR absence has no effect either on virulence factor production or on the gene expression profile, in contrast to a condition of phosphate repletion where the LasR hierarchy is maintained. This work provides evidence on how the QS hierarchy can change from being a strictly LasR-dependent to a LasR-independent RhlR-based hierarchy under phosphate limitation even in the PAO1 type strain.IMPORTANCEPseudomonas aeruginosa is an important pathogen, considered a priority for the development of new therapeutic strategies. An important approach to fight its infections relies on blocking quorum sensing. The Las system is the main regulator of the quorum-sensing response, so many research efforts aim to block this system to suppress the entire response. In this work, we show that LasR is dispensable in a phosphate-limited environment in the PAO1 type strain, which has been used to define the quorum-sensing response hierarchy, and that under this condition RhlR is at the top of the regulation hierarchy. These results are highly significant, since phosphate limitation represents a similar environment to the one that P. aeruginosa faces when establishing infections.

Virulence factors regulation by the quorum-sensing and Rsm systems in the marine strain Pseudomonas aeruginosa ID4365, a natural mutant in lasR.

Pseudomonas aeruginosa is an opportunistic human pathogen that is able to produce several virulence factors such as pyocyanin, rhamnolipids and elastase. In the clinical reference strain PAO1, synthesis of these virulence factors is regulated transcriptionally by quorum sensing (QS) and post-transcriptionally by the Rsm system. Herein, we investigated the role of these systems in the control of the pyocyanin, rhamnolipids and elastase production in the marine strain ID4365. We found that this strain carries a nonsense mutation in lasR that makes it a natural mutant in the Las QS system. However, its QS response is still functional with the Rhl system activating virulence factors synthesis. We found that the Rsm system affects virulence factors production, since overexpression of RsmA reduces pyocyanin production whereas RsmY overexpression increases its synthesis. Unexpectedly, and in contrast to the type strain PAO1, inactivation of rsmA increases pyocyanin but reduces elastase and rhamnolipids production by a reduction of RhlR levels. Thus, QS and Rsm systems are involved in regulating virulence factors production, but this regulation is different to the PAO1 strain even though their genomes are highly conserved. It is likely that these differences are related to the different ecological niches in which these strains lived.