QsrO a novel regulator of quorum-sensing and virulence in Pseudomonas aeruginosa.
ABSTRACT: In Pseudomonas aeruginosa, the production of many secreted virulence factors is controlled by a quorum-sensing (QS) circuit, constituted of transcriptional activators (LasR, RhlR, PqsR) and their cognate signaling molecules (3-oxo-C12-HSL, C4-HSL, PQS). QS is a cooperative behavior that is beneficial to a population but can be exploited by "QS-cheaters", individuals which do not respond to the QS-signal, but can use public goods produced by QS-cooperators. In order to identify QS-deficient clones we designed a genetic screening based on a lasB-lacZ fusion. We isolated one clone (PT1617) deficient in QS-dependent gene expression and virulence factor production despite wild type lasR, rhlR and pqsR alleles. Whole genome sequencing of PT1617 revealed a 3,552 bp deletion encompassing ORFs PA2228-PA2229-PA2230 and the pslA gene. However, complementation of PT1617 by plasmid-encoded copies of these ORFs, did not restore QS. Unexpectedly, gene expression levels of ORFs PA2228, PA2227 (vqsM) and PA2222, located adjacent to the deletion, were 10 to 100 fold higher in mutant PT1617 than in PAO1. When expressed from a constitutive promoter on a plasmid, PA2226, alone was found to be sufficient to confer a QS-negative phenotype on PAO1 as well as on PA14. Co-expression of PA2226 and PA2225 in PAO1 further prevented induction of the type III secretion system. In summary, we have identified a novel genetic locus including ORF2226 termed qsrO (QS-repressing ORF), capable of down-regulating all three known QS-systems in P. aeruginosa.
Project description:The bacterial pathogen Pseudomonas aeruginosa activates expression of many virulence genes in a cell density-dependent manner by using an intricate quorum-sensing (QS) network. QS in P. aeruginosa involves two acyl-homoserine-lactone circuits, LasI-LasR and RhlI-RhlR. LasI-LasR is required to activate many genes including those coding for RhlI-RhlR. P. aeruginosa causes chronic infections in the lungs of people with cystic fibrosis (CF). In these infections, LasR mutants are common, but rhlR-rhlI expression has escaped LasR regulation in many CF isolates. To better understand the evolutionary trajectory of P. aeruginosa QS in chronic infections, we grew LasR mutants of the well-studied P. aeruginosa strain, PAO1, in conditions that recapitulate an environment where QS signal synthesis by other bacteria might still occur. When QS is required for growth, addition of the RhlI product butyryl-homoserine lactone (C4-HSL), or bacteria that produce C4-HSL, to LasR mutants results in the rapid emergence of a population with a LasR-independent RhlI-RhlR QS system. These evolved populations exhibit subsequent growth without added C4-HSL. The variants that emerge have mutations in mexT, which codes for a transcription factor that controls expression of multiple genes. LasR-MexT mutants have a competitive advantage over both the parent LasR mutant and a LasR-MexT-RhlR mutant. Our findings suggest a plausible evolutionary trajectory for QS in P. aeruginosa CF infections where LasR mutants arise during infection, but because these mutants are surrounded by C4-HSL-producing P. aeruginosa, variants rewired to have a LasR-independent RhlIR system quickly emerge.
Project description:The bacterium Pseudomonas aeruginosa has emerged as a central threat in health care settings and can cause a large variety of infections. It expresses an arsenal of virulence factors and a diversity of survival functions, many of which are finely and tightly regulated by an intricate circuitry of three quorum sensing (QS) systems. The las system is considered at the top of the QS hierarchy and activates the rhl and pqs systems. It is composed of the LasR transcriptional regulator and the LasI autoinducer synthase, which produces 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), the ligand of LasR. RhlR is the transcriptional regulator for the rhl system and is associated with RhlI, which produces its cognate autoinducer C4-HSL. The third QS system is composed of the pqsABCDE operon and the MvfR (PqsR) regulator. PqsABCD synthetize 4-hydroxy-2-alkylquinolines (HAQs), which include ligands activating MvfR. PqsE is not required for HAQ production and instead is associated with the expression of genes controlled by the rhl system. While RhlR is often considered the main regulator of rhlI, we confirmed that LasR is in fact the principal regulator of C4-HSL production and that RhlR regulates rhlI and production of C4-HSL essentially only in the absence of LasR by using liquid chromatography-mass spectrometry quantifications and gene expression reporters. Investigating the expression of RhlR targets also clarified that activation of RhlR-dependent QS relies on PqsE, especially when LasR is not functional. This work positions RhlR as the key QS regulator and points to PqsE as an essential effector for full activation of this regulation.IMPORTANCE Pseudomonas aeruginosa is a versatile bacterium found in various environments. It can cause severe infections in immunocompromised patients and naturally resists many antibiotics. The World Health Organization listed it among the top priority pathogens for research and development of new antimicrobial compounds. Quorum sensing (QS) is a cell-cell communication mechanism, which is important for P. aeruginosa adaptation and pathogenesis. Here, we validate the central role of the PqsE protein in QS particularly by its impact on the regulator RhlR. This study challenges the traditional dogmas of QS regulation in P. aeruginosa and ties loose ends in our understanding of the traditional QS circuit by confirming RhlR to be the main QS regulator in P. aeruginosa PqsE could represent an ideal target for the development of new control methods against the virulence of P. aeruginosa This is especially important when considering that LasR-defective mutants frequently arise, e.g., in chronic infections.
Project description:Pseudomonas aeruginosa coordinates the transcription of hundreds of genes, including many virulence genes, through three hierarchically arranged quorum-sensing (QS) systems, namely las, rhl and pqs. Each system consists of genes involved in autoinducer synthesis, lasI, rhlI and pqsABCDH, as well as cognate-regulatory genes, lasR, rhlR and pqsR. In this study, we analyzed the social behavior of signal-blind (?lasR, ?rhlR, ?pqsR) and signal-negative (?lasI, ?rhlI, ?pqsA) mutants from each QS system. As each system controls extracellular common goods but differs in the extent of regulatory control, we hypothesized that all signal-blind mutants can behave as cheaters that vary in their ability to invade a QS-proficient population. We found that lasR and pqsR, but not rhlR, mutants evolve from a wild-type ancestor in vitro under conditions that favor QS. Accordingly, defined lasR and pqsR mutants enriched in wild-type co-culture, whereas rhlR and all signal-negative mutants did not. Both lasR and pqsR mutants enriched with negative frequency dependence, suggesting social interactions with the wild type, although the pqsR mutant also grew well on its own. Taken together, the lasR mutant behaved as a typical cheater, as reported previously. However, the pqsR and rhlR mutants exhibited more complex behaviors, which can be sufficiently explained by positive and negative pleiotropic effects through differential regulation of pqs gene expression in the interconnected QS network. The evolutionary approach adopted here may account for the prevalence of naturally occurring QS mutants.
Project description:Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.
Project description:The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.
Project description:BACKGROUND: Cis-2-dodecenoic acid (BDSF) is well known for its important functions in intraspecies signaling in Burkholderia cenocepacia. Previous work has also established an important role of BDSF in interspecies and inter-kingdom communications. It was identified that BDSF modulates virulence of Pseudomonas aeruginosa. However, how BDSF interferes with virulence of P. aeruginosa is still not clear. RESULTS: We report here that BDSF mediates the cross-talk between B. cenocepacia and P. aeruginosa through interference with quorum sensing (QS) systems and type III secretion system (T3SS) of P. aeruginosa. Bioassay results revealed that exogenous addition of BDSF not only reduced the transcriptional expression of the regulator encoding gene of QS systems, i.e., lasR, pqsR, and rhlR, but also simultaneously decreased the production of QS signals including 3-oxo-C12-HSL, Pseudomonas quinolone signal (PQS) and C4-HSL, consequently resulting in the down-regulation of biofilm formation and virulence factor production of P. aeruginosa. Furthermore, BDSF and some of its derivatives are also capable of inhibiting T3SS of P. aeruginosa at a micromolar level. Treatment with BDSF obviously reduced the virulence of P. aeruginosa in both HeLa cell and zebrafish infection models. CONCLUSIONS: These results depict that BDSF modulates virulence of P. aeruginosa through interference with QS systems and T3SS.
Project description:BACKGROUND: Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors. METHODS AND RESULTS: Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12)HSL and C(4)HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C(4)HSL. F7 also showed antagonistic activity against 3-oxo-C(12)HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract. CONCLUSIONS: This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen using virulence factors and biofilm regulated by quorum sensing (QS) systems to infect patients and protect itself from environmental stress and antibiotics. Interfering with QS systems is a novel approach to combat P. aeruginosa infections without killing the bacteria, meaning that it is much harder for bacteria to develop drug resistance. A marine fungus Cladosporium sp. Z148 with anti-QS activity was obtained from Jiaozhou Bay, China. Cladodionen, a novel QS inhibitor, was isolated from the extracts of this fungus. Cladodionen had a better inhibitory effect than pyocyanin on the production of elastase and rhamnolipid. It also inhibited biofilm formation and motilities. The mRNA expressions of QS-related genes, including receptor proteins (lasR, rhlR and pqsR), autoinducer synthases (lasI, rhlI and pqsA) and virulence factors (lasB and rhlA) were down-regulated by cladodionen. Molecular docking analysis showed that cladodionen had better binding affinity to LasR and PqsR than natural ligands. Moreover, the binding affinity of cladodionen to LasR was higher than to PqsR. Cladodionen exhibits potential as a QS inhibitor against P. aeruginosa, and its structure-activity relationships should be further studied to illustrate the mode of action, optimize its structure and improve anti-QS activity.
Project description:The opportunistic pathogen Pseudomonas aeruginosa damages hosts through the production of diverse secreted products, many of which are regulated by quorum sensing (QS). The lasR gene, which encodes a central QS regulator, is frequently mutated in clinical isolates from chronic infections, and loss of LasR function (LasR-) generally impairs the activity of downstream QS regulators RhlR and PqsR. We found that in cocultures containing LasR+ and LasR- strains, LasR- strains hyperproduce the RhlR/RhlI-regulated antagonistic factors pyocyanin and rhamnolipids in diverse models and media and in different strain backgrounds. Diffusible QS autoinducers produced by the wild type were not required for this effect. Using transcriptomics, genetics, and biochemical approaches, we uncovered a reciprocal interaction between wild-type and lasR mutant pairs wherein the iron-scavenging siderophore pyochelin produced by the lasR mutant induced citrate release and cross-feeding from the wild type. Citrate, a metabolite often secreted in low iron environments, stimulated RhlR signaling and RhlI levels in LasR-but not in LasR+ strains. These studies reveal the potential for complex interactions between recently diverged, genetically distinct isolates within populations from single chronic infections.IMPORTANCE Coculture interactions between lasR loss-of-function and LasR+ Pseudomonas aeruginosa strains may explain the worse outcomes associated with the presence of LasR- strains. More broadly, this report illustrates how interactions within a genotypically diverse population, similar to those that frequently develop in natural settings, can promote unpredictably high virulence factor production.
Project description:Acyl-homoserine lactone (acyl-HSL) and alkyl quinolone (AQ) based quorum-sensing (QS) systems are important for Pseudomonas aeruginosa virulence and biofilm formation. The effect of QS on biofilm formation is influenced by various genetic and environmental factors. Here, we used a colony biofilm assay to study the effect of the central acyl-HSL QS regulator, LasR, on biofilm formation and structure in the representative clinical P. aeruginosa isolate ZK2870.A lasR mutant exhibited wrinkled colony morphology at 37°C in contrast to the smooth colony morphology of the wild-type. Mutational analysis indicated that wrinkling of the lasR mutant is dependent on pel, encoding a biofilm matrix exopolysaccharide. Suppressor mutagenesis and complementation analysis implicated the AQ signaling pathway as the link between las QS and colony morphology. In this pathway, genes pqsA-D are involved in the synthesis of 4-hydroxyalkyl quinolines ("Series A congeners"), which are converted to 3,4-dihydroxyalkyl quinolines ("Series B congeners", including the well-characterized Pseudomonas Quinolone Signal, PQS) by the product of the LasR-dependent pqsH gene. Measurement of AQ in the wild-type, the lasR pqsA::Tn suppressor mutant as well as the defined lasR, pqsH, and lasR pqsH mutants showed a correlation between 4-hydroxyalkyl quinoline levels and the degree of colony wrinkling. Most importantly, the lasR pqsH double mutant displayed wrinkly morphology without producing any 3,4-dihydroxyalkyl quinolines. Constitutive expression of pqsA-D genes in a lasR pqsR::Tnmutant showed that colony wrinkling does not require the AQ receptor PqsR.Taken together, these results indicate that the las QS system represses Pel and modulates colony morphology through a 4-hydroxyalkyl quinoline in a PqsR-independent manner, ascribing a novel function to an AQ other than PQS in P. aeruginosa.