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• W2284129173 abstract "•The Rhl and Pqs QS systems tune virulence factor production to the environment•Small-molecule QS inhibitors have environment-dependent activity•Cocktails of QS inhibitors can best attenuate virulence factor production•Inhibitor cocktails are active in environments that mimic infection (CF sputum) Nutritional cues differentially influence the activities of the three quorum sensing (QS) circuits—Las, Rhl, and Pqs—in the pathogen Pseudomonas aeruginosa. A full understanding of how these systems work together to tune virulence factor production to the environment is lacking. Here, we used chemical probes to evaluate the contribution of each QS circuit to virulence in wild-type P. aeruginosa under defined environmental conditions. Our results indicate that Rhl and Pqs drive virulence factor production in phosphate- and iron-limiting environments, while Las has a minor influence. Consequently, simultaneous inhibition of Rhl and Pqs can attenuate virulence in environments where Las inhibition fails. The activity trends generated in this study can be extrapolated to predict QS inhibitor activity in infection-relevant environments, such as cystic fibrosis sputum. These results indicate that environmental signals can drastically alter the efficacy of small-molecule QS inhibitors in P. aeruginosa and possibly other pathogens. Nutritional cues differentially influence the activities of the three quorum sensing (QS) circuits—Las, Rhl, and Pqs—in the pathogen Pseudomonas aeruginosa. A full understanding of how these systems work together to tune virulence factor production to the environment is lacking. Here, we used chemical probes to evaluate the contribution of each QS circuit to virulence in wild-type P. aeruginosa under defined environmental conditions. Our results indicate that Rhl and Pqs drive virulence factor production in phosphate- and iron-limiting environments, while Las has a minor influence. Consequently, simultaneous inhibition of Rhl and Pqs can attenuate virulence in environments where Las inhibition fails. The activity trends generated in this study can be extrapolated to predict QS inhibitor activity in infection-relevant environments, such as cystic fibrosis sputum. These results indicate that environmental signals can drastically alter the efficacy of small-molecule QS inhibitors in P. aeruginosa and possibly other pathogens. Many common bacterial pathogens can delay virulence factor production until there are a sufficient number of cells such that, working together, the group can overwhelm a host's defenses. To coordinate such an attack, some species use a method of cell-cell communication called quorum sensing (QS) (Camilli and Bassler, 2006Camilli A. Bassler B.L. Bacterial small-molecule signaling pathways.Science. 2006; 311: 1113-1116Crossref PubMed Scopus (752) Google Scholar, Rutherford and Bassler, 2012Rutherford S.T. Bassler B.L. Bacterial quorum sensing: its role in virulence and possibilities for its control.Cold Spring Harb. Perspect. Med. 2012; 2: a012427Crossref Scopus (1142) Google Scholar). In Gram-negative bacteria, QS involves the production of a membrane-diffusible small-molecule signal, often an N-acyl L-homoserine lactone (AHL), that accumulates in the surroundings at a concentration proportional to cell density (Fuqua and Greenberg, 2002Fuqua C. Greenberg E.P. Listening in on bacteria: acyl-homoserine lactone signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (825) Google Scholar). At a threshold concentration, this signal is bound by, and activates, an intracellular LuxR-type receptor that acts as a transcriptional regulator to induce the expression of group-beneficial genes. QS systems are often induced in response to environmental signals (Hense and Schuster, 2015Hense B.A. Schuster M. Core principles of bacterial autoinducer systems.Microbiol. Mol. Biol. Rev. 2015; 79: 153-169Crossref PubMed Scopus (111) Google Scholar, Lee and Zhang, 2015Lee J. Zhang L. The hierarchy quorum sensing network in Pseudomonas aeruginosa.Protein Cell. 2015; 6: 26-41Crossref PubMed Scopus (671) Google Scholar). For example, the plant pathogen Agrobacterium tumefaciens does not express the QS receptor TraR, or produce its cognate AHL, unless the presence of specific plant hormones is detected (White and Winans, 2007White C.E. Winans S.C. Cell-cell communication in the plant pathogen Agrobacterium tumefaciens.Phil. Trans. R. Soc. B. 2007; 362: 1135-1148Crossref PubMed Scopus (120) Google Scholar). Via this mechanism, the bacterium is able to delay the energetically costly production of QS signals and virulence factors until it is in a plant host environment permissive to infection. Because of their association with virulence, QS systems are considered to be potential antivirulence targets (Cegelski et al., 2008Cegelski L. Marshall G.R. Eldridge G.R. Hultgren S.J. The biology and future prospects of antivirulence therapies.Nat. Rev. Microbiol. 2008; 6: 17-27Crossref PubMed Scopus (620) Google Scholar, Allen et al., 2014Allen R.C. Popat R. Diggle S.P. Brown S.P. Targeting virulence: can we make evolution-proof drugs?.Nat. Rev. Microbiol. 2014; 12: 300-308Crossref PubMed Scopus (353) Google Scholar, Gerdt and Blackwell, 2014Gerdt J.P. Blackwell H.E. Competition studies confirm two major barriers that can preclude the spread of resistance to quorum-sensing inhibitors in bacteria.ACS Chem. Biol. 2014; 9: 2291-2299Crossref PubMed Scopus (85) Google Scholar). Accordingly, to both further delineate the connection of QS to virulence and explore possible therapeutic strategies, numerous research groups are actively developing small-molecule and macromolecular agents capable of inhibiting QS receptor activity (Galloway et al., 2011Galloway W.R.J.D. Hodgkinson J.T. Bowden S.D. Welch M. Spring D.R. Quorum sensing in Gram-negative bacteria: small-molecule modulation of AHL and AI-2 quorum sensing pathways.Chem. Rev. 2011; 111: 28-67Crossref PubMed Scopus (456) Google Scholar, Murray et al., 2014Murray E.J. Crowley R.C. Truman A. Clarke S.R. Cottam J.A. Jadhav G.P. Steele V.R. O'Shea P. Lindholm C. Cockayne A. et al.Targeting Staphylococcus aureus quorum sensing with nonpeptidic small molecule inhibitors.J. Med. Chem. 2014; 57: 2813-2819Crossref PubMed Scopus (57) Google Scholar, Amara et al., 2011Amara N. Krom B.P. Kaufmann G.F. Meijler M.M. Macromolecular inhibition of quorum sensing: enzymes, antibodies, and beyond.Chem. Rev. 2011; 111: 195-208Crossref PubMed Scopus (145) Google Scholar, Praneenararat et al., 2012Praneenararat T. Palmer A.G. Blackwell H.E. Chemical methods to interrogate bacterial quorum sensing pathways.Org. Biomol. Chem. 2012; 10: 8189-8199Crossref PubMed Scopus (31) Google Scholar). The opportunistic pathogen Pseudomonas aeruginosa is able to colonize a variety of mammalian tissues including the skin, gut, and perhaps most notoriously, airways of patients suffering from cystic fibrosis (CF) (Lyczak et al., 2000Lyczak J.B. Cannon C.L. Pier G.B. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist.Microbes Infect. 2000; 2: 1051-1060Crossref PubMed Scopus (989) Google Scholar, Folkesson et al., 2012Folkesson A. Jelsbak L. Yang L. Johansen H.K. Ciofu O. Høiby N. Molin S. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective.Nat. Rev. Microbiol. 2012; 10: 841-851Crossref PubMed Scopus (476) Google Scholar). P. aeruginosa is highly adaptable to life in the varied environments found in these tissues (Brown et al., 2008Brown S.A. Palmer K.L. Whiteley M. Revisiting the host as a growth medium.Nat. Rev. Microbiol. 2008; 6: 657-666Crossref PubMed Scopus (138) Google Scholar). For example, the bacterium is able to feed on differing carbon sources (primarily amino acids in the CF airway and fatty acids in burn wounds) (Turner et al., 2014Turner K.H. Everett J. Trivedi U. Rumbaugh K.P. Whiteley M. Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection.PLoS Genet. 2014; 10: e1004518Crossref PubMed Scopus (212) Google Scholar, Turner et al., 2015Turner K.H. Wessel A.K. Palmer G.C. Murray J.L. Whiteley M. Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum.Proc. Natl. Acad. Sci. USA. 2015; 112: 4110-4115Crossref PubMed Scopus (222) Google Scholar) and sense and respond to local changes in the concentration of essential nutrients (such as iron and phosphate), allowing it to adapt its mode of growth and virulence profile to establish either acute or chronic infections (Long et al., 2008Long J. Zaborina O. Holbrook C. Zaborin A. Alverdy J. Depletion of intestinal phosphate after operative injury activates the virulence of P. aeruginosa causing lethal gut-derived sepsis.Surgery. 2008; 144: 189-197Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Markou and Apidianakis, 2014Markou P. Apidianakis Y. Pathogenesis of intestinal Pseudomonas aeruginosa infection in patients with cancer.Front. Cell. Infect. Microbiol. 2014; 3: 115Crossref PubMed Scopus (65) Google Scholar, Crousilles et al., 2015Crousilles A. Maunders E. Bartlett S. Fan C. Ukor E.-F. Abdelhamid Y. Baker Y. Floto A. Spring D.R. Welch M. Which microbial factors really are important in Pseudomonas aeruginosa infections?.Future Microbiol. 2015; 10: 1825-1836Crossref PubMed Scopus (38) Google Scholar). Thus, it is perhaps unsurprising that P. aeruginosa possesses a sophisticated QS system that incorporates a large degree of environmental regulation (Wagner et al., 2003Wagner V.E. Bushnell D. Passador L. Brooks A.I. Iglewski B.H. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment.J. Bacteriol. 2003; 185: 2080-2095Crossref PubMed Scopus (722) Google Scholar, Duan and Surette, 2007Duan K. Surette M.G. Environmental regulation of Pseudomonas aeruginosa PAO1 Las and Rhl quorum-sensing systems.J. Bacteriol. 2007; 189: 4827-4836Crossref PubMed Scopus (187) Google Scholar, Williams and Cámara, 2009Williams P. Cámara M. Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules.Curr. Opin. Microbiol. 2009; 12: 182-191Crossref PubMed Scopus (558) Google Scholar). P. aeruginosa has three distinct QS circuits—Las, Rhl, and Pqs (Figure 1)—whose associated LuxR-type receptors (LasR and RhlR) and LysR-type receptor (PqsR; also known as MvfR) regulate distinct subsets of virulence-associated genes upon activation by their cognate small-molecule signal (Venturi, 2006Venturi V. Regulation of quorum sensing in Pseudomonas.FEMS Microbiol. Rev. 2006; 30: 274-291Crossref PubMed Scopus (340) Google Scholar, Schuster and Greenberg, 2008Schuster M. Greenberg E.P. LuxR-type proteins in Pseuodomonas aeruginosa quorum sensing: distinct mechanisms with global implications.in: Winans S.C. Bassler B.L. Chemical Communication among Bacteria. ASM Press, 2008: 133-144Crossref Google Scholar). In the canonical model of P. aeruginosa QS, there is a regulatory hierarchy between the three QS systems, whereby Las induces the expression and activation of both Rhl and Pqs, while an inverse regulatory relationship exists between the latter systems (Balasubramanian et al., 2013Balasubramanian D. Schneper L. Kumari H. Mathee K. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence.Nucleic Acids Res. 2013; 41: 1-20Crossref PubMed Scopus (254) Google Scholar). Increasing evidence has revealed that nutritional cues found in infection environments can alter this hierarchy (Dekimpe and Déziel, 2009Dekimpe V. Déziel E. Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors.Microbiology. 2009; 155: 712-723Crossref PubMed Scopus (210) Google Scholar, Cabeen, 2014Cabeen M.T. Stationary phase-specific virulence factor overproduction by a lasR mutant of Pseudomonas aeruginosa.PLoS One. 2014; 9: e88743Crossref PubMed Scopus (43) Google Scholar, Lee and Zhang, 2015Lee J. Zhang L. The hierarchy quorum sensing network in Pseudomonas aeruginosa.Protein Cell. 2015; 6: 26-41Crossref PubMed Scopus (671) Google Scholar). For example, cellular factors that sense low levels of iron and phosphate can directly stimulate the Rhl and Pqs systems, bypassing Las (Figure 1A) (Jensen et al., 2006Jensen V. Lons D. Zaoui C. Bredenbruch F. Meissner A. Dieterich G. Munch R. Haussler S. RhlR expression in Pseudomonas aeruginosa is modulated by the Pseudomonas quinolone signal via PhoB-dependent and -independent pathways.J. Bacteriol. 2006; 188: 8601-8606Crossref PubMed Scopus (119) Google Scholar, Oglesby et al., 2008Oglesby A.G. Farrow J.M. Lee J.-H. Tomaras A.P. Greenberg E.P. Pesci E.C. Vasil M.L. The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing.J. Biol. Chem. 2008; 283: 15558-15567Crossref PubMed Scopus (157) Google Scholar, Lee et al., 2013Lee J. Wu J. Deng Y. Wang J. Wang C. Wang J. Chang C. Dong Y. Williams P. Zhang L.-H. A cell-cell communication signal integrates quorum sensing and stress response.Nat. Chem. Biol. 2013; 9: 339-343Crossref PubMed Scopus (279) Google Scholar). In addition, the chemical nature and availability of carbon sources can suppress or induce specific QS systems via the downstream effects of carbon catabolite repression and the stringent response (Figure 1A) (Shrout et al., 2006Shrout J.D. Chopp D.L. Just C.L. Hentzer M. Givskov M. Parsek M.R. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional.Mol. Microbiol. 2006; 62: 1264-1277Crossref PubMed Scopus (416) Google Scholar, Schafhauser et al., 2014Schafhauser J. Lepine F. Mckay G. Ahlgren H.G. Khakimova M. Nguyen D. The stringent response modulates 4-hydroxy-2-alkylquinoline (HAQ) biosynthesis and quorum sensing hierarchy in Pseudomonas aeruginosa.J. Bacteriol. 2014; 196: 1641-1650Crossref PubMed Scopus (46) Google Scholar, Yang et al., 2015Yang N. Ding S. Chen F. Zhang X. Xia Y. Di H. Cao Q. Deng X. Wu M. Wong C.C. et al.The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa.Mol. Microbiol. 2015; 96: 526-547Crossref PubMed Scopus (34) Google Scholar). Therefore, a plausible explanation for the existence of the complex QS network in P. aeruginosa is that it serves to tune the virulence profile of the organism in response to diverse environmental stimuli (Mellbye and Schuster, 2014Mellbye B. Schuster M. Physiological framework for the regulation of quorum sensing-dependent public goods in Pseudomonas aeruginosa.J. Bacteriol. 2014; 196: 1155-1164Crossref PubMed Scopus (58) Google Scholar). Despite considerable recent research, a full understanding of the mechanisms by which Las, Rhl, and Pqs work together to accomplish this regulation remains elusive. Mellbye and Schuster, 2014Mellbye B. Schuster M. Physiological framework for the regulation of quorum sensing-dependent public goods in Pseudomonas aeruginosa.J. Bacteriol. 2014; 196: 1155-1164Crossref PubMed Scopus (58) Google Scholar have shown that Las-responsive genes are primarily induced in a cell density-dependent manner, while Rhl-associated genes are up-regulated in response to environmental cues. Although Pqs is an important regulator of global virulence, how it fits into this model is unknown (Déziel et al., 2005Déziel E. Gopalan S. Tampakaki A.P. Lépine F. Padfield K.E. Saucier M. Xiao G. Rahme L.G. The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones.Mol. Microbiol. 2005; 55: 998-1014Crossref PubMed Scopus (311) Google Scholar, Zaborin et al., 2009Zaborin A. Romanowski K. Gerdes S. Holbrook C. Lepine F. Long J. Poroyko V. Diggle S.P. Wilke A. Righetti K. et al.Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1.Proc. Natl. Acad. Sci. USA. 2009; 106: 6327-6332Crossref PubMed Scopus (160) Google Scholar, Rampioni et al., 2010Rampioni G. Pustelny C. Fletcher M.P. Wright V.J. Bruce M. Rumbaugh K.P. Heeb S. Cámara M. Williams P. Transcriptomic analysis reveals a global alkyl-quinolone-independent regulatory role for PqsE in facilitating the environmental adaptation of Pseudomonas aeruginosa to plant and animal hosts.Environ. Microbiol. 2010; 12: 1659-1673PubMed Google Scholar). The inverse regulation between Rhl and Pqs suggests a close relationship; yet, the relative contribution of Rhl and Pqs to virulence factor production is poorly defined. Furthermore, whether Las remains important for virulence factor production in wild-type (WT) P. aeruginosa under conditions that directly stimulate Rhl and Pqs is unclear. Thus, a greater fundamental understanding of how QS systems work together to coordinate virulence in defined, infection-relevant environments is required. Herein, we utilized a set of chemical probes that target specific QS circuits in WT P. aeruginosa to delineate the role of individual QS receptors in virulence regulation. We examined virulence factor production under a range of defined environmental conditions in the presence of each QS modulator. We discovered that the efficacies of these ligands are highly environment dependent. Our data suggest that the Rhl and Pqs systems work in tandem to drive virulence factor production in nutrient-limiting conditions, while Las is only a minor contributor. We show that LasR inhibitors display reduced activity in low iron and phosphate environments, and, as a result, cocktails of RhlR and PqsR inhibitors can attenuate virulence in a broad range of conditions where Las antagonists are inactive. The activity trends uncovered herein are also predictive of compound activity in infection-relevant environments, including the CF airway. Thus, our study suggests unique roles for the P. aeruginosa QS systems in tailoring virulence factor production to the environment and provides novel insights into pathways that, with further development, could potentially be targeted to fight this pathogen. We initiated our study with a focus on LasR. P. aeruginosa strains carrying nonfunctional lasR mutations are known to produce virulence factors through Rhl and Pqs in stationary phase, when essential nutrients begin to be depleted from the assay medium (Dekimpe and Déziel, 2009Dekimpe V. Déziel E. Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors.Microbiology. 2009; 155: 712-723Crossref PubMed Scopus (210) Google Scholar, Cabeen, 2014Cabeen M.T. Stationary phase-specific virulence factor overproduction by a lasR mutant of Pseudomonas aeruginosa.PLoS One. 2014; 9: e88743Crossref PubMed Scopus (43) Google Scholar). In particular, low phosphate concentrations are known to stimulate Rhl and Pqs via the response regulator PhoB (Figure 1A) (Jensen et al., 2006Jensen V. Lons D. Zaoui C. Bredenbruch F. Meissner A. Dieterich G. Munch R. Haussler S. RhlR expression in Pseudomonas aeruginosa is modulated by the Pseudomonas quinolone signal via PhoB-dependent and -independent pathways.J. Bacteriol. 2006; 188: 8601-8606Crossref PubMed Scopus (119) Google Scholar, Zaborin et al., 2009Zaborin A. Romanowski K. Gerdes S. Holbrook C. Lepine F. Long J. Poroyko V. Diggle S.P. Wilke A. Righetti K. et al.Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1.Proc. Natl. Acad. Sci. USA. 2009; 106: 6327-6332Crossref PubMed Scopus (160) Google Scholar). Therefore, both the contribution of Las to virulence and the ability of a LasR antagonist to attenuate virulence may be conditional. To test this hypothesis, we first grew WT P. aeruginosa (PAO1) in a 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered minimal medium, MOPS Glutamate (Mellbye and Schuster, 2014Mellbye B. Schuster M. Physiological framework for the regulation of quorum sensing-dependent public goods in Pseudomonas aeruginosa.J. Bacteriol. 2014; 196: 1155-1164Crossref PubMed Scopus (58) Google Scholar), with or without phosphate diluted 10-fold from the medium. We treated the cells with compound 1, a strong LasR antagonist reported by Greenberg and coworkers (Müh et al., 2006Müh U. Schuster M. Heim R. Singh A. Olson E.R. Greenberg E.P. Novel Pseudomonas aeruginosa quorum-sensing inhibitors identified in an ultra-high-throughput screen.Antimicrob. Agents Chemother. 2006; 50: 3674-3679Crossref PubMed Scopus (215) Google Scholar, Moore et al., 2015Moore J.D. Rossi F.M. Welsh M.A. Nyffeler K.E. Blackwell H.E. A comparative analysis of synthetic quorum sensing modulators in Pseudomonas aeruginosa: new insights into mechanism, active efflux susceptibility, phenotypic response, and next-generation ligand design.J. Am. Chem. Soc. 2015; 137: 14626-14639Crossref PubMed Scopus (72) Google Scholar) (Figure 1B) and monitored production of the virulence factor pyocyanin over time. Pyocyanin synthesis is directly regulated by Rhl/Pqs and indirectly by Las through induction of the former systems. Hence, LasR inhibitors are thought to attenuate pyocyanin production through suppression of Rhl and Pqs. Under the most nutrient replete conditions (which we refer to as “complete” medium), LasR inhibition by 1 eliminated pyocyanin synthesis, but, strikingly, under phosphate-poor conditions the compound had little to no effect (Figure 2). This result suggests that Las does not contribute to pyocyanin production under low phosphate conditions. Instead, activation of Rhl and Pqs, presumably via PhoB, is able to compensate for the loss of Las-dependent induction of these systems in the presence of 1 under these conditions. Having defined one environmental condition where a QS circuit is unessential for virulence, we sought to examine the individual contributions of all three QS circuits in P. aeruginosa in additional nutrient conditions that feed into QS activation. We replaced glutamate in the above MOPS minimal medium with glucose, a carbon source that is disfavored by carbon catabolite repression relative to glutamate in P. aeruginosa (Figure 1A) (Shrout et al., 2006Shrout J.D. Chopp D.L. Just C.L. Hentzer M. Givskov M. Parsek M.R. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional.Mol. Microbiol. 2006; 62: 1264-1277Crossref PubMed Scopus (416) Google Scholar, Rojo, 2010Rojo F. Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and interactions with the environment.FEMS Microbiol. Rev. 2010; 34: 658-684Crossref PubMed Scopus (342) Google Scholar). We also examined the effect of iron levels in the MOPS media because iron is tightly sequestered in most tissues and can influence Pqs activity (Figure 1A) (Oglesby et al., 2008Oglesby A.G. Farrow J.M. Lee J.-H. Tomaras A.P. Greenberg E.P. Pesci E.C. Vasil M.L. The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing.J. Biol. Chem. 2008; 283: 15558-15567Crossref PubMed Scopus (157) Google Scholar). We grew WT P. aeruginosa and isogenic lasR, rhlR, and pqsR mutants in MOPS Glutamate and MOPS Glucose with or without the iron and phosphate levels diluted 10-fold from the medium, and then quantified the amount of three virulence factors (elastase B, rhamnolipid, and pyocyanin) in the culture supernatant. Each of these virulence factors is closely associated with one QS circuit—elastase B with Las, rhamnolipid with Rhl, and pyocyanin with Pqs—but subject to direct or indirect regulation from all three circuits (Figure 1B) (Cao et al., 2001Cao H. Krishnan G. Goumnerov B. Tsongalis J. Tompkins R. Rahme L.G. A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism.Proc. Natl. Acad. Sci. USA. 2001; 98: 14613-14618Crossref PubMed Scopus (264) Google Scholar, Diggle et al., 2003Diggle S.P. Winzer K. Chhabra S.R. Worrall K.E. Cámara M. Williams P. The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR.Mol. Microbiol. 2003; 50: 29-43Crossref PubMed Scopus (430) Google Scholar, Déziel et al., 2005Déziel E. Gopalan S. Tampakaki A.P. Lépine F. Padfield K.E. Saucier M. Xiao G. Rahme L.G. The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones.Mol. Microbiol. 2005; 55: 998-1014Crossref PubMed Scopus (311) Google Scholar, Gilbert et al., 2009Gilbert K.B. Kim T.H. Gupta R. Greenberg E.P. Schuster M. Global position analysis of the Pseudomonas aeruginosa quorum-sensing transcription factor LasR.Mol. Microbiol. 2009; 73: 1072-1085Crossref PubMed Scopus (162) Google Scholar, Reis et al., 2011Reis R.S. Pereira A.G. Neves B.C. Freire D.M.G. Gene regulation of rhamnolipid production in Pseudomonas aeruginosa – a review.Bioresour. Technol. 2011; 102: 6377-6384Crossref PubMed Scopus (140) Google Scholar, Recinos et al., 2012Recinos D.A. Sekedat M.D. Hernandez A. Cohen T.S. Sakhtah H. Prince A.S. Price-Whelan A. Dietrich L.E.P. Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity.Proc. Natl. Acad. Sci. USA. 2012; 109: 19420-19425Crossref PubMed Scopus (110) Google Scholar). These virulence factors, therefore, are a representative sample of the global QS-controlled virulence regulon of P. aeruginosa. We observed that WT P. aeruginosa exhibits highly distinct virulence profiles under these varied environmental conditions, suggestive of unique QS activity in each medium (Figure 3). Pyocyanin and elastase B production were higher in MOPS Glutamate, but, congruent with a previous report (Shrout et al., 2006Shrout J.D. Chopp D.L. Just C.L. Hentzer M. Givskov M. Parsek M.R. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional.Mol. Microbiol. 2006; 62: 1264-1277Crossref PubMed Scopus (416) Google Scholar), rhamnolipid synthesis was nearly 10-fold higher when growing on glucose. Furthermore, the QS mutants displayed varying abilities to activate virulence factor production in response to nutrient limitation stress (Figure 3). For example, the lasR mutant produced no virulence factors in either complete medium, but depletion of phosphate induced elastase B, rhamnolipid, and pyocyanin production, characteristic of Rhl and Pqs activation in this environment (Dekimpe and Déziel, 2009Dekimpe V. Déziel E. Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors.Microbiology. 2009; 155: 712-723Crossref PubMed Scopus (210) Google Scholar). Furthermore, low iron concentrations uniquely induced rhamnolipid synthesis in the lasR mutant, suggesting that Rhl may respond preferentially to this condition. Notably, rhlR was required for virulence factor production under all conditions tested; nutrient limitation did not induce virulence factor production in the rhlR mutant. Finally, pqsR was essential for full pyocyanin and elastase B production, and nutrient depletion did not induce production of either of these virulence factors beyond levels observed in complete medium in this mutant strain. However, pqsR was only necessary for full rhamnolipid synthesis in low iron media. These data suggest that the Rhl and Pqs systems are required for P. aeruginosa to adapt its virulence profile to nutrient limitation stress, while the Las system plays a lesser role. We next sought to distinguish the relative contribution of Rhl and Pqs to virulence in defined nutrient environments. The genetic knockouts, above, can provide only limited information because gene deletion destroys the inter-regulation between QS circuits. We thus assembled a group of small molecules representing some of the most potent synthetic inhibitors of P. aeruginosa QS circuits known. These included: the LasR antagonist 1, introduced above (Müh et al., 2006Müh U. Schuster M. Heim R. Singh A. Olson E.R. Greenberg E.P. Novel Pseudomonas aeruginosa quorum-sensing inhibitors identified in an ultra-high-throughput screen.Antimicrob. Agents Chemother. 2006; 50: 3674-3679Crossref PubMed Scopus (215) Google Scholar, Moore et al., 2015Moore J.D. Rossi F.M. Welsh M.A. Nyffeler K.E. Blackwell H.E. A comparative analysis of synthetic quorum sensing modulators in Pseudomonas aeruginosa: new insights into mechanism, active efflux susceptibility, phenotypic response, and next-generation ligand design.J. Am. Chem. Soc. 2015; 137: 14626-14639Crossref PubMed Scopus (72) Google Scholar); the RhlR antagonist 2, reported by our laboratory (Welsh et al., 2015Welsh M.A. Eibergen N.R. Moore J.D. Blackwell H.E. Small molecule disruption of quorum sensing cross-regulation in Pseudomonas aeruginosa causes major and unexpected alterations to virulence phenotypes.J. Am. Chem. Soc. 2015; 137: 1510-1519Crossref PubMed Scopus (118) Google Scholar, Eibergen et al., 2015Eibergen N.R. Moore J.D. Mattmann M.E. Blackwell H.E. Potent and selective modulation of the RhlR quorum sensing receptor by using non-native ligands: an emerging target for virulence control in Pseudomonas aeruginosa.Chembiochem. 2015; 16: 2348-2356Crossref PubMed Scopus (46) Google Scholar); and the PqsR antagonist 4, reported by Rahme and coworkers (Figure 1B) (Starkey et al., 2014Starkey M. Lepine F. Ma" @default.
• W2284129173 created "2016-06-24" @default.
• W2284129173 creator A5019702159 @default.
• W2284129173 creator A5072500902 @default.
• W2284129173 date "2016-03-01" @default.
• W2284129173 modified "2023-10-13" @default.
• W2284129173 title "Chemical Genetics Reveals Environment-Specific Roles for Quorum Sensing Circuits in Pseudomonas aeruginosa" @default.
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