A Comparative Analysis of Synthetic Quorum Sensing Modulators in <i>Pseudomonas aeruginosa</i>: New Insights into Mechanism, Active Efflux Susceptibility, Phenotypic Response, and Next-Generation Ligand Design

Moore, Joseph D.; Rossi, Francis M.; Welsh, Michael A.; Nyffeler, Kayleigh E.; Blackwell, Helen E.

doi:10.1021/jacs.5b06728

Cited by 82 publications

(155 citation statements)

References 74 publications

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“…To test this hypothesis, we first grew WT P. aeruginosa (PAO1) in a MOPS buffered minimal medium, MOPS Glutamate (Mellbye and Schuster, 2014), 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, 2006, Moore et al, 2015) (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.…”

Section: Resultsmentioning

confidence: 99%

Chemical Genetics Reveals Environment-Specific Roles for Quorum Sensing Circuits in Pseudomonas aeruginosa

Welsh

Blackwell

2016

Cell Chemical Biology

100

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SUMMARY 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.

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Section: Resultsmentioning

confidence: 99%

Chemical Genetics Reveals Environment-Specific Roles for Quorum Sensing Circuits in Pseudomonas aeruginosa

Welsh

Blackwell

2016

Cell Chemical Biology

100

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“…Due to this prominent role, its not surprising that LasR has been a primary target for the design of small molecule antagonists to block QS in this pathogen (9, 16–18) . Far fewer research efforts have been directed toward the design of non-native ligands for RhlR, likely due to its perceived secondary role in QS.…”

Section: Introductionmentioning

confidence: 99%

Structure–Function Analyses of the N-Butanoyl l-Homoserine Lactone Quorum-Sensing Signal Define Features Critical to Activity in RhlR

et al. 2018

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Pseudomonas aeruginosa is an opportunistic pathogen that coordinates the production of many virulence phenotypes at high population density via quorum sensing (QS). The LuxR-type receptor RhlR plays an important role in the P. aeruginosa QS process, and there is considerable interest in the development of chemical approaches to modulate the activity of this protein. RhlR is activated by a simple, low molecular weight N-acyl L-homoserine lactone signal, N-butanoyl-L-homoserine lactone (BHL). Despite the emerging prominence of RhlR in QS pathways, there has been limited exploration of the chemical features of the BHL scaffold that are critical to its function. In the current study, we sought to systematically delineate the structure-activity relationships (SARs) driving BHL activity for the first time. A focused library of BHL analogs was designed, synthesized, and evaluated in cell-based reporter gene assays for RhlR agonism and antagonism. These investigations allowed us to define a series of SARs for BHL-type ligands and identify structural motifs critical for both activation and inhibition of the RhlR receptor. Notably, we identified agonists that have ~10-fold higher potencies in RhlR relative to BHL, are highly selective for RhlR agonism over LasR, and are active in the P. aeruginosa background. These compounds and the SARs reported herein should pave a route toward new chemical strategies to study RhlR in P. aeruginosa.

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“…There has been significant interest in understanding LasR, as its inhibition represents a promising antivirulence strategy for the treatment of P. aeruginosa. One prevalent mode of inhibition is the development of nonnative small molecules shown to disrupt LasR function through the reduction of virulence factors produced (12)(13)(14). A number of these analogs appear to inhibit LasR through competition with the native 3O-C 12 -HSL because they are often structurally similar.…”

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confidence: 99%

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

Kafle¹,

Amoh²,

Reaves³

et al. 2016

Journal of Biological Chemistry

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The LasR regulator protein functions at the top of the Pseudomonas aeruginosa quorum-sensing hierarchy and is implicated in promoting bacterial virulence. Of note is recent evidence that this transcription factor may also respond to oxidative stress. Here, all cysteines in LasR were inspected to deduce their redox sensitivity and to probe the connection between stress response and LasR activity using purified LasR and individual LasR domains. Cys 79 in the ligand binding domain of LasR appears to be important for ligand recognition and folding of this domain to potentiate DNA binding but does not seem to be sensitive to oxidative stress when bound to its native ligand. Two cysteines in the DNA binding domain of LasR do form a disulfide bond when treated with hydrogen peroxide, and formation of this Cys 201 -Cys 203 disulfide bond appears to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. A cell-based reporter assay linking LasR function with ␤-galactosidase activity gave results consistent with those obtained with purified LasR. This work provides a possible mechanism for oxidative stress response by LasR and indicates that multiple cysteines within the protein may prove to be useful targets for disabling its activity.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that establishes chronic infections in immunocompromised patients, with persistent pulmonary colonization in individuals with cystic fibrosis (1-3). Like many bacteria, P. aeruginosa employs quorumsensing (QS) 4 machinery to communicate local population density through the exchange of self-produced signaling molecules (4). QS contributes to the development of acute infections by coordinating the production of multiple virulence factors, including elastase and pyocyanin, and the formation of biofilms (5-7). QS in P. aeruginosa is largely mediated by N-acyl L-homoserine lactone autoinducer molecules, biosynthesized by LuxI-type synthases, and detected by LuxR-type regulator proteins (8). The two major systems responsive to acylhomoserine lactones (AHLs) in P. aeruginosa are LasI/LasR and RhlI/RhlR. LasI and RhlI are LuxI-type synthases, whereas LasR and RhlR are LuxR-type regulatory proteins. These AHL-dependent systems are also closely connected to signaling governed by 2-alkyl-4-quinolones in P. aeruginosa (9).The regulator protein LasR, with its cognate synthase LasI, functions at the top of the P. aeruginosa quorum-sensing hierarchy (10). Like other LuxR-type proteins, this transcription factor is composed of two domains (11). The amino-terminal ligand binding domain (LBD) specifically recognizes its autoinducer, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C 12 -HSL), promoting protein folding and dimerization, whereas the DNA binding domain (DBD) recognizes target sites to activate downstream gene expression. There has been significant interest in understanding LasR, as its inhibition represents a...

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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

Cited by 82 publications

References 74 publications

Chemical Genetics Reveals Environment-Specific Roles for Quorum Sensing Circuits in Pseudomonas aeruginosa

Chemical Genetics Reveals Environment-Specific Roles for Quorum Sensing Circuits in Pseudomonas aeruginosa

Structure–Function Analyses of the N-Butanoyl l-Homoserine Lactone Quorum-Sensing Signal Define Features Critical to Activity in RhlR

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

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