Potent Irreversible Inhibitors of LasR Quorum Sensing in <i>Pseudomonas aeruginosa</i>

O’Brien, Kevin T.; Noto, Joseph; Nichols-O’Neill, Luke; Perez, Lark J.

doi:10.1021/ml500459f

Cited by 53 publications

(42 citation statements)

References 34 publications

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“…Third, we have provided a considerable number of co-crystal structures (nine in total) for the LasR-LBD bound to non-native ligands. In view of the growing interest in the TP scaffold (Capilato et al, 2017; O’Brien et al, 2015; O’Reilly and Blackwell, 2016) and QS in P. aeruginosa in general (Papenfort and Bassler, 2016; Welsh and Blackwell, 2016b), these structures are of fundamental interest. Fourth, we propose a mechanism for LasR activation and signal transduction that implicates the L3 loop as a possible gatekeeper for productive protein folding, dynamically opening and closing in the absence of ligand to provide entry to the ligand-binding pocket.…”

Section: Discussionmentioning

confidence: 99%

“…By scrutinizing the LasR-LBD structural data in concert with prior activity data for AHL-derived LasR antagonists (Amara et al, 2009; Galloway et al, 2011; Geske et al, 2007), various laboratories have sought to develop TP-derived compounds that antagonize LasR (Fig. 1B) (Capilato et al, 2017; O’Brien et al, 2015; O’Reilly and Blackwell, 2016). The antagonists identified in these past studies are among the most potent LasR antagonists known; however, they are orders of magnitude less potent than LasR’s native ligand or TP1 (i.e., micromolar IC 50 values relative to nanomolar EC 50 values for OdDHL and TP1 ).…”

Section: Introductionmentioning

confidence: 99%

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Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

O’Reilly

Dong

Rossi

et al. 2018

Cell Chemical Biology

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SUMMARY Chemical strategies to block quorum sensing (QS) could provide a route to attenuate virulence in bacterial pathogens. Considerable research has focused on this approach in Pseudomonas aeruginosa, which uses the LuxR-type receptor LasR to regulate much of its QS network. Non-native ligands that antagonize LasR have been developed, yet we have little understanding of the mode by which these compounds interact with LasR and alter its function, as the receptor is unstable in their presence. Herein, we report an approach to circumvent this challenge though the study of a series of synthetic LasR agonists with varying levels of potency. Structural investigations of these ligands with the LasR ligand-binding domain reveal that certain agonists can enforce a conformation that deviates from that observed for other, often more potent agonists. These results, when combined with cell-based and biophysical analyses, suggest a functional model for LasR that could guide future ligand design.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

O’Reilly

Dong

Rossi

et al. 2018

Cell Chemical Biology

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“…The proximity of Cys 79 to the terminal carbon of 3O-C 12 -HSL (22) (Fig. 1B) suggests that this thiol might be important for ligand recognition, and others have reported covalent modification of Cys 79 when LBD is expressed in the presence of electrophilic probes derived from the native AHL structure, placing reactive functional groups proximal to the thiol (34,35). Cys 79 appears to be most sensitive to oxidative stress when not bound to its native ligand, and perhaps the dimer observed by SDS-PAGE ( Fig.…”

Section: Impact Of Oxidative Stress On the Lasr Ligand Binding Domainmentioning

confidence: 94%

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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“…Our results add to this intriguing interkingdom chemical communication cross-talk by demonstrating that LXA 4 , with primary activity in the resolution of inflammatory signaling in the host, inhibits the activity of a bacterial QS receptor, LasR, and decreases the expression of virulence factors in this bacteria. The potency of this inhibition by LXA 4 as evaluated by comparing IC 50 values is several orders of magnitude greater than any reported LasR antagonist (77) and occurs at physiologically relevant concentrations of LXA 4 (64). Of interest, LXA 4 is not only an antagonist but it also has partial agonist activity against LasR (Fig.…”

Section: Discussionmentioning

confidence: 87%

Lipoxin A₄augments host defense in sepsis and reducesPseudomonas aeruginosavirulence through quorum sensing inhibition

Capilato

Pham

et al. 2016

FASEB j.

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Bacterial infections can quickly turn into sepsis, with its attendant clinical sequelae of inflammation, tissue injury, and organ failure. Paradoxically, sustained inflammation in sepsis may lead to immune suppression, because of which the host is unable to clear the existing infection. Use of agents that suppress the inflammatory response may accelerate host immune suppression, whereas use of traditional antibiotics does not significantly affect inflammation. In this study, we investigated whether lipoxin A4 (LXA4), a specialized, proresolution lipid mediator, could increase neutrophil phagocytic activity as well as reduce bacterial virulence. Using the mouse cecal ligation and puncture (CLP) model of sepsis, the administration of LXA4 (7 μg/kg i.v.) 1 h after surgery increased neutrophil phagocytic ability and Fcγ receptor I (CD64) expression. Ex vivo studies have confirmed that the direct addition of LXA4 to CLP neutrophils increased phagocytic ability but not CD64 expression. LXA4 did not affect neutrophils taken from control mice in which CD64 expression was minimal. Taken together with in vivo data, these results suggest that LXA4 directly augments CD64-mediated neutrophil phagocytic ability but does not directly increase neutrophil CD64 expression. Bacterial communication and virulence is regulated by quorum sensing inducers. In Pseudomonas aeruginosa, virulence is induced with release of various virulence factors, by N-3-oxododecanolyl homoserine lactone binding to the quorum sensing receptor, LasR. We show that LXA4 is an inhibitor of LasR in P. aeruginosa and that it decreases the release of pyocyanin exotoxin. These results suggest that LXA4 has the novel dual properties of increasing host defense and decreasing pathogen virulence by inhibiting quorum sensing.-Wu, B., Capilato, J., Pham, M. P., Walker, J., Spur, B., Rodriguez, A., Perez, L. J., Yin, K. Lipoxin A4 augments host defense in sepsis and reduces Pseudomonas aeruginosa virulence through quorum sensing inhibition.

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Potent Irreversible Inhibitors of LasR Quorum Sensing in Pseudomonas aeruginosa

Cited by 53 publications

References 34 publications

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

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

Lipoxin A₄augments host defense in sepsis and reducesPseudomonas aeruginosavirulence through quorum sensing inhibition

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Potent Irreversible Inhibitors of LasR Quorum Sensing in Pseudomonas aeruginosa

Cited by 53 publications

References 34 publications

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

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

Lipoxin A4augments host defense in sepsis and reducesPseudomonas aeruginosavirulence through quorum sensing inhibition

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Product

Resources

About

Lipoxin A₄augments host defense in sepsis and reducesPseudomonas aeruginosavirulence through quorum sensing inhibition