Mutational Analysis of the Quorum-Sensing Receptor LasR Reveals Interactions that Govern Activation and Inhibition by Nonlactone Ligands

Gerdt, Joseph P.; McInnis, Christine E.; Schell, Trevor L.; Rossi, Francis M.; Blackwell, Helen E.

doi:10.1016/j.chembiol.2014.08.008

Cited by 40 publications

(68 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2D). Our results join previous findings suggesting that autoinducer acyl side chain length is particularly important in dictating the stimulation of LasR activity (36) and recent work cataloging hydrogen-bonding interactions in the LasR LBD critical for LasR activation and inhibition (37,38).…”

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

confidence: 89%

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

View full text Add to dashboard Cite

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...

show abstract

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

confidence: 89%

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

View full text Add to dashboard Cite

show abstract

“…[39][40][41][42][43][44] Both Suga and Blackwell have shown that this residue determines whether a ligand acts as an agonist or antagonist. 45,46 Ligands that exhibit unfavorable interactions with Trp60 have antagonistic profiles. Recently Blackwell also revealed that the interactions between a ligand and Tyr56 and Ser129 in LasR are also important in determining whether a ligand acts as an antagonist or agonist since these residues bond to the carbonyl of the 3-oxo-C12-HSL ligand to position the lactone head group towards Tyr 60, which is a key residue.…”

Section: Resultsmentioning

confidence: 99%

“…Recently Blackwell also revealed that the interactions between a ligand and Tyr56 and Ser129 in LasR are also important in determining whether a ligand acts as an antagonist or agonist since these residues bond to the carbonyl of the 3-oxo-C12-HSL ligand to position the lactone head group towards Tyr 60, which is a key residue. 45,47 Docking experiments [48][49][50] revealed that the docked poses of 3-oxo-C12-HSL and of the 3-aminooxazolidinone analog (1) are similar, with the exception of the orientation of the 3-oxo group (see Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

3-Aminooxazolidinone AHL analogs as hydrolytically-stable quorum sensingagonists in Gram-negative bacteria

et al. 2015

View full text Add to dashboard Cite

Synthetic molecules that modulate quorum sensing, QS, in bacteria have great potential for use in synthetic biology applications as well as acting as anti-virulence and anti-biofilm agents. Acylhomoserine lactone (AHL)-based autoinducer analogs have been extensively developed as QS modulators but these suffer from both chemical and enzymatic degradations. Here, we reveal that 3-aminooxazolidinone acylhomoserine lactone analogs are hydrolytically stable and are as potent in activating LuxR-type receptors.Docking analysis revealed that 3-oxo-C12-3-aminooxazolidinone docked in LasR of P. aeruginosa, making similar interactions with the protein's active-site residues to the native ligand, 3-oxo-C12 HSL. Experimentally, 3-oxo-C12-3-aminooxazolidinone was equally as potent as the natural ligand in inducing bioluminescence in E. coli carrying a bioluminescent gene that was under the control of LasR. In C. violaceum CV026, the 3-aminooxazolidinone analogs could also modulate pigment (violacein) formation, albeit this time not as potent as the natural AHL ligands.

show abstract

“…It has been proposed that targeting LasR may have the largest impact on QS-related virulence in P. aeruginosa (Galloway et al, 2012), since LasR activation directly upregulates certain virulence phenotypes (e.g., proteases, biofilm) and indirectly upregulates other virulence phenotypes (e.g., pyocyanin, rhamnolipid) through positive regulation of both the RhlR and PqsR systems (Asfahl and Schuster, 2018; Welsh and Blackwell, 2016a, b). Therefore, considerable efforts have been directed toward designing molecules to antagonize LasR and thereby block its associated virulence phenotypes, with notable contributions from the Bassler (O’Loughlin et al, 2013), Blackwell (Gerdt et al, 2014; Geske et al, 2007; Moore et al, 2015; O’Reilly and Blackwell, 2016), Greenberg (Muh et al, 2006; Müh et al, 2006), Meijler (Amara et al, 2011; Amara et al, 2009), Spring (Galloway et al, 2011; Hodgkinson et al, 2012), and Suga (Smith et al, 2003a; Smith et al, 2003b) laboratories.…”

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

View full text Add to dashboard Cite

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.

show abstract

Mutational Analysis of the Quorum-Sensing Receptor LasR Reveals Interactions that Govern Activation and Inhibition by Nonlactone Ligands

Cited by 40 publications

References 50 publications

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

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

3-Aminooxazolidinone AHL analogs as hydrolytically-stable quorum sensingagonists in Gram-negative bacteria

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

Contact Info

Product

Resources

About