Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator

ABSTRACT New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important i...

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Autores principales:	Tomoe Kitao, Francois Lepine, Seda Babloudi, Frederick Walte, Stefan Steinbacher, Klaus Maskos, Michael Blaesse, Michele Negri, Michael Pucci, Bob Zahler, Antonio Felici, Laurence G. Rahme
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	MvfR PqsR Pseudomonas aeruginosa structural analysis X-ray crystallography antivirulence Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/3a5377cb41f442beb617a5562a681113
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spelling	oai:doaj.org-article:3a5377cb41f442beb617a5562a6811132021-11-15T15:53:26ZMolecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator10.1128/mBio.02158-172150-7511https://doaj.org/article/3a5377cb41f442beb617a5562a6811132018-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02158-17https://doaj.org/toc/2150-7511ABSTRACT New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation. IMPORTANCE Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by this pathogen. The Pseudomonas aeruginosa quorum sensing transcription factor MvfR regulates functions important in both acute and persistent infections. We used recently identified inhibitors of MvfR to perform structural studies and reveal important insights that would benefit the optimization of anti-MvfR compounds. Altogether, the results reported here provide critical detailed mechanistic insights into the function of MvfR domains that may benefit the optimization of the chemical, pharmacological, and safety properties of MvfR antagonist series.Tomoe KitaoFrancois LepineSeda BabloudiFrederick WalteStefan SteinbacherKlaus MaskosMichael BlaesseMichele NegriMichael PucciBob ZahlerAntonio FeliciLaurence G. RahmeAmerican Society for MicrobiologyarticleMvfRPqsRPseudomonas aeruginosastructural analysisX-ray crystallographyantivirulenceMicrobiologyQR1-502ENmBio, Vol 9, Iss 1 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	MvfR PqsR Pseudomonas aeruginosa structural analysis X-ray crystallography antivirulence Microbiology QR1-502
spellingShingle	MvfR PqsR Pseudomonas aeruginosa structural analysis X-ray crystallography antivirulence Microbiology QR1-502 Tomoe Kitao Francois Lepine Seda Babloudi Frederick Walte Stefan Steinbacher Klaus Maskos Michael Blaesse Michele Negri Michael Pucci Bob Zahler Antonio Felici Laurence G. Rahme Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator
description	ABSTRACT New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation. IMPORTANCE Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by this pathogen. The Pseudomonas aeruginosa quorum sensing transcription factor MvfR regulates functions important in both acute and persistent infections. We used recently identified inhibitors of MvfR to perform structural studies and reveal important insights that would benefit the optimization of anti-MvfR compounds. Altogether, the results reported here provide critical detailed mechanistic insights into the function of MvfR domains that may benefit the optimization of the chemical, pharmacological, and safety properties of MvfR antagonist series.
format	article
author	Tomoe Kitao Francois Lepine Seda Babloudi Frederick Walte Stefan Steinbacher Klaus Maskos Michael Blaesse Michele Negri Michael Pucci Bob Zahler Antonio Felici Laurence G. Rahme
author_facet	Tomoe Kitao Francois Lepine Seda Babloudi Frederick Walte Stefan Steinbacher Klaus Maskos Michael Blaesse Michele Negri Michael Pucci Bob Zahler Antonio Felici Laurence G. Rahme
author_sort	Tomoe Kitao
title	Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator
title_short	Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator
title_full	Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator
title_fullStr	Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator
title_full_unstemmed	Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator
title_sort	molecular insights into function and competitive inhibition of <italic toggle="yes">pseudomonas aeruginosa</italic> multiple virulence factor regulator
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/3a5377cb41f442beb617a5562a681113
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Molecular Insights into Function and Competitive Inhibition of <italic toggle="yes">Pseudomonas aeruginosa</italic> Multiple Virulence Factor Regulator

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