Abstract
The virulence factor pyocyanin and the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP) play key roles in regulating biofilm formation and multi-drug efflux pump expression in Pseudomonas aeruginosa. However, the crosstalk between these two signaling pathways remains unclear. Here we show that BrlR (PA4878), previously identified as a c-di-GMP responsive transcriptional regulator, acts also as a receptor for pyocyanin. Crystal structures of free BrlR and c-di-GMP-bound BrlR reveal that the DNA-binding domain of BrlR contains two separate c-di-GMP binding sites, both of which are involved in promoting brlR expression. In addition, we identify a pyocyanin-binding site on the C-terminal multidrug-binding domain based on the structure of the BrlR-C domain in complex with a pyocyanin analog. Biochemical analysis indicates that pyocyanin enhances BrlR-DNA binding and brlR expression in a concentration-dependent manner.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Anti-Bacterial Agents / pharmacology
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Anti-Bacterial Agents / therapeutic use
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Bacterial Proteins / chemistry
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Bacterial Proteins / isolation & purification
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Bacterial Proteins / metabolism*
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Binding Sites
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Biofilms / drug effects
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Crystallography, X-Ray
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Cyclic GMP / analogs & derivatives*
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Cyclic GMP / chemistry
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Cyclic GMP / metabolism
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Drug Resistance, Multiple, Bacterial / genetics
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Gene Expression Regulation, Bacterial
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Humans
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Microbial Sensitivity Tests
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Molecular Docking Simulation
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Protein Domains
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Pseudomonas Infections / drug therapy*
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Pseudomonas Infections / virology
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Pseudomonas aeruginosa / pathogenicity
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Pseudomonas aeruginosa / physiology*
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Pyocyanine / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Transcription Factors / chemistry
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Transcription Factors / isolation & purification
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Transcription Factors / metabolism*
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Virulence Factors / metabolism*
Substances
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Anti-Bacterial Agents
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Bacterial Proteins
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Recombinant Proteins
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Transcription Factors
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Virulence Factors
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bis(3',5')-cyclic diguanylic acid
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Pyocyanine
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Cyclic GMP