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.
Highlights
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
The structure of apoBrlR is determined by molecular replacement (MR) using a protein monomer of BrlR–c-di-GMP complex as the search model
BrlR is a member of MerR-family transcriptional regulators from P. aeruginosa, which is activated by c-di-GMP5
Summary
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. To c-di-GMP, pyocyanin enhances the resistance of P. aeruginosa to many antimicrobial reagents by upregulating the expression of multidrug efflux pumps[34,35]. Whether the C-terminal domain of BrlR contains a c-di-GMP binding site, and whether BrlR acts as a pyocyanin receptor to activate the expression of multidrug efflux pumps, remain to be determined. These structural studies combined with biochemical data support that BrlR is a receptor for both c-di-GMP and pyocyanin, establishing a link between biofilm formation and multidrug efflux pumps expression
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