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Ligand responses of Vfr, the virulence factor regulator from Pseudomonas aeruginosa.

ABSTRACT: Vfr, a transcription factor homologous to the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP), regulates many aspects of virulence in Pseudomonas aeruginosa. Vfr, like CRP, binds to cAMP and then recognizes its target DNA and activates transcription. Here we report that Vfr has important functional differences from CRP in terms of ligand sensing and response. First, Vfr has a significantly higher cAMP affinity than does CRP, which might explain the mysteriously unidirectional functional complementation between the two proteins (S. E. H. West et al., J. Bacteriol. 176:7532-7542, 1994). Second, Vfr is activated by both cAMP and cGMP, while CRP is specific to cAMP. Mutagenic analyses show that Thr133 (analogous to Ser128 of CRP) is the key residue for both of these distinct Vfr properties. On the other hand, substitutions that cause cAMP-independent activity in Vfr are similar to those seen in CRP, suggesting that a common cAMP activation mechanism is present. In the course of these analyses, we found a remarkable class of Vfr variants that have completely reversed the regulatory logic of the protein: they are active in DNA binding without cAMP and are strongly inhibited by cAMP. The physiological impact of Vfr's ligand sensing and response is discussed, as is a plausible basis for the fundamental change in protein allostery in the novel group of Vfr variants.

SUBMITTER: Serate J

PROVIDER: S-EPMC3165676 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Ligand responses of Vfr, the virulence factor regulator from Pseudomonas aeruginosa.

Serate Jose J Roberts Gary P GP Berg Otto O Youn Hwan H

Journal of bacteriology 20110715 18

Vfr, a transcription factor homologous to the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP), regulates many aspects of virulence in Pseudomonas aeruginosa. Vfr, like CRP, binds to cAMP and then recognizes its target DNA and activates transcription. Here we report that Vfr has important functional differences from CRP in terms of ligand sensing and response. First, Vfr has a significantly higher cAMP affinity than does CRP, which might explain the mysteriously unidirectional functiona ...[more]

PMID: 21764924

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Project description: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.IMPORTANCEPseudomonas 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.

Project description:The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [Keq] of ?2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers.IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Dataset Information

Ligand responses of Vfr, the virulence factor regulator from Pseudomonas aeruginosa.

Publications

Ligand responses of Vfr, the virulence factor regulator from Pseudomonas aeruginosa.

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