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The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence.

ABSTRACT: Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen Pseudomonas aeruginosa Macrophage infection with the atvR mutant strain resulted in higher levels of tumor necrosis factor alpha secretion as well as increased bacterial clearance compared to those for macrophages infected with the wild-type strain. In an acute pneumonia model, mice infected with the atvR mutant presented increased amounts of proinflammatory cytokines, increased neutrophil recruitment to the lungs, reductions in bacterial burdens, and higher survival rates in comparison with the findings for mice infected with the wild-type strain. Further, several genes involved in hypoxia/anoxia adaptation were upregulated upon atvR overexpression, as seen by high-throughput transcriptome sequencing (RNA-Seq) analysis. In addition, atvR was more expressed in hypoxia in the presence of nitrate and required for full expression of nitrate reductase genes, promoting bacterial growth under this condition. Thus, AtvR would be crucial for successful infection, aiding P. aeruginosa survival under conditions of low oxygen tension in the host. Taken together, our data demonstrate that the atypical response regulator AtvR is part of the repertoire of transcriptional regulators involved in the lifestyle switch from aerobic to anaerobic conditions. This finding increases the complexity of regulation of one of the central metabolic pathways that contributes to Pseudomonas ubiquity and versatility.

SUBMITTER: Kaihami GH

PROVIDER: S-EPMC5520423 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence.

Kaihami Gilberto Hideo GH Breda Leandro Carvalho Dantas LCD de Almeida José Roberto Fogaça JRF de Oliveira Pereira Thays T Nicastro Gianlucca Gonçalves GG Boechat Ana Laura AL de Almeida Sandro Rogério SR Baldini Regina Lúcia RL

Infection and immunity 20170719 8

Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen <i ...[more]

PMID: 28533471

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Project description:In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. In addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, we compared the transcriptional profile generated using DNA microarrays of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought, with the deletion of ampR influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Other virulence mechanisms including biofilm formation and QS-regulated acute virulence factors are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the microarray data. Further, using a Caenorhabditis elegans model, we demonstrate that a functional AmpR is required for P. aeruginosa pathogenicity. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. Further, we show differential regulation of other transcriptional regulators and sigma factors by AmpR, accounting for the extensive AmpR regulon. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating biofilm formation, a chronic infection phenotype. Unraveling this complex regulatory circuit will provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors in response to antibiotic exposure.

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.

Dataset Information

The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence.

Publications

The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence.

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