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Muropeptide Binding and the X-ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa.

ABSTRACT: A complex link exists between cell-wall recycling/repair and the manifestation of resistance to ?-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of ?-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was purified to homogeneity. We document that the EBD exists exclusively as a dimer, even at a concentration as low as 1 ?M. The EBD binds to the suppressor ligand UDP-N-acetyl-?-d-muramyl-l-Ala-?-d-Glu-meso-DAP-d-Ala-d-Ala and binds to two activator muropeptides, N-acetyl-?-d-glucosamine-(1?4)-1,6-anhydro-N-acetyl-?-d-muramyl-l-Ala-?-d-Glu-meso-DAP-d-Ala-d-Ala and 1,6-anhydro-N-acetyl-?-d-muramyl-l-Ala-?-d-Glu-meso-DAP-d-Ala-d-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-?-d-muramyl-l-Ala-?-d-Glu-meso-DAP. This binding selectivity revises the dogma in the field. The crystal structure of the EBD dimer was solved to 2.2 Å resolution. The EBD crystallizes in a "closed" conformation, in contrast to the "open" structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules.

SUBMITTER: Dik DA

PROVIDER: S-EPMC5436579 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Muropeptide Binding and the X-ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa.

Dik David A DA Domínguez-Gil Teresa T Lee Mijoon M Hesek Dusan D Byun Byungjin B Fishovitz Jennifer J Boggess Bill B Hellman Lance M LM Fisher Jed F JF Hermoso Juan A JA Mobashery Shahriar S

Journal of the American Chemical Society 20170117 4

A complex link exists between cell-wall recycling/repair and the manifestation of resistance to β-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of β-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD ...[more]

PMID: 28079369

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Project description:In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal ?-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, PAO?ampR. 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 ?-lactam antibiotics via AmpC, AmpR also regulates non-?-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.

Dataset Information

Muropeptide Binding and the X-ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa.

Publications

Muropeptide Binding and the X-ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa.

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