4 results on '"Federle MJ"'
Search Results
2. A novel chemical inducer of Streptococcus quorum sensing acts by inhibiting the pheromone-degrading endopeptidase PepO.
- Author
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Pérez Morales TG, Ratia K, Wang DS, Gogos A, Bloem L, Driver TG, and Federle MJ
- Subjects
- Gene Expression Regulation, Bacterial, Gram-Positive Bacteria metabolism, Neprilysin metabolism, Signal Transduction genetics, Signal Transduction physiology, Streptococcus pyogenes genetics, Streptococcus pyogenes metabolism, Bacterial Proteins metabolism, Endopeptidases metabolism, Pheromones metabolism, Quorum Sensing physiology
- Abstract
Bacteria produce chemical signals (pheromones) to coordinate behaviors across a population in a process termed quorum sensing (QS). QS systems comprising peptide pheromones and their corresponding Rgg receptors are widespread among Firmicutes and may be useful targets for manipulating microbial behaviors, like suppressing virulence. The Rgg2/3 QS circuit of the human pathogen Streptococcus pyogenes controls genes affecting resistance to host lysozyme in response to short hydrophobic pheromones (SHPs). Considering that artificial activation of a QS pathway may be as useful in the objective of manipulating bacteria as inhibiting it, we sought to identify small-molecule inducers of the Rgg2/3 QS system. We report the identification of a small molecule, P516-0475, that specifically induced expression of Rgg2/3-regulated genes in the presence of SHP pheromones at concentrations lower than typically required for QS induction. In searching for the mode of action of P516-0475, we discovered that an S. pyogenes mutant deficient in pepO , a neprilysin-like metalloendopeptidase that degrades SHP pheromones, was unresponsive to the compound. P516-0475 directly inhibited recombinant PepO in vitro as an uncompetitive inhibitor. We conclude that this compound induces QS by stabilizing SHP pheromones in culture. Our study indicates the usefulness of cell-based screens that modulate pathway activities to identify unanticipated therapeutic targets contributing to QS signaling., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
- Full Text
- View/download PDF
3. Activating mutations in quorum-sensing regulator Rgg2 and its conformational flexibility in the absence of an intermolecular disulfide bond.
- Author
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Wilkening RV, Capodagli GC, Khataokar A, Tylor KM, Neiditch MB, and Federle MJ
- Subjects
- Amino Acid Substitution, Bacterial Proteins antagonists & inhibitors, Bacterial Proteins chemistry, Bacterial Proteins genetics, Binding Sites, Crystallography, X-Ray, Cyclosporine pharmacology, Dimerization, Drug Resistance, Bacterial, Kinetics, Mutagenesis, Site-Directed, Pheromones chemistry, Pheromones metabolism, Pheromones pharmacology, Protein Conformation, Protein Interaction Domains and Motifs, Protein Isoforms antagonists & inhibitors, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Stability drug effects, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Static Electricity, Streptococcus pyogenes drug effects, Trans-Activators antagonists & inhibitors, Trans-Activators chemistry, Trans-Activators genetics, Transcription Factors antagonists & inhibitors, Transcription Factors chemistry, Transcription Factors genetics, Bacterial Proteins metabolism, Cysteine chemistry, Models, Molecular, Point Mutation, Streptococcus pyogenes metabolism, Trans-Activators metabolism, Transcription Factors metabolism
- Abstract
Rap/Rgg/NprR/PlcR/PrgX (RRNPP) quorum-sensing systems use extracellular peptide pheromones that are detected by cytoplasmic receptors to regulate gene expression in firmicute bacteria. Rgg-type receptors are allosterically regulated through direct pheromone binding to control transcriptional activity; however, the receptor activation mechanism remains poorly understood. Previous work has identified a disulfide bond between Cys-45 residues within the homodimer interface of Rgg2 from Streptococcus dysgalactiae (Rgg2
Sd ). Here, we compared two Rgg2Sd (C45S) X-ray crystal structures with that of wild-type Rgg2Sd and found that in the absence of the intermolecular disulfide, the Rgg2Sd dimer interface is destabilized and Rgg2Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2Sd secondary and tertiary structures, but another displayed more extensive rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify mutations in rgg2 of Streptococcus pyogenes ( rgg2Sp ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2Sp *, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2Sd (C45S) crystal structures. The Rgg2Sp * mutations increased Rgg2Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2Sp * mutations invoke shifts in free-energy bias to favor the active state of the protein. Finally, we present evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2Sp ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
- Full Text
- View/download PDF
4. Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation.
- Author
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Aggarwal C, Jimenez JC, Nanavati D, and Federle MJ
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Gene Expression Regulation, Bacterial, Genetic Variation, Humans, Pheromones chemistry, Protein Binding, Quorum Sensing genetics, Quorum Sensing physiology, Streptococcus pyogenes pathogenicity, Bacterial Proteins genetics, Bacterial Proteins metabolism, Pheromones genetics, Pheromones metabolism, Streptococcus pyogenes genetics, Streptococcus pyogenes metabolism, Trans-Activators genetics, Trans-Activators metabolism
- Abstract
Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
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