title: Inhibition of virulence-promoting disulfide bond formation enzyme DsbB is blocked by mutating residues in two distinct regions.
abstract: Disulfide bonds contribute to protein stability, activity, and folding in a variety of proteins, including many involved in bacterial virulence such as toxins, adhesins, flagella, and pili, among others. Therefore, inhibitors of disulfide bond formation enzymes could have profound effects on pathogen virulence. In theEscherichia colidisulfide bond formation pathway, the periplasmic protein DsbA introduces disulfide bonds into substrates, and then the cytoplasmic membrane protein DsbB reoxidizes DsbA's cysteines regenerating its activity. Thus, DsbB generates a protein disulfide bondde novoby transferring electrons to the quinone pool. We previously identified an effective pyridazinone-related inhibitor of DsbB enzymes from several Gram-negative bacteria. To map the protein residues that are important for the interaction with this inhibitor, we randomly mutagenized by error-prone PCR theE. coli dsbBgene and selecteddsbBmutants that confer resistance to this drug using two approaches. We characterizedin vivoandin vitrosome of these mutants that map to two areas in the structure of DsbB, one located between the two first transmembrane segments where the quinone ring binds and the other located in the second periplasmic loop of DsbB, which interacts with DsbA. In addition, we show that a mutant version of a protein involved in lipopolysaccharide assembly,lptD4213, is synthetically lethal with the deletion ofdsbBas well as with DsbB inhibitors. This finding suggests that drugs decreasing LptD assembly may be synthetically lethal with inhibitors of the Dsb pathway, potentiating the antibiotic effects.
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Cristina
authors.initials: C
authors.lastname: Landeta
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Meehan
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Laura
authors.initials: L
authors.lastname: McPartland
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Linda
authors.initials: L
authors.lastname: Ingendahl
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Feras
authors.initials: F
authors.lastname: Hatahet
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Ngoc Q
authors.initials: NQ
authors.lastname: Tran
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Dana
authors.initials: D
authors.lastname: Boyd
authors.affiliation: From the Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115 Jon_Beckwith@hms.harvard.edu.
authors.forename: Jon
authors.initials: J
authors.lastname: Beckwith
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D015723:Gene Library; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics; D011724:Pyridazines / Q000737:chemistry; D011809:Quinones / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D013696:Temperature; D014774:Virulence
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; C475958:LptD protein, E coli; D008565:Membrane Proteins; D011724:Pyridazines; D011809:Quinones; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli
keywords: Escherichia coli (E. coli); bacteria; bacterial genetics; disulfide; inhibition mechanism; lptD; pyridazinone; redox; synthetic lethality