title: A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.
abstract: The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St. Kitts KrBaker@rossvet.edu.kn LGuardabassi@rossvet.edu.kn.
authors.forename: Kristin R
authors.initials: KR
authors.lastname: Baker
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St. Kitts.
authors.forename: Bimal
authors.initials: B
authors.lastname: Jana
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Franzyk
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St. Kitts KrBaker@rossvet.edu.kn LGuardabassi@rossvet.edu.kn.
authors.forename: Luca
authors.initials: L
authors.lastname: Guardabassi
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002473:Cell Wall / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005672:Fusidic Acid / Q000494:pharmacology; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006868:Hydrolysis; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009598:Nitrophenylgalactosides / Q000737:chemistry / Q000378:metabolism; D009675:Novobiocin / Q000494:pharmacology; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D057786:Peptidomimetics / Q000737:chemistry / Q000494:pharmacology*; D010539:Permeability / Q000187:drug effects; D012293:Rifampin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D019151:Peptide Library; D010455:Peptides; D057786:Peptidomimetics; D009675:Novobiocin; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D005672:Fusidic Acid; D004917:Erythromycin; D001616:beta-Galactosidase; C473109:beta-lactamase CTX-M-15; D001618:beta-Lactamases; D012293:Rifampin