title: High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae.
abstract: BACKGROUND
  Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen.
  
  
  RESULTS
  A high-throughput screening assay with a Vibrio cholerae reporter strain constitutively expressing green fluorescent protein (GFP) was developed and applied in the investigation of the growth inhibitory effect of approximately 28,300 structurally diverse natural compounds and synthetic small molecules. Several compounds with activities in the low micromolar concentration range were identified. The most active structure, designated vz0825, displayed a minimal inhibitory concentration (MIC) of 1.6 μM and a minimal bactericidal concentration (MBC) of 3.2 μM against several strains of V. cholerae and was specific for this pathogen. Mutants with reduced sensitivity against vz0825 were generated and whole genome sequencing of 15 pooled mutants was carried out. Comparison with the genome of the wild type strain identified the gene VC_A0531 (GenBank: AE003853.1) as the major site of single nucleotide polymorphisms in the resistant mutants. VC_A0531 is located on the small chromosome of V. cholerae and encodes the osmosensitive K+-channel sensor histidine kinase (KdpD). Nucleotide exchange of the major mutation site in the wild type strain confirmed the sensitive phenotype.
  
  
  CONCLUSIONS
  The reporter strain MO10 pG13 was successfully used for the identification of new antibacterial compounds against V. cholerae. Generation of resistant mutants and whole genome sequencing was carried out to identify the histidine kinase KdpD as a novel antimicrobial target.
authors.forename: Galina
authors.initials: G
authors.lastname: Sergeev
authors.forename: Sambit
authors.initials: S
authors.lastname: Roy
authors.forename: Michael
authors.initials: M
authors.lastname: Jarek
authors.forename: Viktor
authors.initials: V
authors.lastname: Zapolskii
authors.forename: Dieter E
authors.initials: DE
authors.lastname: Kaufmann
authors.affiliation: Department of Chemical Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstraße 7, D-38124 Braunschweig, Germany. nandy_rk@hotmail.com.
authors.forename: Ranjan K
authors.initials: RK
authors.lastname: Nandy
authors.forename: Werner
authors.initials: W
authors.lastname: Tegge
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D004252:DNA Mutational Analysis; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*; D016680:Genome, Bacterial; D057166:High-Throughput Screening Assays; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D011494:Protein Kinases / Q000235:genetics; D014734:Vibrio cholerae / Q000187:drug effects* / Q000254:growth & development* / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011494:Protein Kinases; C075616:kdpD protein, Bacteria