title: Targeting aphA : a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae.
abstract: A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (KmR) gene under the control of the aphA -like promoter for conferring a KmR phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml-1), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced KmR phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .
authors.affiliation: Department of Chemical Biology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
authors.forename: Galina
authors.initials: G
authors.lastname: Bolger
authors.affiliation: Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India.
authors.forename: Sambit
authors.initials: S
authors.lastname: Roy
authors.affiliation: Institute of Organic Chemistry, Technical University of Clausthal, Clausthal-Zellerfeld, Germany.
authors.forename: Viktor A
authors.initials: VA
authors.lastname: Zapol'skii
authors.affiliation: Institute of Organic Chemistry, Technical University of Clausthal, Clausthal-Zellerfeld, Germany.
authors.forename: Dieter E
authors.initials: DE
authors.lastname: Kaufmann
authors.affiliation: Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Schnürch
authors.affiliation: Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria.
authors.forename: Marko D
authors.initials: MD
authors.lastname: Mihovilovic
authors.affiliation: Division of Bacteriology, National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India.
authors.forename: Ranjan K
authors.initials: RK
authors.lastname: Nandy
authors.affiliation: Department of Chemical Biology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Tegge
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002771:Cholera / Q000473:pathology / Q000517:prevention & control; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D057166:High-Throughput Screening Assays; D051379:Mice; D016019:Survival Analysis; D015534:Trans-Activators / Q000037:antagonists & inhibitors*; D014734:Vibrio cholerae / Q000187:drug effects* / Q000472:pathogenicity*; D037521:Virulence Factors / Q000037:antagonists & inhibitors*
chemical_list: D000900:Anti-Bacterial Agents; C501058:AphA protein, Vibrio cholerae; D015534:Trans-Activators; D037521:Virulence Factors