A rapid thin layer chromatography autographic method for the detection of inhibitors of the Salmonella PhoP/PhoQ regulatory system in chemically engineered extracts

SALAZAR, M.; VIARENGO, G.; SCIARA, M.I.; KIEFFER, P.; GARCÍA VÉSCOVI, E.; FURLAN, R. L.

PHYTOCHEMICAL ANALYSIS

JOHN WILEY & SONS LTD

Lugar: Londres; Año: 2014 vol. 25 p. 155 - 160

0958-0344

Introduction The PhoP/PhoQ system from Salmonella enterica serovar Typhimurium constitutes an interesting target to search for compounds that would prevent and/or control Salmonella virulence. Localization of inhibitors of this system in complex matrixes such us crude o chemically engineered plant extracts could be facilitated by TLC-bioautography that combines chromatographic separation and in situ activity determination.  Objective To develop a TLC-bioautography to detect inhibitors of the PhoP/PhoQ regulatory system in complex matrixes.  Methodology TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), kanamycin and a S. Typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 hours. Results A bioautographic assay suitable for the localization of inhibitors of the PhoP/PhoQ regulatory system activity in Salmonella enterica serovar Typhimurium present in a complex matrix is described: activity of the PhoP/PhoQ system was detected using X-gal as the substrate to produce 5-bromo-4-chloro-3-hydroxyindole, which spontaneously dimerizes and oxidizes into 5,5?-dibromo-4,4?-dichloro-indigo, an intensely sky-blue product. The assay was used to analyze a series of chemically engineered extracts prepared by alkaline treatment of crude plant extracts, and to assign the observed bioactivity to unsaturated fatty acids.  Conclusion  A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP/PhoQ regulatory system. The assay convenience is illustrated with the rapid identification of the first two organic compounds that down-regulate PhoP/PhoQ regulatory system from a chemically engineered extract prepared through alkaline treatment.