title: A thin-layer chromatography autographic method for the detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system.
abstract: BACKGROUND
  The PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography.
  
  
  OBJECTIVE
  To develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes.
  
  
  METHODS
  The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-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 h.
  
  
  RESULTS
  A bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system.
  
  
  CONCLUSIONS
  A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract.
authors.affiliation: Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, S2002LRK, Rosario, Argentina.
authors.forename: Mario O
authors.initials: MO
authors.lastname: Salazar
authors.forename: Gaston
authors.initials: G
authors.lastname: Viarengo
authors.forename: Mariela I
authors.initials: MI
authors.lastname: Sciara
authors.forename: Pablo M
authors.initials: PM
authors.lastname: Kieffer
authors.forename: Eleonora
authors.initials: E
authors.lastname: Garcia Vescovi
authors.forename: Ricardo L E
authors.initials: RL
authors.lastname: Furlan
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D002855:Chromatography, Thin Layer / Q000379:methods*; D019281:Dimerization; D005697:Galactosides; D017930:Genes, Reporter; D006868:Hydrolysis; D007211:Indoles; D019787:Linoleic Acid / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D019684:Magnoliopsida / Q000737:chemistry; D013058:Mass Spectrometry; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000378:metabolism; D014774:Virulence; D017962:alpha-Linolenic Acid / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D005697:Galactosides; D007211:Indoles; C079792:PhoQ protein, Bacteria; D010936:Plant Extracts; D017962:alpha-Linolenic Acid; C061064:PhoP protein, Bacteria; D019787:Linoleic Acid; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside
keywords: Bioautography; PhoP-PhoQ; Salmonella; linoleic acid; linolenic acid