title: Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum.
abstract: BACKGROUND
  Fatty acid synthase (FAS) is a promising antifungal target due to its marked structural differences between fungal and mammalian cells. The aim of this study was to evaluate the antifungal activity of flavonoids described in the scientific literature as FAS inhibitors (quercetin, trans-chalcone, ellagic acid, luteolin, galangin, and genistein) against the dermatophyte Trichophyton rubrum and their effects on fatty acid and ergosterol synthesis.
  
  
  METHODS
  The antifungal activity of the natural products was tested by the microdilution assay for determination of the minimum inhibitory concentration (MIC). The effect of the compounds on the cell membrane was evaluated using a protoplast regeneration assay. Ergosterol content was quantified by spectrophotometry. Inhibition of FAS by flavonoids was evaluated by an enzymatic assay to determine IC50 values. Quantitative RT-PCR was used to measure transcription levels of the FAS1 and ERG6 genes involved in fatty acid and ergosterol biosynthesis, respectively, during exposure of T. rubrum to the flavonoids tested.
  
  
  RESULTS
  The flavonoids quercetin and trans-chalcone were effective against T. rubrum, with MICs of 125 and 7.5 μg/mL for the wild-type strain (MYA3108) and of 63 and 1.9 μg/mL for the ABC transporter mutant strain (ΔTruMDR2), respectively. The MICs of the fluconazole and cerulenin controls were 63 and 125 μg/mL for the wild-type strain and 30 and 15 μg/mL for the mutant strain, respectively. Quercetin and trans-chalcone also reduced ergosterol content in the two strains, indicating that interference with fatty acid and ergosterol synthesis caused cell membrane disruption. The MIC of quercetin reduced the number of regenerated protoplasts by 30.26% (wild-type strain) and by 91.66% (mutant strain). Half the MIC (0.5 MIC) of quercetin did not reduce the number of regenerated wild-type fungal colonies, but caused a 36.19% reduction in the number of mutant strain protoplasts. In contrast, the MIC and 0.5 MIC of trans-chalcone and cerulenin drastically reduced protoplast regeneration in the two strains. The FAS1 gene was repressed in the presence of MICs of quercetin, trans-chalcone, fluconazole and cerulenin. The ERG6 gene was induced in the presence of MICs of fluconazole and cerulenin and was repressed in the presence of MICs of trans-chalcone and quercetin. Trans-chalcone and quercetin inhibited the enzymatic activity of FAS, with IC50 values of 68.23 and 17.1 μg/mL, respectively.
  
  
  CONCLUSIONS
  Trans-chalcone and quercetin showed antifungal activity against T. rubrum, reducing ergosterol levels and modulating the expression of FAS1 and ERG6.
authors.affiliation: Unidade de Biotecnologia, Universidade de Ribeirão Preto, Av: Costábile Romano 2201, 14096-900, Ribeirão Preto, SP, Brasil. afachin@unaerp.br.
authors.forename: Tamires Aparecida
authors.initials: TA
authors.lastname: Bitencourt
authors.forename: Tatiana Takahasi
authors.initials: TT
authors.lastname: Komoto
authors.forename: Bruna Gabriele
authors.initials: BG
authors.lastname: Massaroto
authors.forename: Carlos Eduardo Saraiva
authors.initials: CE
authors.lastname: Miranda
authors.forename: Rene Oliveira
authors.initials: RO
authors.lastname: Beleboni
authors.forename: Mozart
authors.initials: M
authors.lastname: Marins
authors.forename: Ana Lúcia
authors.initials: AL
authors.lastname: Fachin
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D002599:Chalcone / Q000494:pharmacology*; D004875:Ergosterol / Q000032:analysis*; D064429:Fatty Acid Synthases / Q000032:analysis / Q000378:metabolism*; D005656:Fungal Proteins / Q000032:analysis / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D008780:Methyltransferases / Q000032:analysis / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D011794:Quercetin / Q000494:pharmacology*; D014249:Trichophyton / Q000187:drug effects*
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D002599:Chalcone; D011794:Quercetin; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D064429:Fatty Acid Synthases; D004875:Ergosterol
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