Malassezia spp. extracts and metabolites induce the AhR dependent genes in HaCaT cells

In previous works [1,2] we compared Malassezia furfur isolates from healthy and seborrhoic dermatitis skin for the production of indole derivatives and identified the preferential biosynthesis of malassezin, indolo[3,2-b]carbazol (ICZ) and indirubin by M. furfur strains isolated from diseased skin. These compounds, which are known highly active Aryl hydrocarbon receptor (AhR) inducers, were synthesized and used as standards for their quantification in extracts of M.furfur strains grown on L-tryptophan agar. HPLC analysis revealed that ICZ (1.2–6.0µg/mg), indirubin (0.1–1.7µg/mg), malassezin (2.3–40.9µg/mg) are produced in significant quantities especially in the extracts of the clinical strains. The extracts were normalized according to ICZ content and cytotoxicity tests (MTS assay) were performed after exposure of HaCaT cells to different concentrations for 24, 48 and 72 hours. Subsequently, expression of the AhR battery of enzymes was assessed by Reverse Transcriptase Real-Time PCR (exposure for 24h). The results showed statistically significant alterations in AhRR, CYP1A1, CYP1B1, GSTT1 and GSTP1 mRNA. In addition to this, malassezin (0.04–1.5µg/mg), indirubin (0.03µg/mg) and ICZ (0.2µg/mg) were identified and quantified for the first time, in extracts of other species like reference strains of M.japonica, M. sympodialis and M.yamatoensis grown on L-tryptophan agar. The concentration of the measured compounds was significantly lower than that of M.furfur clinical strains but their widespread presence implies the importance of these metabolites in the skin physiology and disease development. Finally, in an effort to identify the biosynthetic pathway for indirubin which is the most active AhR ligand among the studied metabolites we have found a new reaction for the one-step transformation of indol-3-carboxaldehyde (the major Malassezia metabolite of tryptophan) to indirubin using hydrogen peroxide in acid medium or selenium based catalysts. References: [1] Gaitanis, G. et al. (2008)J. Invest. Dermatol. 128:1620–1625. [2] Giakoumaki, D. et al. (2008) Planta Med. 74:1081.