Session: Poster Session: Fungal Biofilms
Sunday, October 26, 2008: 12:00 AM
Room: Hall C
Background: A. fumigatus is able to form biofilm- like structures A. fumigatus biofilm was analyzed by 2D-gel electrophoresis to elucidate regulation mechanisms and relevant protein regulations and drug resistance of the biofilm-grown cultures was tested. Methods: A. fumigatus strain ATCC #46645 was adjusted to 106 conidia /ml MEM +5% FCS for biofilm formation. MICs of non-biofilm and biofilm cultures were determined after 48h, and after 48h exposure to Amphotericin B, Itraconazole, Voriconazole and Micafungin visually and by an XTT-based assay. Aspergillus biofilm was grown for 24h / 48h and broken by grinding in a precooled mortar. Proteins were isolated using phenol extraction. For 2D-gel electrophoresis, proteins were labeled using DIGE. Isoelectric focusing was carried out with IPG-strips followed by SDS-PAGE using gradient gels. Differentially regulated proteins were analyzed by MALDI-TOF/TOF. Results: A trend to higher MICs for 24h biofilm in comparison to non-biofilm was observed. Proteins of the glycolysis (GpdA), pyruvate metabolism (PdcA), the TCA-cycle (Cit1) and oxidative phosphorylation (FI ATPase subunit) showed a higher abundance in 24 h-old biofilm, indicating a higher respiratory metabolism during early biofilm formation. At 48h biofilm, A. fumigatus developed resistance against all tested antifungals. At this stage the NAD-dependent formate dehydrogenase was significantly up-regulated that is induced by many stress conditions and that is involved in the regulation of the NAD+/NADH equilibrium. Furthermore, some proteins involved in the biosynthesis of secondary metabolites were also differentially regulated during biofilm formation. Conclusions: During A. fumigatus biofilm formation antifungal drug resistance increases and the metabolic status changes significantly. At later stages the respiratory activity decreases, whereas pathways involved in the consumption of reduction equivalent are induced.