IADR Abstract Archives
Overexpression of Ifd6p Increased Farnesol Level in Candida Dubliniensis Biofilms
Objectives: To investigate the in vitro effects of overexpression of ADH5, CSH1, and IFD6 on levels of excreted alcohols and virulence in Candida dubliniensis biofilms.
Methods: Overexpression of ADH5, CSH1, and IFD6 in C. dubliniensis Wü284 was achieved using a PCR-based gene targeting method. Overexpression was verified by real time quantitative PCR (RT-qPCR). C. dubliniensis biofilms were prepared in microtiter plates (polystyrene, flat bottom 24-well). The cell suspension (1 mL, 107cells/mL in PBS) was transferred to the wells and incubated at 37oC, 80 rpm for 1.5 h. Unattached fungal cells were washed away, and added with fresh YPD (1 mL) with doxycycline (50 µg/mL) and further incubated for 48 h. Composition of excreted alcohols in culture supernatant was evaluated by gas chromatograph-mass spectrometer equipped with an HP 19091B-005 50-m capillary column at a flow rate of 1 mL/min. Peaks were identified and quantified by comparing with the retention times and MS spectra of authenticated alcohol standards. Virulence was evaluated using oral reconstituted human epithelial (RHE) tissues. Sections of 1 µm-thick were cut and stained with hematoxylin and eosin, and viewed under light microscope. Released lactate dehydrogenase from the RHE tissues was determined using CytoTox 96 nonradioactive cytotoxicity assay.
Results: Four kinds of alcohols (isoamyl alcohol, phenethyl alcohol, nerolidol, and farnesol) were detected and authenticated. Level of farnesol was increased by ~23% (P < 0.05) in IFD6-overexpressed C. dubliniensis when compared with their control. There was no significant difference in alcohol levels in ADH5-overexpressed and CSH1-overexpressed C. dubliniensis. The virulence of all C. dubliniensis biofilms was similar to that of the reference strains.
Conclusions: IFD6 is a gene that is involved in biosynthesis of farnesol in C. dubliniensis biofilms. Overexpression of IFD6 in C. dubliniensis leads to increased level of farnesol without any effect on fungal virulence.
Methods: Overexpression of ADH5, CSH1, and IFD6 in C. dubliniensis Wü284 was achieved using a PCR-based gene targeting method. Overexpression was verified by real time quantitative PCR (RT-qPCR). C. dubliniensis biofilms were prepared in microtiter plates (polystyrene, flat bottom 24-well). The cell suspension (1 mL, 107cells/mL in PBS) was transferred to the wells and incubated at 37oC, 80 rpm for 1.5 h. Unattached fungal cells were washed away, and added with fresh YPD (1 mL) with doxycycline (50 µg/mL) and further incubated for 48 h. Composition of excreted alcohols in culture supernatant was evaluated by gas chromatograph-mass spectrometer equipped with an HP 19091B-005 50-m capillary column at a flow rate of 1 mL/min. Peaks were identified and quantified by comparing with the retention times and MS spectra of authenticated alcohol standards. Virulence was evaluated using oral reconstituted human epithelial (RHE) tissues. Sections of 1 µm-thick were cut and stained with hematoxylin and eosin, and viewed under light microscope. Released lactate dehydrogenase from the RHE tissues was determined using CytoTox 96 nonradioactive cytotoxicity assay.
Results: Four kinds of alcohols (isoamyl alcohol, phenethyl alcohol, nerolidol, and farnesol) were detected and authenticated. Level of farnesol was increased by ~23% (P < 0.05) in IFD6-overexpressed C. dubliniensis when compared with their control. There was no significant difference in alcohol levels in ADH5-overexpressed and CSH1-overexpressed C. dubliniensis. The virulence of all C. dubliniensis biofilms was similar to that of the reference strains.
Conclusions: IFD6 is a gene that is involved in biosynthesis of farnesol in C. dubliniensis biofilms. Overexpression of IFD6 in C. dubliniensis leads to increased level of farnesol without any effect on fungal virulence.