IADR Abstract Archives
Overexpression of Cd36_03760 in Candida dubliniensis Abrogates Development of Biofilms
Objectives: To investigate the in vitro effects of overexpression of Cd36_03760 on the development of biofilms in Candida dubliniensis
Methods: C. dubliniensis strains that overexpress Cd36_03760 were constructed using a PCR-based gene targeting method. Overexpression was verified by real time quantitative PCR (RT-qPCR) using gene-specific primers upon addition of doxycycline. C. dubliniensis biofilms were prepared in polystyrene, flat bottom 96-well microtiter plates. The cell suspension (1 mL, 107cells/mL in PBS) was transferred to the wells and incubated at 37oC, 75 rpm for 90 min. After the removal of loosely adherent cells, fresh YPD (1 mL) with doxycycline (50 µg/mL) was added to the wells and incubated for 48 h for biofilm formation. The amount of protein in the extracellular matrix of the C. dubliniensis biofilms was determined using a BCA protein assay. The development of biofilms was qualitatively assessed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).
Results: BCA protein assay indicated that the amount of protein in C. dubliniensis biofilms that overexpressed Cd36_03760 was reduced by ~35% (P < 0.05) compared to its control. The SEM and CLSM results confirmed that overexpression of Cd36_03760 in C. dubliniensis abrogated the biofilm architecture: the biofilms were scanty, less dense, and less organised compared to its control which was relatively confluent.
Conclusions: Cd36_03760 is a gene that is involved in biofilm development in C. dubliniensis. Overexpression of Cd36_03760 in C. dubliniensis disintegrates the biofilms. It sheds light on further development of novel antifungal interventions against this emerging human fungal pathogen by selectively inhibiting the activity of enzymes that are involved in biofilm development.
Methods: C. dubliniensis strains that overexpress Cd36_03760 were constructed using a PCR-based gene targeting method. Overexpression was verified by real time quantitative PCR (RT-qPCR) using gene-specific primers upon addition of doxycycline. C. dubliniensis biofilms were prepared in polystyrene, flat bottom 96-well microtiter plates. The cell suspension (1 mL, 107cells/mL in PBS) was transferred to the wells and incubated at 37oC, 75 rpm for 90 min. After the removal of loosely adherent cells, fresh YPD (1 mL) with doxycycline (50 µg/mL) was added to the wells and incubated for 48 h for biofilm formation. The amount of protein in the extracellular matrix of the C. dubliniensis biofilms was determined using a BCA protein assay. The development of biofilms was qualitatively assessed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).
Results: BCA protein assay indicated that the amount of protein in C. dubliniensis biofilms that overexpressed Cd36_03760 was reduced by ~35% (P < 0.05) compared to its control. The SEM and CLSM results confirmed that overexpression of Cd36_03760 in C. dubliniensis abrogated the biofilm architecture: the biofilms were scanty, less dense, and less organised compared to its control which was relatively confluent.
Conclusions: Cd36_03760 is a gene that is involved in biofilm development in C. dubliniensis. Overexpression of Cd36_03760 in C. dubliniensis disintegrates the biofilms. It sheds light on further development of novel antifungal interventions against this emerging human fungal pathogen by selectively inhibiting the activity of enzymes that are involved in biofilm development.