| Objective: C.albicans is a human opportunistic pathogen in the human oral cavity,upper respiratory tract,intestinal and vaginal mucosa tissue.The disease can be caused by the decrease of host immunity or the imbalance of normal flora.With the abuse of antifungals,antifungal resistance of C.albicans biofilms is becoming a growing health problem worldwide.Hyphae and biofilms formation of C.albicans were associated with the increased resistance of biofilms.RAS1 and CEK1 genes were involved in hyphae formation of C.albicans and could be involved in the resistance of C.albicans biofilms to antifungals.Farnesol had the effect of reducing the resistance of C.albicans to antifungals.The study showed that farnesol regulated hyphae formation through RAS1 and CEK1 genes.This study hypothesizes that RAS1 and CEK1 genes were associated with the resistance of C.albicans to antifungals.Farnesol reduced the resistance of C.albicans biofilms via RAS1 and CEK1 genes.Methods: To construct RAS1 and CEK1 gene knockout strains in C.albicans by using HIS-LEU-ARG knocking-out strategy with fusion PCR.The antifungal susceptibilities of the planktonic and biofilms to antifungals were compared between the RAS1 and CEK1 gene knockout strains and wild strain using spot assay and XTT reduction assay,respectively.Furthermore,the effect of farnesol on antifungal susceptibilities of the biofilms to antifungals were compared between the RAS1 and CEK1 gene knockout strains and wild strain using XTT reduction assay.Additionally,the morphological changes were compared between the RAS1 and CEK1 gene knockout strains and wild strain were observed by laser scanning confocal microscopy（LSCM）and scanning electron microscopy（SEM）.Results: The identification of agarose gel electrophoresis and nutritional type showed that the RAS1 and CEK1 knockout strains of C.albicans were successfully constructed.RAS1 knockout strains decreased the resistance to fluconazole,amphotericin B,caspofungin,itraconazole,terbinafine,nystatin and 5-fluocytosine in planktonic mode,as compared to wild strains.Besides,CEK1 knockout strains decreased the resistance to fluconazole,caspofungin,itraconazole in planktonic mode,as compared to wild strains.RAS1 knockout strains decreased the resistance to fluconazole,caspofungin,itraconazole,terbinafine,nystatin and 5-fluocytosine in biofilm mode,as compared to wild strains.However,RAS1 knockout strains increased the resistance to amphotericin B in biofilm mode,as compared to wild strains.Additionally,CEK1 knockout strains decreased the resistance to fluconazole,caspofungin,terbinafine,nystatin itraconazole,amphotericin B and 5-fluocytosine in biofilm mode,as compared to wild strains.The reduce effect of farnesol on the resistance of RAS1 and CEK1 knockout strains to fluconazole,itraconazole,caspofungin and 5-fluorcytosine was lower than that of farnesol on the resistance of wild strains to resistance of antifungals.LSCM and SEM showed that RAS1 and CEK1 genes were involved in hyphae formation of C.albicans.The hyphae formation of RAS1 knockout strain was significantly less than that of CEK1 knockout strain.The inhibition of farnesol on the formation of hyphae of RAS1 and CEK1 knockout strains was weaker than that of farnesol on wild strains.Conclusions: RAS1 and CEK1 genes were not only associated with antifungals resistance of planktonic,but also associated with biofilm mode.The regulation of farnesol on RAS1 and CEK1 genes were related to the antifungals resistance of C.albicans biofilms.The RAS1 and CEK1 genes were related to the hyphae formation of biofilm.Farnesol inhibited the hyphae and biofilm formation of C.albicans via regulating the expression of resistance genes RAS1 and CEK1. |
