Study On The Regulatory Mechanism Of Farnesol On Candida Albicans Biofilm RAS1 And Its Drug Resistance Correlation

Objective:Farnesol enhanced the drug suscepitibility to fluconzole of Candida albicans,This research investgated the relationship between the drug resistance of Candida albicans biofilms and RAS1 gene of MAPK singal pathway regulated by farnesol,also the effects of RAS1 gene on the drug suscepitibility of Candida albicans biofilms regulated by farnesol.Methods:The possible drug resistance gene RAS1of MAPK singal pathway was screened by farnesol through q-PCR and Western blot.We put RAS1 gene under the control ofplasmid pCaEXP to construct recombinant plasmid pCaEXP-RAS1 which can overexpression RAS1 gene.The recombinant plasmid pCaEXP-RAS1 was introduced into Candida albicans CAI4 by lithium acetate to construct the RAS1-overexpression strain pCaEXP-RAS1-CAI4.The experiments were performed mainly in four groups:farnesol-treated pCaEXP-CAI4 biofilm,farnesol-untreated pCaEXP-CAI4 biofilm,farnesol-treated pCaEXP-RAS1-CAI4 biofilm,farnesol-untreated pCaEXP-RAS1-CAI4 biofilm.XTT reduction assay was used to test the antifungal suscepitibility between farnesol-untreated pCaEXP-RAS1-CAI4 biofilm and farnesol-untreated pCaEXP-CAI4 biofilm as well as the antifungal suscepitibility between farnesol-treated pCaEXP-RAS1-CAI4 biofilm and farnesol-treated pCaEXP-CAI4 biofilm.Moreover,the antifungal resistance gene RAS1 expressionwas analyzed by q-PCR,and the RAS1 protein expression was analyzed by Western blot.Result:The possible drug resistance gene RAS1 of Candida albicans biofilm was successfully screened by farnesol.RAS1-overexpression strain was exactly conducted and selected as confirmed by q-PCR.The XTT analysis suggested that compared to farnesol-untreated pCaEXP-CAI4 biofilm,farnesol decreased antifungal suscepitibility of farnesol-untreated pCaEXP-RAS1-CAI4 biofilm to fluconazole at 12h and 24h biofilms.Compared to farnesol-untreated pCaEXP-RAS1-CAI4 biofilm,farnesol increased drug suscepitibility of farnesol-treated pCaEXP-RAS1-CAI4 biofilm to fluconazole at 6 h,12h and 24h biofilms.Compared to farnesol-untreated pCaEXP-CAI4 biofilm,farnesol increased drug suscepitibility of farnesol-treated pCaEXP-CAI4 biofilm to fluconazole at 24h biofilm.The q-PCR showed that compared to the farnesol-untreated pCaEXP-RAS1-CAI4 biofilm,the expression of farnesol-treated pCaEXP-RAS1-CAI4 biofilm RAS1 gene was down-regulated at 12h,24 h and 36 h biofilms.Western blot results demonstrated that the expression of RAS1 protein level was decreased at 12 h,24 h and 36 h in farnesol-treated pCaEXP-RAS1-CAI4 biofilm when compared to the farnesol-untreated pCaEXP-RAS1-CAI4 biofilms.Conclusion:RAS1 is the drug resistance gene of Candida albicans biofilm.Farnesol increased drug suscepitibility of candida albicans biofilm to fluconazole by regulating the expression of RAS1gene and protein.And this conclusion was further confirmed by the decreased expression of RAS1gene and protein by farnesol.