Possible Inhibitory Molecular Mechanism Of Farnesol On The C AMP-PKA Pathway Of Candida Albicans Biofilms

Objective: People are more and more concerned about antifungal resistance for it is slowing the cure rate,and farnesol weakened the sensitivity to antifungal of many fungi.This research investigated the effect of farnesol on sensitivity to fluconazole of Candida albicans(C. albicans) planktonic and biofilms. Through the construction of Candida albicans resistant strains illustratiing the molecular mechanism of farnesol on the c AMP-PKA pathway of Candida albicans SC5314 and fluconazole-resistance biofilms and their relevence to antifungal resistance.Methods:Experiment 1 : The research included four groups: farensol-treated planktonic group,farnesol-untreated planktonic group,farnesol-treated biofilms group and farnesol-untreated biofilms group. XTT reduction assay was used to test the optical density value and analyze the difference of antifungal suscepitibility of farnesol between the C.albicans in planktonic state and biofilms state in vitro. Experiment 2 : The experimental subject are:Candida albicans SC5314 and fluconazole-resistance strain constructed by the Candida albicans SC5314. The experimental groups were as follows: farnesol-treated C.albicans SC5314 biofilms, farnesol-untreated C.albicans SC5314 biofilms,farnesol-treated fluconazole-resistance biofilms and farnesol-untreated luconazole-resistance biofilms. Total RNA was isolated from the C.albicans biofilms,the Real-time Quantitative PCR was used to analyze the expression of possible targeting genes below to investgate the effect on farnesol on c AMP-PKA pathway gene: Ras1,Gpa2,Cap1,Sgt1,Cyr1,Pde1,Pde2,Pka1,Bcy1,Tpk2,Efg1. Total proteins were isolated from the C.albicans biofilms,Western blot was used to analyze RAS1,CYR1, PDE2 and BCY1 protein expression in c AMP-PKA pathway of different stages of biofilms treated by farnesol.Results:. Experiment 1:The XTT analysis suggested the antifungal suscepitibility of farnesol-untreated planktonic is 8~61 fold high to biofilms(P<0.001).And there is no significant difference on the antifungal suscepitibility of farnesol-treated planktonic compared to farnesol-untreated planktonic when exposed to fluconazole( P>0.05),but there is remarkable difference on farnesol-treated biofilms compared to farnesol-untreated biofilms when treated with flucoazole( P<0.05). Experiment 2:The Real-time Quantitative PCR showed that in farnesol-treated C.albicans SC5314 group to the control the expression of Ras1,Cap1,Sgt1,Cyr1,Pde1, Pka1,Bcy1,Tpk2,Efg1 was down-regulated, Pde2 was up-regulated and the rest had no significant changes at 6 h. The expression of Ras1 was down-regulated,Cap1,Cyr1,Pde1,Pde2,Pka1,Bcy1,Tpk2 was up-regulated and the rest had no significant changes at 12 h. The expression of Ras1,Cyr1 was down-regulated,Pka1,Pde1,Pde2 was up-regulated and the rest had no significant changes at 24 h. The expression of Ras1,Cyr1 was down-regulated,Pka1 was up-regulated and the rest had no significant changes at 36 h. The expression of genes which were directly regulated by farnesol was below: Ras1 expression was down-regulated at 6,12,24 and 36 h; Pde2 expression was up-regulated at 6 h,12 h and 24 h, and there was no significant changes at 36 h; Cyr1 expression was down-regulated at 6 h, 24 h and 36 h,and it was up-regulated at 12 h(P<0.01). In farnesol-treated C.albicans fluconazole-resistance group,the expression of Ras1, Pka1,Bcy1,Tpk2 was down-regulated,the expression of Cap1,Pde1, Pde2 was up-regulated and the rest had no significant changes at 6 h.The expression of Ras1,Sgt1 was down-regulated, Cyr1,Pde2,Pka1,Bcy1 was up-regulated and the rest had no significant changes at 12 h.The expression of Ras1,,Cap1 was down-regulated, Pde1,Pde2,Bcy1 was up-regulated and the rest had no significant changes at 24 h. The expression of Ras1,Cyr1,Pde2 was down-regulated, Cap1,Pka1,Bcy1,Tpk2,Efg1 was up-regulated and the rest had no significant changes at 36 h. The expression of genes which were directly regulated by farnesol was below: Ras1 expression was down-regulated at 6,12,24 and 36 h; Pde2 expression wasup-regulated at 6,12 and 24 h, and there was no significant changes at 36 h; Cyr1 expression was down-regulated at 36 h and it was up-regulated at 12 h,there was no significant changes at 6 and 12 h(P<0.01). In Candida albicans fluconazole-resistance group to C.albicans SC5314 group the expression of Pde1,Pde2 was down-regulated,Cyr1,Tpk2 was up-regulated and the rest had no significant changes at 6 h.The expression of Pde1,Pde2 was down-regulated, Cyr1, Tpk2 was up-regulated and the rest had no significant changes at 12 h.The expression of Cyr1,Tpk2,Efg1 was up-regulated and the rest had no significant changes at 24 h. The expression of Sgt1,Pde1,Pde2 was down-regulated, Bcy1 was up-regulated and the rest had no significant changes at 36 h. The expression of genes which were directly regulated by farnesol was below: Ras1 expression was not significantly changed at 6,12,24 and 36 h; Pde2 expression was up-regulated at 6 h,12 h and 36 h, and there was no significant changes at 24 h; Cyr1 expression was up-regulated at 6 h,12 h and 24 h,there was no significant changes at 36 h(P<0.01). Western blot results : Farnesol-treated C.albicans SC5314 group compared to the farnesol-untreated C.albicans SC5314 group: The expression of RAS1,CYR1,BCY1 protein level was decreased,PDE2 was increased at 6 h.The expression of RAS1 protein level was decreased,CYR1,PDE2 and BCY1 was increased at 12 h.The expression of RAS1,CYR1,PDE2 and BCY1 protein level was all decreased at 24 and 36 h. The expression of proteins which were directly regulated by farnesol was below:RAS1 protein level was decreased at 6 h,12 h,24 h and 36 h; PDE2 was increased at 6 h,12 h and decreased at 24 h,36 h; CYR1 was decreased at 6 h,24 h,36 h and increased at 12 h. Farnesol-treated fluconazole-resistance group compared to the farnesol-untreated fluconazole-resistance group: The expression of RAS1,CYR1,BCY1 protein level was decreased,PDE2 was increased at 6 h.The expression of RAS1 protein level was decreased,CYR1,PDE2 and BCY1 was increased at 12 h.The expression of RAS1,CYR1,PDE2 and BCY1 protein level was all decreased at 24 h and 36 h. The expression of proteins which were directly regulated by farnesol was below:RAS1protein level was decreased at 6 h,12 h,24 h and 36 h;PDE2 was increased at 6 h,12 h and decreased at 24 h,36 h;CYR1 was decreased at 6 h,24 h,36 h and increased at 12 h. Farnesol-untreated fluconazole-resistance group compared to the farnesol-untreated C.albicans SC5314 group. The expression of RAS1,CYR1,BCY1 protein level was not significantly changed,PDE2 was decreased at 6 h and 12 h.The expression of RAS1,BCY1,PDE2 protein level was not significantly changed,CYR1 was increased at 24 h and 36 h. The expression of proteins which were directly regulated by farnesol was below:RAS1 protein level was not significantly changed at 6 h,12 h,24 h and 36 h;PDE2 was increased at 6 h,12 h and was not significantly changed at 24 h,36 h;CYR1 was decreased at 24 h,36 h and was not significantly changed at 6 h and 12 h.Conclusions: Experiment 1:Planktonic state C.albicans is much more suscepious to farnesol against to candida albicans biofilms.The effect of farnesol on the antifungal suscepitibility of farnesol-treated planktonic and biofilms is different.The antifungal suscepitibility was not significantly changed of C.albicans planktonic treated with farnesol compared to their control group.The antifungal suscepitibility was remarkably enhanced of candida albicans biofilms treated with farnesol. Experiment 2:The regulation of farnesol to C.albicans biofilms is relevent to the different biofilms stages,the expression of genes and proteins regulated by farnesol is not totally the same at different biofilms stages.Farnesol may affects the C.albicans bofilm antifungal resistance through regulating partial genes and proteins in the c AMP-PKA pathway and its effect was bidirectional regulation。The effect can be positive and negative depend on the biofilms stage.And the diffence on gene and protein expression between C.albicans SC5314 and C.albicans fluconazole-resistance group may hint that the The emergence of antifungal resistant strains may be caused by the change of expression of molecules which resistant to antifungal.