The Regulation Mechanism Of Farnesol On CYR1 And PDE2 In Candida Albicans Biofilm And Its Drug Resistance Correlation

Objective:Candida albicans,commonly forming biofilms,is a common member of the microbiota in the human gastrointestinal tract,mouth,and genital tract.With the abuse of antifungals,antifungal resistance of C.albicans biofilms is becoming a growing health problem worldwide.Farnesol,the first quorum-sensing molecule discovered in a eukaryote,has a potential antifungal activity against C.albicans biofilms,while the mechanism of this activity is still unclear.Farnesol inhibited hyphae growth by regulating cyclic AMP(cAMP)/protein kinase A(PKA)signaling pathway in C.albicans.We hypothesizes that farnesol enhances the susceptibility of C.albicans biofilms to antifungals by regulating gene CYR1 or PDE2 in cAMP/PKA signaling pathway.This research aimed at the molecular mechanism of farnesol on the antifungal resistance via the regulation of CYR1 and PDE2 genes.Methods:Overexpressing strains(pCaEXP-CYR1-CAI4 and pCaEXP-PDE2-CAI4)were generated using plasmids(pCaEXP-CYR1 and pCaEXP-PDE2)by the lithium acetate method and detected by agarose gel electrophoresis and q-PCR.The experiments were performed in six groups:farnesol treated biofilms of CYR1 overexpressing strain,farnesol untreated biofilms of CYR1 overexpressing strain,farnesol treated biofilms of PDE2 overexpressing strain,farnesol untreated biofilms of PDE2 overexpressing strain,farnesol treated biofilms of wild strain,farnesol untreated biofilms of wild strain.XTT reduction assay and spot assay were used to test susceptibility of C.albicans in biofilm and planktonic forms,respectively.Moreover,the morphological changes were observed by confocal laser scanning electron microscopy.Additionally,the expression of gene CYR1 and PDE2 was examined by q-PCR,and the expression of proteins was analyzed by Western blot.Results:After overexpressing strains were constructed successfully,we demonstrated that the overexpressing strains(pCaEXP-CYR1-CAI4 and pCaEXP-PDE2-CAI4)in planktonic form were more resistant to antifungals(fluconazole,caspofungin,itraconazole and terbinafine)while biofilms of overexpressing strains were less hypersensitive to amphotericin B.Moreover,exogenous farnesol could both decrease the antifungal resistance of C.albicans biofilms.Besides,confocal laser scanning electron microscopy revealed that the overexpressing strains showed more hypha and farnesol repressed hypha formation in C.albicans biofilms.Additionally,in biofilms of CYR1 overexpressing strain,expression of gene CYR1 was downregulated at 6,12,24h and the expression of protein CYR1 was downregulated at 6,12,24h by farnesol.Meanwhile,in biofilms of PDE2 overexpressing strain,expression of gene PDE2 was upregulated at 12,24,36h and the expression of protein PDE2 was upregulated at 6,12,24,36h by farnesol.Conclusions:The CYR1 and PDE2 overexpressing strains were more resistant to amphotericin B in biofilms and showed less hypersensitivity to most antifungals in planktonic form.It revealed that gene CYRl and PDE2 played a positive role in the mechanism of resistance to antifungals in C.albicans biofilms and also behaved as positive regulators of drug resistance in planktonic form.Besides,the regulation of genes and proteins by farnesol provided further evidence that farnesol decreased the antifungal resistance of C.albicans biofilms via down-regulating the expression of gene CYR1 rather than up-regulating the expression of gene PDE2.