Farnesol Impact On Some Ergosterol Biosynthetic Genes In Giant Panda Source Trichosporon Asahii Induced Fluconazole-Resistant Strains

Objective:In recent years,with the antifungal drug use in giant pandas fungal disease,caused giant pandas faced with antifungal drug resistance.Therefore,to study the drug resistance mechanism of fungi isolated from giant pandas,it’s important for giant pandas protection.Investigate the effect of farnesol on reducing fluconazole resistance in fungi isolated from giant pandas.To lays a foundation for the study of new drug research of fungal diseases in giant pandas.Method: In this study,a strain of suspected Trichosporon asahii isolated from the vagina of giant panda(YAN0802),which proceed molecular biology and morphologic identification and built phylogenetic tree.The sub-dose fluconazole was used to induce YAN0802 into fluconazole resistance step by step,to get a strain(YAN0802-PB)which resistant to fluconazole.The 96-well microtiter plates was used to measure the MIC of antifungal drugs and farnesol for YAN0802 and YAN0802-PB,compared the colony phenotype and morphologic development.Compared the pathogenicity by fungal load quentity,PAS and HE tissue staining of KM mice organs after caudal vein.q RT-PCR was used to measure the ergosterol synthesis pathway related genes(Erg4、Erg5、Erg6、Erg7、Erg11、Erg25、Erg26)expression in YAN0802-PB;q RT-PCR was used to measure these genes expression in different concentration farnesol treated the fluconazole-resistance mutant strains.Rerults: YAN0802 was identified as T.asahii.We got a fluconazole-resistance strain YAN0802-PB by YAN0802 fluconazole induction in vitro,and the drug resistance spectrum and fungal form were stable after continuous passage for 10 times without fluconazole.The colony of YAN0802-PB swtich into yeast.Scanning electron microscope showed that the surface of YAN0802-PB was more rough than YAN0802,a thicker biofilm is formed.Therefore,YAN0802-PB is more difficult to be cleared by the host,and the pathogenicity of YAN0802-PB stronger than YAN0802.YAN0802 as the reference,ergosterol synthesis pathway genes(Erg4、Erg5、Erg6、Erg11、Erg25)of YAN0802-PB expression was significantly up-regulated except Erg7 and Erg26.Among them,the expression of Erg4 was upregulated 3.26 folds,the expression of Erg5 was upregulated15.45 folds,the expression of Erg6 was upregulated 3.99 folds,Erg11 upregulated 5.66 folds,and the expression of Erg25 was upregulated 4.79 folds(P<0.01).The farnesol MIC of YAN0802 and YAN0802-PB are 400μM and 800μM respectively,biofilms were significantly reduced and more than half of YAN0802-PB apoptosis induced by 400μM farnesol.The expression of Erg4、Erg5、Erg6、Erg7、Erg11、Erg25、Erg26 were significantly down-regulation for YAN0802-PB,when treated with 25μM-50μM farnesol,(P<0.001).Conclusion: This is the first report of T.asahii isolate from the vagina of a giant panda.Fluconazole resistance was associated with T.asahii morphological transformation.T.asahii fluconazole resistance not only weaken the ability of the host to clear it,but also weaken the virulence,up-regulated the Erg genes.T.asahii has a strong tolerance to farnesol,meanwhile,farnesol could down-regulated the Erg genes and reduce biofilm formation.Infered that farnesol could alleviate the fluconazole resistance in T.asahii,and farnesol is the potential antifungal agent.