| Background and ObjectiveTricrosporon asahii is the most common pathogen of the trichosporonosis.Recently,with the abuse of antifungals,the widespread use of immunosuppressive drugs and the increase number of AIDS patients,the incidence of infection of T.asahii is increased constantly.Azole drugs have been widely used in the treatment of mycosis,as well in the treatment of T.asahii infections due to their broad antifungal spectrum and overall safety profiles.However,in the last years,there were reports about high MIC,even resistance of Trichosporon spp.to azole drugs.The ERG11 gene encoding lanosterol14-alpha demethylase,which is the target enzyme of azoles,and was inhibited after combined with the azole drug's specific activity,and thereby prevent the synthesis of ergosterol,which is essential for fungal cell membrane.Previous studies have shown that mutation and overexpression of ERG11 gene may lead to resistance to azole drugs.And has been discovered in Candida albicans,Candida glabrata,Candida tropicalis,Cryptococcus neoformans and other fungi species.However there is no research reports about the role of ERG11 gene overexpression in the resistance of T.asahii to azoles.In this study,we used Real-time PCR to detect the expression of ERG11 gene in several couples of fluconazole-susceptible and-resistant isolates,and to explore the role of ERG11 gene in the resistance of T.asahii to azoles,and the relationship between gene expression and drug concentration.To lay the foundation for additional research of the T.asahii's resistance mechanism and exploring new drug targets.MethodsTested fluconazole-susceptible isolates were serially subcultured in YEPD medium containing 0 ~ 64 g/ml fluconazole to induce fluconazole-resistant strains(MIC?64?g/ml).Then,both the fluconazole-susceptible and-resistant isolates were exposed to drugs at the indicated concentrations at 35°C with shaking.ERG11 genes were tested by Real-time quantitative polymerase chain reaction detecting system(QPCR)for their expression in Trichosporon asahii cells at the m RNA level.Then compared both the expression of the gene in fluconazole sensitive and resistant strains,and the expression in different concentrations of fluconazole.The changes of ERG11 gene expression during the induction of fluconazole in vitro were tested and analyzed.Results6 isolates of fluconzole-resistant T.asahii were successfully induced.In fluconazole-free medium higher expression of ERG11 gene was observed in the fluconazole-resistant isolates(7.542±5.311)than that in the fluconazole-susceptible isolates(1.014±0.012),the difference was statistically significant(t=-3.002,P=0.03).In different concentration of fluconazole,the levels of ERG11 m RNA of fluconazole-resistant isolates(respectively as 9.183±3.226,13.657±5.428,15.292±7.007,13.720±8.550,13.949±2.960,13.123±6.429)also higher than the fluconazole-susceptible isolates(respectively as 3.281±2.068,3.459±1.923,3.242±2.530,3.651±0.728,3.969±1.924,3.824±1.875)(P<0.05).However,there's no significant correlation between the expression of ERG11 and the fluconazole concentrations.(fluconazole-resistant isolates: rs=0.229,P=0.096;fluconazole-susceptible isolates: rs=0.166,P=0.357).And during the induction,although the expression of ERG11 increased with its rised resistance to fluconazole,but there is no significant correlation between the expression level of ERG11 and its resistance to fluconazole except two isolates(BZP902:rs=0.865,p=0.003;BZP705:rs=0.847,p=0.002).ConclusionsThe overexpression of ERG11 gene is relevant to fluconazole resistance in Trichosporon asahii.However,wether the overexpression of ERG11 can lead to the T.asahii be resistant to fluconazole and it's mechanism needs to be further studied. |
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