Preliminary Research On MIC Of Fluconazole Against Candida Albicans ADH1 Gene Knockout Strain And The Correlation With Efflux Pump

Purposes:The purposes of our study were to explore whether the minimum inhibitory concentration(MIC) of fluconazole(FLC) against Candida albicans strains changed after ADH1 genes were knocked out, and if so, whether it was correlated with the drug efflux pump.Methods:We chose C. albicans strain SC5314, and ADH1 gene deletion strain EX2 and its revertant strain RE1(both were former built by other researcher of our group,SC5314 as the parental strain) as our study subject. Then, we examined the MICs of FLC against these three strains treated with FLC by microdilution method according to the Clinical and Laboratory Standards Institute(CLSI)M27-A3 guidelines. At the same time, we tested their sensitivity towards FLC by spot assay method, to examine whether there were any differences of FLC-sensitivity between the three strains. Next, the changes of efflux pump function were measured through rhodamine123(Rh123) accumulation/efflux experiments and m RNA expression levels of the drug efflux pump genes CDR1, CDR2 and MDR1 were measured through RT-PCR method in all these three strains treated with FLC, in order to explore the effects of ADH1 gene to the drug efflux pump function and drug efflux pump genes expression. To explore whether energy metabolism level of the strain also changed after ADH1 gene deletion, we measured the mitochondrial membrane potential and intracellular ATPlevelsResults:1. The results of microdilution method showed that, the MICs of FLC against ADH1 gene deletion strain EX2 got lower than the parental strain SC5314 and the revertant strain RE1, namely the MIC50 and MIC80 decreased by two degrees after cultured for 72 hours and decreased by one degree after cultured for 48 hours compared with the parental strain SC5314 and revertant strain RE1, while the latter two strains shared no differences. The spot assay method also showed that the ADH1 gene strain EX2 was more sensitive than the other two strains, while these two strains shared no differences.2. After accumulation experiment, the percentage of Rh123 positive cells in ADH1 gene deletion strain EX2 was 2.09-fold higher than the parental strain SC5314 and 1.67-fold higher than the revertant strain RE1(both P<0.05),the ADH1 gene deletion strain EX2 got increased accumulation of Rh123. After efflux experiment,the percentage of Rh123 positive cells in ADH1 gene deletion EX2 strain were2.27-fold higher than the parental strain SC5314 and 2.13-fold higher than therevertant strain RE1(both P<0.05), the ADH1 gene deletion strain EX2 got decreased efflux of Rh123. While the parental strain SC5314 and revertant strain RE1 shared no differences in the accumulation and efflux of Rh123(P>0.05).3. The results of RT-PCR showed that the expression of MDR1 in ADH1 gene deletion strain EX2 was respectively 0.32-fold and 0.29-fold compared to the parental strain SC5314 and revertant strain RE1(both P<0.05), the expression of CDR2 in ADH1 gene deletion strain EX2 was respectively 3.10-fold and 2.12-fold compared to the parental strain SC5314 and revertant strain RE1(both P<0.05), and the expression of CDR1 in ADH1 gene deletion strain EX2 was respectively1.42-fold and 1.18-fold compared to the parental strain SC5314 and revertant strain RE1(both P>0.05). An up-regulation levels of drug efflux pump genes CDR2 and down-regulation of MDR1 were discovered in ADH1 gene deletion strain EX2, while the expressions of all these three genes in the other two strains shared no differences(P>0.05).4. The ratios of mitochondrial membrane potential levels of ADH1 gene deletion strain EX2, the parental strain SC5314 and revertant strain RE1 were 7.93, 12.84, and 16.38.The ADH1 gene deletion strain EX2 got lower mitochondrial membrane potential than the other two strains(P<0.05), while the mitochondrial membrane potential between these two strains shared no differences(P>0.05).5. The intracellular ATP levels of ADH1 gene deletion strain EX2, the parental strain SC5314 and revertant strain RE1 were 374.51 n M, 568.82 n M and 517.94 n M. The ADH1 gene deletion strain EX2 got lower intracellular ATP level than the other two strains.While these two strains shared no differences(P>0.05).Conclusions:ADH1 may be a gene related with FLC- resistance in C.albicans, and perhaps involves a mechanism by affecting the intracellular ATP levels and mitochondrial membrane potential, then further up-regulating the expression of efflux pump gene MDR1 and thus affecting efflux pump function.