Studies On The CYP51 Activity In Azole-resistant Candida Albicans

The cytochrome P450 14ot-lanosterol demethylase (CYP51) of fungi is involved in an important step in the biosynthesis of ergosterol which is essential for fungi to build their plasma membrane and is the target enzyme of azole drugs (fluconazole, ketoconazole and itraconazole, etc.), but it is prone to alterations leading to resistance to these agents. Azole resistance in the pathogenic yeast Candida albicans is an emerging problem in the HIV-infected population.We have been interested in understanding the molecular mechanisms of azole resistance in Candida albicans , so it is important to study the structure and function of CYPSl.Two strategies were adopted to research for it: Site-directed mutagenesis was performed to estimate effect of each of those mutations on resistance to azole derivatives.T315A, R467K, G464S mutant CYP51 have been researched by this mothod. (2) To find the mutagenesis from resistant clinical isolates or from induced-resistant strains in vitro.In this study, several Candida albicans isolates (include azole-resistant and susceptible strains) were tested. We obtained fluconazole-resistant strains(MIC 128ug/ml) induced by serial subcultures in YEPD containing different concentration of fluconazole. Sterol isolation and identification were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the cause for the drug tolerance. When grown in the absence of fluconazole, two sensitive strains and one resistant strain were found to contain ergosterol as their predominant sterol.In contrast, the ergosterol levels in two resistant isolates were comparatively low. When exposure to fluconazole, the decrease in the ergosterol level and the rise in the trimethyl(24-methylenelanost-8-en-3-ol) level were dosage-dependence, but there were many differences between azole-resistant and susceptible strains. It suggested the CYP51 of resistant isolates was less sensitive to fluconazole.The inhibitory effect of fluconazole on ergosterol biosynthesis in whole-cell fungi was measured by the incorporation of [l-14C]acetate into nonsaponifiable5lipids(NSLs). Compared to sensitive isolates, the half inhibitory concentration(IC5o)of fluconazole for ergosterol rise obviously in resistant isolates. IC5o to 010213A was 313.3 ng/ml. While the effect in cell-free extracts was measured by the incorporation of [2-'4C]mevalonate into NSLs. Compared to azole-susceptible strains, ICjo to CYP51 rised significant in azole-resistant strains. y01.09A from 28.5 ng/ml to 40.0 ng/ml, but others rised more significant .There were differences between azole- resistant strains. It suggested that the CYP51 of y01 .09A distinct from other azole-resistant strains.The aim of this topic is to establish a reliable method for the research of CYP51 on biosynthesis of ergosterol, settle foundation for the research of mutagenesis in resistant fungi and the structure and function of CYP51. In successive experiments, we will sequence and compare the CYP51 genes of the resistant isolates to inspect the molecular mechanisms of azole resistance in Candida albicans.