Studies on the Molecular Mechanisms of the Formation of Azole Resistant Mutants of Pathogenic Fungi

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Molecular and genetic mechanisms emerging an azole-resistant fungi were studied focusing on the alteration of the target enzyme, CYP51, by using an azole resistant isolate of Candida albicans named DUMC136 as a material. Analysis of CYP51 genes of DUMC136 and an azole-sensitive C.albicans ATCC 90028 revealed that DUMC136 was a homozygote of an altered CYP51, whereas CYP51 of ATCC 90028 showed allelic variation. This fact suggests that formation of the homozygote of an altered CYP51, which encodes azole-resistant CYP51 enzyme, may be the reason for azole resistance of DUMC136. The CYP51 alleles of ATCC 90028 named CYP51_<ATCC 90028-1> and CYP51_<ATCC 90028-2> and the altered CYP51 of DUMC136 named CYP51_<DUMC> were cloned and expressed in E.coli. The expressed CYP51s, CYP51_<ATCC 90028-1>, CYP51_<ATCC 90028-2> and CYP51_<DUMC>, were purified and characterized. No essential difference was observed between the enzymatic and spectrophotometric characteristics of CYP51_<ATCC 90028-1> and CY … More P51_<ATCC 90028-2>. However, catalytic activity and fluconazole susceptibility of CYP51_<DUMC> were significantly lower than those of CYP51_<ATCC 90028-1> and CYP51_<ATCC 90028-2>. It was also found that spectrophotometric characteristics of CYP51_<DUMC> such as the absorption maximum of the Soret band of the oxidized form and the spectral change induced by the interaction with fluconazole were different from those of the wild-type enzyme. These findings clearly indicate that the structural change caused by the two amino acid substitution observed between CYP51_<DUMC> and CYP51_<ATCC 90028-2> reduced the azole susceptibility of CYP51_<DUMC>. Recent X-ray crystallographic analysis of CYP51 of Mycobacterium tuberculosis revealed that the helix C region that includes above-mentioned two amino acid residues is a critical region affecting the structure of heme pocket of this enzyme. Consequently, azole resistance of CYP51_<DUMC> may be due not to the interaction between the substituted amino acids with azole compound but to the amino acid change in the C helix region. Less