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

以一株耐唑白色念珠菌DUMC136为材料，研究了抗唑真菌的分子和遗传机制，重点研究了目标酶CYP51的改变。对DUMC136的CYP51基因和对唑敏感的白色念珠菌ATCC 90028的CYP51基因进行分析发现，DUMC136是CYP51基因突变的纯合子，而ATCC 90028的CYP51基因存在等位基因变异。这一事实表明，编码抗唑CYP51酶的变异CYP51纯合子的形成可能是DUMC136耐唑的原因。克隆并在大肠杆菌中表达了ATCC 90028的CYP51等位基因CYP51_<ATCC 90028-1>和CYP51_<ATCC 90028-2>和DUMC136的CYP51等位基因CYP51_<DUMC>。表达的CYP51s分别为CYP51_<ATCC 90028-1>、CYP51_<ATCC 90028-2>和CYP51_<DUMC>。CYP51_<ATCC 90028-1>和cy…More P51_<ATCC 90028-2>的酶学和分光光度特性无本质差异。CYP51_<DUMC>的催化活性和氟康唑敏感性显著低于CYP51_<ATCC 90028-1>和CYP51_<ATCC 90028-2>。同时发现，CYP51_<DUMC>的氧化型Soret波段的最大吸收和与氟康唑相互作用引起的光谱变化等光谱特征与野生型酶不同。这些结果清楚地表明，CYP51_<DUMC>和CYP51_<ATCC 90028-2>之间的两个氨基酸取代引起的结构变化降低了CYP51_<DUMC>的唑敏感性。最近对结核分枝杆菌CYP51的x射线晶体分析显示，包含上述两个氨基酸残基的螺旋C区是影响该酶血红素袋结构的关键区域。因此，CYP51_<DUMC>的抗唑性可能不是由于取代的氨基酸与唑类化合物相互作用所致，而是由于C螺旋区氨基酸的变化所致。少

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