| Project/Area Number |
10672081
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Mukogawa Women's University |
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Principal Investigator |
YOSHIDA Yuzo Mukogawa Women's University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (70085281)
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| Co-Investigator(Kenkyū-buntansha) |
AOYAMA Yuri Soka University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (00158718)
工藤 万起子 武庫川女子大学, 薬学部, 副手
山下 稚加 武庫川女子大学, 薬学部, 助手
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Azole antifungal agent / Cytochrome P450 / Drug resistance / Mutation / Sterol biosynthesis inhibitor / Azole resistant fungus / CYP51 / Candida albicans / 抗真菌剤 / 耐性獲得機構 / P450 / 大腸菌内発現 / フルコナゾール / 病原性真菌 / 薬剤耐性遺伝子 / ステロール代謝 / 異種発現 / クローニング |
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| Research Abstract |
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
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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
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