Niimi Masakazu, Nagai Yuki, Niimi Kyoko, Wada Shun ichi, Cannon Richard D, Uehara Yoshimasa, Monk Brian C
Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Dec 25;782(1-2):245-52. doi: 10.1016/s1570-0232(02)00668-2.
Candida glabrata is an increasingly important cause of opportunistic fungal infection of humans and appears to be intrinsically resistant to the triazole antifungal fluconazole. However, the mechanisms responsible for reduced susceptibility to azole drugs are not understood. Fluconazole exposure rapidly induced expression of a 169-kDa protein band in plasma membrane fractions of C. glabrata cells. Mass spectrometry of trypsin-digested peptide fragments showed that the induced protein band comprised the ATP binding cassette-type drug efflux transporter CgCdr1p. CgCdr1p was also functionally overexpressed in S. cerevisiae and similarly identified by mass spectrometry. A 61-kDa protein band in the plasma membrane fraction from C. glabrata was also induced by fluconazole exposure. Mass spectrometric peptide fingerprinting identified this band as lanosterol 14alpha-demethylase, the enzyme in the ergosterol biosynthesis pathway targeted by fluconazole. The rapid induction of a multidrug efflux pump and/or overproduction of lanosterol 14alpha-demethylase are mechanisms that could make C. glabrata appear intrinsically resistant to fluconazole. Mass spectrometric fingerprint analysis of SDS-PAGE separated plasma membrane fractions combined with heterologous hyper-expression provides a convenient method for protein identification and functional evaluation of induced proteins, even in an organism where the genome sequence database is incomplete.
光滑念珠菌是人类机会性真菌感染日益重要的病因，且似乎对三唑类抗真菌药氟康唑具有内在抗性。然而，其对唑类药物敏感性降低的机制尚不清楚。氟康唑暴露可迅速诱导光滑念珠菌细胞膜组分中一条169 kDa蛋白条带的表达。对胰蛋白酶消化后的肽段进行质谱分析表明，诱导产生的蛋白条带包含ATP结合盒式药物外排转运蛋白CgCdr1p。CgCdr1p在酿酒酵母中也有功能过表达，并通过质谱得到类似鉴定。氟康唑暴露也可诱导光滑念珠菌细胞膜组分中一条61 kDa蛋白条带的产生。质谱肽指纹图谱鉴定该条带为羊毛甾醇14α-去甲基酶，即氟康唑作用的麦角甾醇生物合成途径中的酶。多药外排泵的快速诱导和/或羊毛甾醇14α-去甲基酶的过量产生可能是使光滑念珠菌对氟康唑表现出内在抗性的机制。对SDS-PAGE分离的细胞膜组分进行质谱指纹分析并结合异源过表达，为蛋白质鉴定以及诱导蛋白的功能评估提供了一种便捷方法，即使是在基因组序列数据库不完整的生物体中。
