Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole.
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.
Niimi
Masakazu
M
Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan. niimi@nih.go.jp
Nagai
Yuki
Y
Niimi
Kyoko
K
Wada
Shun ichi
Si
Cannon
Richard D
RD
Uehara
Yoshimasa
Y
Monk
Brian C
BC
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D002175:Candida / Q000737:chemistry / Q000187:drug effects*; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015203:Reproducibility of Results
D000935:Antifungal Agents; D005656:Fungal Proteins; D015725:Fluconazole