title: Effects of fluconazole on the secretome, the wall proteome, and wall integrity of the clinical fungus Candida albicans.
abstract: Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of (14)N-labeled query walls relative to a reference standard mixture of (15)N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.
authors.affiliation: Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, Science Park 904, 1098 XH Amsterdam, Netherlands.
authors.forename: Alice G
authors.initials: AG
authors.lastname: Sorgo
authors.forename: Clemens J
authors.initials: CJ
authors.lastname: Heilmann
authors.forename: Henk L
authors.initials: HL
authors.lastname: Dekker
authors.forename: Martijn
authors.initials: M
authors.lastname: Bekker
authors.forename: Stanley
authors.initials: S
authors.lastname: Brul
authors.forename: Chris G
authors.initials: CG
authors.lastname: de Koster
authors.forename: Leo J
authors.initials: LJ
authors.lastname: de Koning
authors.forename: Frans M
authors.initials: FM
authors.lastname: Klis
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism*; D015725:Fluconazole / Q000494:pharmacology*; D005583:Fourier Analysis; D005656:Fungal Proteins / Q000378:metabolism*; D025301:Hyphae / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D010446:Peptide Fragments; D015725:Fluconazole