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 14␣-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14␣-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.

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

“…Sample preparation for mass spectrometry (MS) analysis was performed as described previously (14,16,36). For quadrupole time of flight liquid chromatography-tandem mass spectrometry (Q-TOF LC-MS/MS) analysis of secreted proteins, alkylated proteins were digested for 18 h using 2 g of Trypsin Gold (Promega, Madison, WI) and subsequently desalted using a C 18 tip column (Varian, Palo Alto, CA) according to the supplier's instructions.…”

“…Secretome analysis was performed as described before (14,36). Cells were separated from the growth medium by centrif- ugation.…”

“…Reinforcement of the cell wall in response to antifungal stresses is well described, especially with respect to an increase in chitin content as a result of increased chitin synthesis (11,12). The cell wall proteome itself is highly dynamic (13) and adaptable in response to external conditions (14,15) as well as morphological changes (16).…”

“…Relative quantification revealed a set of wall-remodeling proteins that was induced during thermal stress. Intriguingly, the same set of proteins was shown previously to be involved in the response to other surface stressors (14,30), suggesting a general stress response. Therefore, we investigated the phosphorylation status of three stress-responsive mitogen-activated protein (MAP) kinases, Hog1, Cek1, and Mkc1.…”

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms

“…In this study, there was complementarity in the activities of xanthochymol and fluconazole. Fluconazole inhibits production of hyphae and hypha-specific wall proteins (18,45). Our data show similarity in that xanthochymol-inhibited hyphal development ( Fig.…”