Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma − phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

Using a multivariate model, the presence of polymorphic variants in DNA-repair genes are powerful prognosis factors and response to cisplatin predictors among SCCHN patients.

The identification of FKS1 mutations in Candida albicans associated with echinocandin resistance has raised concerns over the spread of drug-resistant strains. We studied the impact of fks1 mutations on C. albicans virulence and fitness. Compared with wild-type strains for FKS1, echinocandin-resistant C. albicans strains with homozygous fks1 hot-spot mutations had reduced maximum catalytic capacity of their glucan synthase complexes and thicker cell walls attributable to increased cell wall chitin content. The fks1 mutants with the highest chitin contents had reduced growth rates and impaired filamentation capacities. Fks1 mutants were hypovirulent in fly and mouse models of candidiasis, and this phenotype correlated with the cell wall chitin content. In addition, we observed reduced fitness of echinocandin-resistant C. albicans in competitive mixed infection models. We conclude that fks1 mutations that confer echinocandin resistance come at fitness and virulence costs, which may limit their epidemiological and clinical impact.

The antiarrhythmic drug amiodarone has been found to have fungicidal activity. In Saccharomyces cerevisiae, its antifungal activity is mediated by calcium overload stress, which leads to a rapid nuclear accumulation of the calcineurin-regulated transcription factor CRZ1. In addition, low doses of amiodarone have been reported to be synergistic with fluconazole in fluconazole-resistant Candida albicans. To establish its mechanism of toxicity in C. albicans, we used expression profiling of key pathway genes to examine cellular responses to amiodarone alone and in combination with fluconazole. Gene expression profiling of 59 genes was done in five C. albicans strains (three fluconazole-susceptible strains and two fluconazole-resistant strains) after amiodarone and/or fluconazole exposure. Of the 59 genes, 27 analyzed showed a significant change (>2-fold) in expression levels after amiodarone exposure. The up-or downregulated genes included genes involved in Ca 2؉ homeostasis, cell wall synthesis, vacuolar/lysosomal transport, diverse pathway regulation, stress response, and pseudohyphal morphogenesis. As expected, fluconazole induces an increase in ergosterol pathway genes expression levels. The combination treatment significantly dampened the transcriptional response to either drug, suggesting that synergism was due to an inhibition of compensatory response pathways. This dampening resulted in a decrease in total ergosterol levels and decreased pseudohyphal formation, a finding consistent with decreased virulence in a murine candidiasis model.Candida albicans is the most frequently observed opportunistic human fungal pathogen causing mucosal and systemic infections in individuals with compromised immune defenses (44). Antifungal therapy is limited by the paucity of chemical classes, toxicity, drug resistance, moderate response rates, and substantial interpatient variation in serum drug levels. Thus, candidiasis remains a challenging opportunistic infection with high mortality, despite current available treatment. There is a pressing need for alternative treatments with new drug classes representing novel drug targets. One promising new antifungal class is represented by amiodarone (AMD), a drug now in clinical use as an antiarrhythmic. AMD has shown fungicidal activity against yeasts and a range of clinically important fungi, including C. albicans, Cryptococcus neoformans, Fusarium oxysporum, and Aspergillus nidulans (9, 53). In addition, low doses of AMD have been reported to be synergistic with different azoles in itraconazole-resistant A. fumigatus strains (1) and also in the protozoans Trypanosoma cruzi (4) and Leishmania mexicana (49).In Saccharomyces cerevisiae, it is known that AMD affects calcium homeostasis (10), leading to an immediate influx of Ca 2ϩ and a rapid activation of the calcineurin pathway, including nuclear accumulation of the calcineurin-regulated Crz1p. Transcriptional profiling also revealed an apparent disruption of nutrient sensing/signaling within minutes based on the upregulation of ge...

bAspergillus fumigatus intrinsic fluconazole resistance has been demonstrated to be linked to the CYP51A gene, although the precise molecular mechanism has not been elucidated yet. Comparisons between A. fumigatus Cyp51Ap and Candida albicans Erg11p sequences showed differences in amino acid residues already associated with fluconazole resistance in C. albicans. The aim of this study was to analyze the role of the natural polymorphism I301 in Aspergillus fumigatus Cyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue in A. fumigatus Cyp51Ap was replaced with a threonine (analogue to T315 at Candida albicans fluconazole-susceptible Erg11p) by changing one single nucleotide in the CYP51A gene. Also, a CYP51A knockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 g/ml) than the parental strain (MIC, 640 g/ ml), while no changes in MIC were observed for other azole-and non-azole-based drugs. These data strongly implicate the A. fumigatus Cyp51Ap I301 residue in the intrinsic resistance to fluconazole. Aspergillus fumigatus is the most common hyphomycete to cause disease in humans (1-3). It is intrinsically resistant to ketoconazole and fluconazole but normally susceptible to the other available azole antifungal agents (itraconazole, posaconazole, voriconazole, and isavuconazole) (4-8). The molecular mechanism for fluconazole intrinsic resistance has not been described yet. However, a hypothetical molecular mechanism has been proposed by Edlind et al., who linked A. fumigatus fluconazole intrinsic resistance with a naturally occurring amino acid substitution in Cyp51Ap (14-␣ sterol demethylase A) (9). These authors carried out an in silico comparison of the Candida albicans Erg11p and A. fumigatus Cyp51Ap sequences and found that among the residues most commonly implicated in fluconazole resistance in C. albicans (Y132, T315, S405, G464, and R467) (10, 11), only the T315 residue is not conserved in A. fumigatus Cyp51Ap and is naturally replaced by a nonpolar isoleucine (I301). In C. albicans, the replacement of the polar T315 residue by the nonpolar alanine (T315A) is enough to confer fluconazole resistance on the yeast (10).The aim of this study was to molecularly confirm that the natural polymorphism I301 in the Cyp51Ap is necessary and sufficient to explain the intrinsic reduced fluconazole susceptibility of A. fumigatus. An A. fumigatus mutant harboring the I301T substitution was generated, and susceptibilities to fluconazole and other antifungals were tested. Also, a CYP51A-defective mutant was obtained using the same parental strain in order to compare their antifungal susceptibility patterns. MATERIALS AND METHODS Strains.Aspergillus fumigatus akuB KU80 (12) was considered the wild-type strain, and its DNA was used as the template for all PCRs. It was the recipient strain for electroporation assays. Escherichia coli TOP10 (Promega) wa...