No abstract

Plant fungal pathogens can have devastating effects on a wide range of crops, including cereals and fruit (such as wheat and grapes), causing losses in crop yield, which are costly to the agricultural economy and threaten food security. Azole antifungals are the treatment of choice; however, resistance has arisen against these compounds, which could lead to devastating consequences. Therefore, it is important to understand how these fungicides are used and how the resistance arises in order to tackle the problem fully. Here, we give an overview of the problem and discuss the mechanisms that mediate azole resistance in agriculture (point mutations in the CYP51 amino acid sequence, overexpression of the CYP51 enzyme and overexpression of genes encoding efflux pump proteins). © 2015 Society of Chemical Industry.

Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol ⌬ 5,6 -desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, >256, 16, 16, 8, and 1 g ml ؊1 , respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 g ml ؊1 ); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 g ml ؊1 ). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14␣-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.

Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated ⌬60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K s , 14 to 18 M) and catalyzed the 14␣-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K d s], 42 to 131 nM) but bound fluconazole (K d , ϳ30,500 nM) and voriconazole (K d , ϳ2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K d s, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over ⌬60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and ⌬60HsCYP51, producing atypical type I UV-visible difference spectra (K d s, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and ⌬60HsCYP51 (K d , ϳ40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC 50 ) values, which were 9-to 2,000-fold higher for ⌬60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC 50 , ϳ150 M) and did not significantly inhibit ⌬60HsCYP51. Sterol 14␣-demethylase (CYP51) is an ancestral activity of the cytochrome P450 superfamily and is required for ergosterol biosynthesis in fungi and cholesterol biosynthesis in mammals (1). Fungal CYP51 (Erg11) is the main target for therapeutic azole antifungal drugs and agricultural azole fungicides. This has led to the development of azole inhibitors that are selective for the fungal CYP51 enzyme over the human homolog and are commonly used to treat fungal infections, including those caused by Candida albicans and Aspergillus fumigatus (2-4). Agricultural azoles, however, were developed primarily for selectivity against the fungal CYP51 over the plant homolog. The mode of action of azole antifungals involves the nucleophilic nitrogen of the azole heterocyclic ring directly coordinating as the sixth ligand of the heme ferric ion and the azole drug side chains interacting with the CYP51 polypeptide structure (5).Many yeasts and fungi that are causative agents of clinical infections, such as Candida species and Aspergillus species, are also present in the general environment and are exposed to the selective pressure of agricultural azoles in the field. This has led to concerns that azole-resistant strains of yeasts and fungi responsible for clinical infections are emerging due to the use of agricultural azole fungicides on crops raising azole tole...

Purified Candida albicans sterol 14-␣ demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K s values of 11 and 25 M, respectively, and a K m value of 6 M for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (⌬A max ) and the azole concentration required to obtain a half-⌬A max being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K d values of 10 to 26 M under oxidative conditions, compared with 47 M for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4-and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min ؊1 ), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.Fungal sterol 14-␣ demethylase (CYP51) is required for ergosterol biosynthesis, an ancestral activity in the cytochrome P450 (CYP) superfamily of hemoproteins, and is the main target for azole antifungal drugs (15). CYP51 has been shown to be essential for viability in Saccharomyces cerevisiae (14). Azole inhibitors that are selective for the fungal enzyme over the human homologue have been developed and are commonly used to treat fungal infections, including those caused by Candida albicans (23,39). The mode of action of azole antifungal drugs involves the selective inhibition of the fungal CYP51, involving the nucleophilic nitrogen of the azole heterocyclic ring coordinating as the sixth ligand of the heme iron in the ferric state and the azole drug side chains interacting with the polypeptide structure (12, 57). However, due to prolonged and prophylactic use of azole drugs in the clinic, the emergence of azole-resistant C. albicans strains and other Candida species has become an increasing problem, especially among hospitalized immunocompromised patients, such as HIV and AIDS, cancer, and transplant patients, leading to a growing need to develop new effective antifungal strategies against drug-resistant stra...