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...

A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 g ml ؊1 , respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 g ml ؊1 for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14␣-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 g ml ؊1 ). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.Several mechanisms can contribute to azole resistance in pathogenic fungi, such as Candida albicans (4,11,14,32,33). There has been an increase in research surrounding the potential importance of drug efflux transporters (26, 30) and changes in sterol 14␣-demethylase (ERG11 [CYP51]), the target of azole antifungals (10,12,13,14,25). Biofilm formation (2) and the possibility for azole sequestration mechanisms (15) have also attracted attention. Following the identification of defective sterol ⌬ 5,6 -desaturase (encoded by ERG3) as a mechanism of azole resistance in Saccharomyces cerevisiae some 20 years ago (31), this mechanism has also been reported in clinical C. albicans isolates (3,8,9,19,21). There remains sustained interest in the regulation of the ERG genes and proteins that mediate fungal sterol (specifically ergosterol) biosynthesis ( Fig. 1) in azole-resistant isolates. Many antifungal compounds that are currently available to clinicians target either ergosterol (e.g., polyene antifungals) or the enzymes central to its synthesis (e.g., azole inhibitors of sterol 14␣-demethylase), and hence, the potential for the emergence of cross-resistant strains exists.Data suggest that azole inhibitors of sterol 14␣-demethylase (here called Erg11p) also bind to C22 desaturase (here called Erg5p) (6); however, the possibility that the latter could constitute a target for new antifungal compounds (6, 7) remains understudied. Here, we present information on the phenotypic and genotypic characteristics of a novel azole-resistant strain (CA108) that was initially identified as an erg5 mutant completely lacking ergosterol, using gas chromatography and mass spectrometry (GC/MS) (24). Given the importance of ergosterol for maintaining fungal cell membrane integrity (1), we were keen to investigate how CA108 was able to...

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...

bWe identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 g ml ؊1 , respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14␣-demethylase (Erg11p) mutant, wherein 14␣-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14␣-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium ( glc YM). However, when grown on sterol-supplemented glc YM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, ⌬ 7 -cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented glc YM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using glc YM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using glc YM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 g AMB ml ؊1 , respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.

The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC50s], 0.23 μM and 0.22 μM, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to >8 μg/ml, while those of voriconazole ranged from 0.064 to 4 μg/ml. Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and -resistant A. fumigatus growth yielded IC50 (determined based on optical density [OD]) values of 0.0012 to 0.034 μg/ml, whereas voriconazole (0.019 to >1 μg/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 μg/ml), including Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 μg/mouse, while posaconazole showed similar effects (44%) at 14 μg/mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung.