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...

Aspergillus fumigatus sterol 14-␣ demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (K s The sterol pathways of eukaryotes are highly conserved and are part of a larger biosynthetic pathway that includes the formation of dolichols, coenzyme Q, heme A, and isoprenylated proteins. In Saccharomyces cerevisiae, the first step exclusively involved in sterol synthesis is the formation of squalene, with the first sterol intermediate in the pathway being lanosterol, culminating in ergosterol some 15 enzymatic steps later. In fungi, these reactions are governed by individual enzymes, but closer examination of other fungal genome sequences has revealed that there is often duplication (29) and, in some instances, triplicate versions of the same gene (1). We are interested as to why fungi have kept multiple copies of these genes, the roles of the proteins in vivo, and their contribution to both sterol biosynthesis and fungal resistance.,14-␣ Demethylase (CYP51) is an ancestral activity of the cytochrome P450 superfamily, which is also the target of azole antifungals (18). The isolation of CYP51 was initially from Saccharomyces cerevisiae (17), and in the fungal pathogen Aspergillus fumigatus, cytochrome P450 was first observed in 1990 (5, 6). Evidence that alteration of CYP51 activity might contribute to azole resistance first emerged in 1997 (11). In this particular study, we have looked in detail at the biochemical properties of the two CYP51 forms in Aspergillus fumigatus (29) encoded by CYP51A (Afu4g06890) and CYP51B (Afu7g03740). A comparison of the deduced amino acid sequences show 63% identity between them; and both orthologues in A. fumigatus have been shown to act in a compensatory manner in the ergosterol pathway; i.e., neither is essential individually, but a double knockdown is lethal (13). It is postulated that CYP51A may encode the major sterol 14-␣ demethylase activity required for growth on the basis of accumulation of multiple missense mutations linked to azole resistance (31), with CYP51B either being functionally redundant or having an alternative function under particular growth conditions still to be defined. We expressed both proteins in Escherichia coli to investigate their azole binding properties. MATERIALS AND METHODSConstruction of pSPORT AF51A and pSPORT AF51B expression vectors. The coding regions of the A. fumigatus (strain Af293 [http://www.aspergillusgenome .org/gbrowse/afum_af293]) CYP51 isoenzyme A (AF51A) and B (AF51B) genes (ExPASy protein database accession numbers Q4WNT5 and Q96W81, respectively) were synthesized commercially by GeneCust Europe (Dudelange, Luxembourg) in pUC57 with codon optimization for expression in E. coli. NdeI and HindIII sites were included at the 5Ј and 3Ј ends, r...

Candida albicans is a major human pathogen whose treatment is challenging due to antifungal drug toxicity, drug resistance and paucity of antifungal agents available. Myrocin (MYR) inhibits sphingosine synthesis, a precursor of sphingolipids, an important cell membrane and signaling molecule component. MYR also has dual immune suppressive and antifungal properties, potentially modulating mammalian immunity and simultaneously reducing fungal infection risk. Wax moth (Galleria mellonella) larvae, alternatives to mice, were used to establish if MYR suppressed insect immunity and increased survival of C. albicans-infected insects. MYR effects were studied in vivo and in vitro, and compared alone and combined with those of approved antifungal drugs, fluconazole (FLC) and amphotericin B (AMPH). Insect immune defenses failed to inhibit C. albicans with high mortalities. In insects pretreated with the drug followed by C. albicans inoculation, MYR+C. albicans significantly increased mortality to 93% from 67% with C. albicans alone 48 h post-infection whilst AMPH+C. albicans and FLC+C. albicans only showed 26% and 0% mortalities, respectively. MYR combinations with other antifungal drugs in vivo also enhanced larval mortalities, contrasting the synergistic antifungal effect of the MYR+AMPH combination in vitro. MYR treatment influenced immunity and stress management gene expression during C. albicans pathogenesis, modulating transcripts putatively associated with signal transduction/regulation of cytokines, I-kappaB kinase/NF-kappaB cascade, G-protein coupled receptor and inflammation. In contrast, all stress management gene expression was down-regulated in FLC and AMPH pretreated C. albicans -infected insects. Results are discussed with their implications for clinical use of MYR to treat sphingolipid-associated disorders.

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min ؊1 and a k s of 26 M. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56⌬ mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

This study examines how the dynamics of fungus-insect interactions can be modulated by temperature. The wax moth, Galleria mellonella, is a well-studied and important model insect whose larvae in the wild develop optimally at around 34 °C in beehives. However, surprisingly little research on wax moths has been conducted at relevant temperatures. In this study, the entomopathogenic fungus Metarhizium robertsii inflicted rapid and substantial mortality on wax moth larvae maintained at a constant temperature of 24 °C, but at 34 °C a 10 fold higher dose was required to achieve an equivalent mortality. The cooler temperature favored fungal pathogenicity, with condial adhesion to the cuticle, germination and hemocoel invasion all significantly enhanced at 24 °C, compared with 34 °C. The wax moth larvae immune responses altered with the temperature, and with the infective dose of the fungus. Enzyme-based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. A dramatic upregulation in the basal expression of galiomicin and gallerimycin was triggered by cooling, and this was augmented in the presence of the fungus. Profiling of the predominant insect epicuticular fatty acids revealed a 4-7 fold increase in palmetic, oleic and linoleic acids in larvae maintained at 24 °C compared with those at 34 °C, but these failed to exert fungistatic effects on topically applied fungus. This study demonstrates the importance of choosing environmental conditions relevant to the habitat of the insect host when determining the dynamics and outcome of insect/fungus interactions, and has particular significance for the application of entomopathogens as biocontrol agents.