A Sterol-Regulatory Element Binding Protein Is Required for Cell Polarity, Hypoxia Adaptation, Azole Drug Resistance, and Virulence in Aspergillus fumigatus

At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds

Characterization of a cytochrome P450 deficient mutant of Candida albicans

A previously described Candida albicans nystatin resistant mutant blocked in 14 alpha-demethylation of lanosterol was shown to also lack all traces of cytochrome P450 as determined by carbon monoxide difference spectra. This strain does not require ergosterol for growth and reverted to an ergosterol producing, cytochrome P450 containing strain indicating no other lesions. Cytochrome P450 mutants described in Saccharomyces cerevisiae are auxotrophic for ergosterol or contain a second mutation in 5,6 desaturation of the sterol B ring. These results suggest that a cytochrome P450 lesion in these yeasts have different phenotypes and may reflect different sterol requirements for the two organisms

Candida albicans Sterol C-14 Reductase, Encoded by the ERG24 Gene, as a Potential Antifungal Target Site

The incidence of fungal infections has increased dramatically, which has necessitated additional and prolonged use of the available antifungal agents. Increased resistance to the commonly used antifungal agents, primarily the azoles, has been reported, thus necessitating the discovery and development of compounds that would be effective against the major human fungal pathogens. The sterol biosynthetic pathway has proved to be a fertile area for antifungal development, and steps which might provide good targets for novel antifungal development remain. The sterol C-14 reductase, encoded by the ERG24 gene, could be an effective target for drug development since the morpholine antifungals, inhibitors of Erg24p, have been successful in agricultural applications. The ERG24 gene of Candida albicans has been isolated by complementation of a Saccharomyces cerevisiae erg24 mutant. Both copies of the C. albicans ERG24 gene have been disrupted by using short homologous regions of the ERG24 gene flanking a selectable marker. Unlike S. cerevisiae, the C. albicans ERG24 gene was not required for growth, but erg24 mutants showed several altered phenotypes. They were demonstrated to be slowly growing, with doubling times at least twice that of the wild type. They were also shown to be significantly more sensitive to an allylamine antifungal and to selected cellular inhibitors including cycloheximide, cerulenin, fluphenazine, and brefeldin A. The erg24 mutants were also slightly resistant to the azoles. Most importantly, erg24 mutants were shown to be significantly less pathogenic in a mouse model system and failed to produce germ tubes upon incubation in human serum. On the basis of these characteristics, inhibitors of Erg24p would be effective against C. albicans

Disruption of the Candida albicans CYB5 Gene Results in Increased Azole Sensitivity

Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme. Cytochrome b(5) is required for sterol C5-6 desaturation and the encoding gene, CYB5, is nonessential in Saccharomyces cerevisiae. Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable. The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals. Based on this phenotype, the CYB5 gene in the human pathogen Candida albicans was investigated to determine whether the cyb5 genotype was viable and would also demonstrate azole sensitivity. Sequential disruption of the CYB5 alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the CYB5 gene. Subsequent disruption procedures with a pMAL2-CYB5 rescue cassette and a CYB5-URA3 blaster cassette resulted in viable cyb5 strains with no third copy. The C. albicans CYB5 gene is concluded to be nonessential. Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol. The C. albicans cyb5 strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the S. cerevisiae cyb5. The C. albicans cyb5 shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the ERG2 and ERG24 gene products. These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment