David C. Lamb

Resistance to fluconazole and cross‐resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol Δ5,6‐desaturation

Fluconazole resistance occurs in >10% of cases of candidosis during the late stages of AIDS. We show here in two clinical isolates that resistance was caused by defective sterol Δ5,6‐desaturation. This altered the type of sterol accumulating under fluconazole treatment from 14α‐methylergosta‐8,24(28)‐dien‐3β,6α‐diol to 14α‐methylfecosterol which is capable of supporting growth. A consequence of this mechanism of azole resistance is that an absence of ergosterol causes cross‐resistance to the other major antifungal agent available, amphotericin B. The results also show that growth arrest after fluconazole treatment of C. albicans in clinical conditions is caused by 14α‐methylergosta‐8,24(28)‐dien‐3β,6α‐diol accumulation.

The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity.

Sterol 14α-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.

The Mechanism of the Acyl-Carbon Bond Cleavage Reaction Catalyzed by Recombinant Sterol 14α-Demethylase of Candida albicans (Other Names Are: Lanosterol 14α-Demethylase, P-45014DM, and CYP51)

The Candida albicans sterol 14α-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 μmol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14α-demethylase, 3β-hydroxylanost-7-en-32-al and 3β-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3β-hydroxylanost-7-en-32-al with the recombinant 14α-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17α-hydroxylase-17,20-lyase (P-450,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14α-demethylase is considered.

Purification and reconstitution of activity of Saccharomyces cerevisiae P450 61, a sterol Δ22-desaturase

P450 was purified from microsomal fractions of a strain of Saccharomyces cerevisiae which contained detectable P450 despite the disruption of CYP51A1. The P450 had a molecular mass of 58 kDa, similar to P450 51A1, and in a reconstituted assay with rabbit NADPH‐P450 reductase and dilauryl phosphotidylcholine exhibited activity for conversion of ergosta‐5,7‐dienol into ergosterol. N‐Terminal amino acid sequencing of the purified protein corresponded to the translated sequence of P450 61 which was recently identified during sequencing of chromosome XIII. This allowed the function of this family of P450 to be identified as sterol Δ 22‐desaturation in the pathway of ergosterol biosynthesis.

Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients

Azole antifungal compounds are important in the treatment of Cryptococcosis, a major cause of mortality in AIDS patients. The target of the azole drugs is P450 mediated sterol 14α‐demethylase. We have investigated the P450 system of Cryptococcus neoformans with respect to azole tolerance observed in clinical isolates which were obtained following the failure of fluconazole therapy. The clinical failure was correlated with in vitro tolerance of azole antifungal when compared to wild‐type strains. The microsomal P450 system was typical of yeast and fungi and fluconazole tolerance was not associated with defective sterol biosynthesis. The strains had slightly elevated P450 content and slightly reduced azole levels in the cells, but a clear cause for resistance was the increased level of drug needed to inhibit the sterol 14α‐demethylase in vitro.

Sterol 22-desaturase, cytochrome P45061, possesses activity in xenobiotic metabolism

CYP61 was revealed in the sequencing of the yeast genome on chromosome XIII and was the last member of the CYP superfamily in yeast to be discovered. We show here that besides the housekeeping role in 22‐desaturation during ergosterol biosynthesis the enzyme is also that responsible for benzo(a)pyrene metabolism/promutagen activation by yeast in genotoxicity assays. This enzyme may represent an ancestral activity for the superfamily which allowed xenobiotic metabolism for the first time.