Brian C. Baldwin

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.

Inhibition of ergosterol biosynthesis in saccharomyces cerevisiae and Ustilago maydis by tridemorph, fenpropimorph and fenpropidin

Abstract The structurally related fungicides, tridemorph, fenpropimorph and fenpropidin have been shown to inhibit the sterol Δ 14 -reductase and Δ 8 →Δ 7 -isomerase during ergosterol biosynthesis in Saccharomyces cerevisiae and Ustilago maydis . However, although the three fungicides are able to inhibit both enzymes, tridemorph inhibits the Δ 8 -Δ 7 -isomerase better than the Δ 14 -reductase whilst the reverse is true for fenpropidin and to a lesser extent for fenpropimorph.

Alternative Respiration: a Biochemical Mechanism of Resistance to Azoxystrobin (ICIA 5504) in Septoria tritici

The mechanism of resistance to ICIA 5504 (azoxystrobin) in a Septoria tritici mutant raised in the laboratory has been investigated. This mutant was approximately 10 times less sensitive than the wild-type strain in in-vitro tests towards spore germination or fungal growth. Glucose oxidation in whole cells was inhibited in the wild type (80% inhibition at 0.1 μg ml -1 ), whereas in the resistant mutant, oxygen uptake was stimulated (50% stimulation at 1.0 μg ml -1 ). Respiration of the wild-type strain was inhibited by antimycin A and cyanide but not that of the mutant. These results indicate the existence of an efficient alternative respiratory pathway in the mutant, which was inhibited by the addition of 2 mM salicylhydroxamate (SHAM). Using mitochondria, antimycin A and ICIA 5504 did not completely inhibit NADH oxidation in either strain. Addition of SHAM inhibited part of the antimycin- and ICIA 5504-insensitive oxygen uptake only in mutant mitochondria. For complete inhibition of oxygen reduction, SHAM and cyanide need to be present. Thus, three systems of electron transfer from exogenous NADH to oxygen are present in S. tritici mitochondria: the cytochrome pathway which is sensitive to ICIA 5504 and antimycin A inhibition in both strains, the system of NADH-cytochrome c reductase which bypasses the methoxyacrylate inhibition at the cytochrome bc 1 complex, and the alternative oxidase which is inhibited by SHAM, and which is partially functioning only in mitochondria isolated from the ICIA 5504-resistant mutant. When the S. tritici isolates were tested for their in-vivo sensitivity to ICIA 5504 on wheat, the resistant strain was controlled better than the wild type. This indicates that the decreased ATP formation by the alternative pathway of respiration was inadequate for efficient parasitic growth on the host.

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.

Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents.

The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14alpha-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC(50) values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0. 2 microM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC(50) values of 0.008 and 0.0076 microM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(-) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra.

The Relationship between the Development of Haustoria of Erysiphe graminis and the Energy Status of Leaves

Powdery mildews are obligate pathogens. The fungus grows externally on leaf tissues and forms haustoria only in host epidermal cells. The haustorium is believed to be the absorptive organ through which the fungus acquires photosynthates for growth. The intimate interface established between fungus and host and subsequent alterations made to the region of the host plasmalemma in direct contact with the haustorium [1] may aid this function.