Cytochrome P450 enzyme systems in fungi

This paper reviews the current status of our understanding of azole antifungal resistance mechanisms at the molecular level and explores their implications. Extensive biochemical studies have highlighted a significant diversity in mechanisms conferring resistance to azoles, which include alterations in sterol biosynthesis, target site, uptake and efflux. In stark contrast, few examples document the molecular basis of azole resistance. Those that do refer almost exclusively to mechanisms in laboratory mutants, with the exception of the role of multi-drug resistance proteins in clinical isolates of Candida albicans. It is clear that the technologies required to examine and define azole resistance mechanisms at the molecular level exist, but research appears distinctly lacking in this most important area.

Molecular mechanisms of azole resistance in fungi

During their foraging activity, honey bees are often exposed to direct and residual contacts with pesticides, especially insecticides, all substances specifically designed to kill, repel, attract or perturb the vital functions of insects Insecticides may elicit lethal and sublethal effects of different natures that may affect various biological systems of the honey bee The first step in the induction of toxicity by a chemical is the interaction between the toxic compound and its molecular target The action on the molecular target can lead to the induction of observable or non-visible effects The toxic substance may impair important processes involved in cognitive functions, behaviour or integrity of physiological functions This review is focused on the neural effects of insecticides that have repercussions on (a) cognitive functions, including learning and memory, habituation, olfaction and gustation, navigation and orientation; (b) behaviour, including foraging and (c) physiological functions, including thermoregulation and muscle activity

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Neural effects of insecticides in the honey bee

Expression of the ABC transporter BcatrD from Botrytis cinerea reduces sensitivity to sterol demethylation inhibitor fungicides

The biosynthesis of steroids and triterpenoids

The selective fungitoxic actions of prochloraz (an imidazole) and a triazole fungicide, quinconazole (3-(2,4-dichlorophenyl)-2-(1 H- 1,2,4-triazol-1-yl)- 4(3H)-quinazolinone: II), were studied with selected phytopathogenic fungi. With the exception of Ustilago maydis, all the fungi tested were more sensitive to prochloraz than to II. A number of DMI-resistant mutants of Penicillium digitatum and P. italicum showed positive cross-resistance to both DMIs, but except for P. italicum isolate H17, the levels of resistance to II were much higher than to prochloraz.



The generally higher toxicity of prochloraz to the fungi investigated, as compared to II, could not be ascribed to the slightly higher accumulation of prochloraz. With regard to prochloraz, there was no general correlation between the sensitivity of the fungi tested and the amount of fungicide accumulated. A similar situation was evident for II. However, the DMI-resistant mutants of P. italicum did show a reduced accumulation of both azoles, which may account for a low level of acquired DMI-resistance in this fungus. Since accumulation levels of the test compounds in the isolates with different degrees of resistance were the same, additional mechanisms of resistance may be involved in isolates with relatively high degrees of DMI-resistance.



No detectable amounts of fungicide metabolites were found in most fungi tested over a 16-hour incubation period. Therefore, fungal metabolism is not generally responsible for the differences in sensitivity between fungi to each azole tested. It also does not generally explain the differential toxicities of prochloraz and II to each individual species. The exception to this was Rhizoctonia solani which metabolized prochloraz to a non-fungitoxic compound. This correlated with its low prochloraz sensitivity.

Biochemical Mechanisms Involved in Selective Fungitoxicity of Two Sterol 14α‐Demethylation Inhibitors, Prochloraz and Quinconazole: Accumulation and Metabolism Studies

An assay for measuring ergosterol synthesis in cell-free extracts of the filamentous plant pathogen Botrytis cinerea is described. The extracts capable of synthesizing C4-desmethyl sterols from [2- 14 C]mevalonate were derived by mechanical disruption of young conidial germlings in a Bead-Beater apparatus. The C4-desmethyl sterol fraction consisted of three distinct compounds and totalled 39% of the non-saponifiable lipids formed. Ergosterol accounted for 63% of the C4-desmethyl sterols. Only small amounts of C4-monomethyl sterols were synthesized, while C4, 4-dimethyl sterols made up 29% of the non-saponifiable lipids. The latter fraction mainly consisted of lanosterol (54%) and eburicol (28%). The cell-free system had a narrow pH optimum for synthesis of C4-desmethyl sterols of pH 7.3–7.4. Cell-free synthesis of C4-desmethyl sterols was inhibited by the imidazole fungicide imazalil, concomitant with an accumulation of eburicol. The IC50 value (concentration of fungicide which inhibited cell-free synthesis of C4-desmethyl sterols by 50%) was 9.1 × 10 −9 M. These results are consistent with the hypothesis that imazalil is a potent inhibitor of the cytochrome P450-dependent sterol 14x-demethylase of B. cinerea. The method described may be used to screen compounds biochemically for inhibition of sterol synthesis in an agriculturally important plant pathogen.

Development of a cell-free assay from Botrytis cinerea as a biochemical screen for sterol biosynthesis inhibitors.

A procedure for the isolation of microsomes containing cytochrome-P450 isozymes from Ustilago maydis is described. Yields of P450 amount to approximately 19(±+ 6) pmol mg−1 of microsomal protein. The wavelength of maximum absorbance of the reduced carbon monoxide difference spectrum is 448-449 nm. The azole fungicides prochloraz, etaconazole, imazalil, triadimefon and 3-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-4(3H)-quinazoline, which differ markedly in toxicity to U. maydis, all induce type II binding difference spectra at extremely low concentrations (10−9-10−8 M). The DMI concentrations which cause half saturation of type II binding difference spectra (IC50) do not correlate with the fungicidal activities of the azoles. Binding of carbon monoxide to ferrous cytochrome-P450 was only slightly inhibited to different degrees by the DMIs tested. However, the inhibition of carbon monoxide binding also does not correlate with fungitoxicity of the DMIs. The results in this paper suggest that the spectrophotometric studies with this preparation are not useful for evaluating selective toxicity of DMIs to intact sporidia of U. maydis.

Isolation of microsomal cytochrome-P450 isozymes from Ustilago maydis and their interaction with sterol demethylation inhibitors.

The sensitivity of cytochrome-P450-dependent sterol 14α-demethylase (P45014DM) to prochloraz and several prochloraz analogues was studied in a cell-free assay of Botrytis cinerea Pers. ex Fr. The EC50 values (concentrations which inhibited radial growth of B. cinerea by 50%) of the compounds tested ranged from 3.3 × 10 −8 to 1.7 × 10 −5 M. The IV50 values (concentrations which inhibited cell-free C4-demethyl sterol synthesis by 50%) in cell-free assays of B. cinerea ranged from 2.6 × 10 −9 to 4.4 × 10 −7 M. Ranking compounds in terms of their relative inhibitory potencies showed quite similar trends to the order of fungitoxicity, but the IC50 values did not quantitatively reflect the differences in toxicity. Therefore, the differential inhibition of cell-free P45014DM activity by these compounds cannot fully account for their differences in activity towards B. cinerea. Additional mechanisms must be involved.



The compounds tested were generally more potent in the B. cinerea assay than in similar assays developed for Penicillium italicum Wehmer and, in particular, Saccharomyces cerevisiae Meyen. This correlated with the relatively higher activity of most test compounds to B. cinerea. Results suggest that the cell-free assay of B. cinerea is more useful to evaluate candidate fungicides as inhibitors of sterol 14α-demethyiase activity than similar assays from model organisms.



The present study confirms that the affinity of prochloraz analogues for P45014DM depends on the nature of the N-1 substituent of the imidazole and the azole ring. It was also found that addition of an amino group at C-2 of the imidazole moiety of prochloraz gave a compound (6) which inhibited 4, 4-demethyl sterol biosynthesis in B. cinerea at a different site from the P45014DM. This was confirmed by the observation that laboratory-generated triadimenol-resistant isolates of B. cinerea showed reduced sensitivity to triadimenol and prochloraz, but not to compound 6.

Inhibition of sterol biosynthesis in cell-free extracts of Botrytis cinerea by prochloraz and prochloraz analogues

Differential accumulation of [/4C]imazalil and [14 C]fenarimol by germlings of wild-type and DMI-resistant isolates of Penicillium italicum was studied at various pH values. At pH 7 and 8 the low-resistant isolate E 300-3 accumulated 22~ and 35 %, respectively, less imazalil than the wild-type isolate W 5 . Imazalil accumulation at pH 5 and 6 was similar. Isolate E300_ 3 also accumulated less fenarimol as compared with the wild-type isolate. This difference was much more obvious than for imazalil and was observed at all pH values tested. Differences in accumulation of both imazalil and fenarimol between low (E30o.3) , medium (H 17) and high resistant (I 33) isolates were not observed. These results suggest that decreased accumulation of DMIs is responsible for a low level of resistance only and that additional mechanisms of resistance might operate in isolates with a medium and high degree of resistance. With all isolates fenarimol accumulation was energy-dependent. This was not obvious for imazalil. The wild-type and DMI-resistant isolates had a similar plasma membrane potential as determined with the probe [lac]tetraphenylphosphonium bromide ([14C]TPP +). Various test compounds, among which ATPase inhibitors, ionophoric antibiotics and calmodulin antagonists, affected the accumulation of [14C]TPP+, [14C]imazalil and [14C]fenarimol. No obvious correlation between the effects of the test compounds on accumulation levels of the fungicides and [14C]TPP+ could be observed. These results indicate that the plasma membrane potential does not mediate the efflux of DMI fungicides by P. italicum. Additional keywords: ATPase inhibitors, calmodulin antagonists, fenarimol, imazalil, ionophoric antibiotics, sterol 14a-demethylation inhibitors, tetraphenylphosphonium bromide.

Characterization of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low, medium and high degree of resistance to DMI-fungicides.

J.C. Kapteyn

Papers

Biochemical Mechanisms Involved in Selective Fungitoxicity of Two Sterol 14α‐Demethylation Inhibitors, Prochloraz and Quinconazole: Accumulation and Metabolism Studies

Development of a cell-free assay from Botrytis cinerea as a biochemical screen for sterol biosynthesis inhibitors.

Isolation of microsomal cytochrome-P450 isozymes from Ustilago maydis and their interaction with sterol demethylation inhibitors.

Inhibition of sterol biosynthesis in cell-free extracts of Botrytis cinerea by prochloraz and prochloraz analogues

Characterization of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low, medium and high degree of resistance to DMI-fungicides.