Fluconazole was observed to inhibit sterol 14␣-demethylase in the human pathogen Cryptococcus neoformans, and accumulation of a ketosteroid product was associated with growth arrest. A novel mechanism(s) of azole and amphotericin B cross-resistance was identified, unrelated to changes in sterol biosynthesis, as previously identified in Saccharomyces cerevisiae. Reduced cellular content of drug could account for the resistance phenotype, indicating the possible involvement of a mechanism similar to multidrug resistance observed in higher eukaryotes. Infection withCryptococcus neoformans leading to cryptococcal meningitis has been associated with up to 8% of all AIDS cases (7). Treatment failures and recurrence of infection in AIDS patients on current chemotherapeutic programs involving the use of azole antifungal agents or the polyene antibiotic amphotericin B are increasing (1,3,12,21).Ergosterol is the principal sterol in most fungi (for a review, see reference 9). Amphotericin B has been shown to bind ergosterol in the membrane, with resistance occurring through mutation in the biosynthetic pathway (3). Mode-of-action studies have shown that azole antifungal agents bind to cytochrome P-450 (P-450 14␣-dm ), preventing sterol 14␣-demethylation (20). Subsequent 4-demethylation may still occur, with formation of abnormal 14␣-methyl sterols such as obtusifoliol, 14␣-methylfecosterol, and the presumed product of attempted ⌬ 5(6) desaturation of 14␣-methylfecosterol, 14␣-methyl-3,6-diol (17). The accumulation of this last sterol has been shown genetically and biochemically to be associated with the arrest of growth in azole-treated Saccharomyces cerevisiae (17).Lesions in P-450 14␣-dm confer resistance to polyene antibiotics by preventing the formation of ergosterol, which is the target molecule for polyene action. For viability, these strains require a second defect in sterol ⌬ 5(6) -desaturase which prevents the formation of 14␣-methyl-3,6-diol (14). Azole-resistant mutants generated directly in S. cerevisiae are also defective in sterol ⌬ 5(6) -desaturase (18); these mutants do not accumulate ergosterol and are also cross-resistant to polyene antifungal agents.In the study described in this report we examined the potential for cross-resistance between azole and polyene antifungal agents in the fungal pathogen C. neoformans. A different pattern of sterol accumulation was observed following azole treatment of C. neoformans compared with that observed in S. cerevisiae, suggesting that the mechanisms for avoiding the formation of 14␣-methyl-3,6-diol were not applicable (17). However, mutants which were cross-resistant to the azoles and amphotericin B were isolated, indicating an important consideration for future antifungal therapy. MATERIALS AND METHODSCulture conditions. Two strains of the human pathogen C. neoformans (B4476 and B4500) were obtained from K. J. Kwon-Chung, National Institutes of Health, Bethesda, Md. Growth was supported on 2% (wt/vol) glucose, 2% (wt/vol) Difco peptone, and 1% (wt/vol) Difco yeast e...

Azole antifungals inhibit CYP51Al-mediated sterol 14c~-demethylation and the mechanism(s) of resistance to such compounds in Ustilago maydis were examined. The inhibition of growth was correlated with the accumulation of the snbstrate, 24-methylene-24,25-dihydrolanosterol (eburicol), and depletion of ergosterol. Mutants overcoming the effect of azole antifungal treatment exhibited a unique phenotype with leaky CYP51A1 activity which was resistant to inhibition. The results demonstrate that alterations at the level of inhibitor binding to the target site can produce azole resistance. Similar changes may account for fungal azole resistance phenomena in agriculture, and also in medicine where resistance has become a problem in immunocompromised patients suffering from AIDS.

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Amphotericin B resistant mutants of Cryptococcus neoformans were isolated accumulating mainly ergosterol. Cross-resistance to azole antifungals was not observed. Together with previous data this indicates that at least three categories of amphotericin B resistance can arise: sterol mutants, amphotericin B and azole cross-resistant mutants and amphotericin B resistant mutants with no azole cross-resistance.

Ustilago maydis mutants resistant to polyene antibiotics were screened for defects in ergosterol biosynthesis. Slowgrowing mutants recovered after selecting for amphotericin B resistance were devoid of ergosterol and accumulated the methylated sterols, 14a-methylfecosterol, obtusifoliol and eburid, indicating that these isolates were impaired in C-14 sterol demethylation and were similar to the Erg40 mutant of U. m y & By contrast, nystatin-and pimaricin-resistant isolates which exhibited reduced growth rates showed a dysfunction in C-8 sterol isomerization. In these mutants (Erg2) ergosterol was replaced by the A*-sterols, ergosta-5,8,22-trienol, ergosta-8,22-dienol, fecosterol and ergost-8-enol. Analysis of a random sample of polyene-resistant isolates that grew normally revealed that although most retained a typical wild-type sterol profile, two of the isolates failed to accumulate ergosterol. The msjor sterols detected in these isolates were ergosta-7,22-dienol and ergosta-7-eno1, suggesting a lesion in C-5 sterol desaturation in these mutants (Erg3). Of four Erg2 mutants recovered, one mutant, selected on nystatin, contained low but detectable amounts of ergosterol. Ergosterol was not detected immediately after selection in the three other pimaricin-resistant Erg2 mutants. Although the growth of three of the Erg2 mutants remained unchanged during non-selective culture, one mutant reverted and began to grow at a greater rate than the rest; analysis of the sterols produced by this strain revealed that ergosterol was now present, but at lower concentrations than those in the wild-type strain. No changes in the type of sterol formed were observed in the other slower-growing Erg2 mutants even after prolonged culture. All the Erg2 mutants exhibited morphological abnormalities; sporidia were swollen and distorted, and the inability of sporidia to separate after cell division led to the development of highly branched, multicellular groups of cells.