Cross-resistance to polyene and azole drugs in Cryptococcus neoformans

Abstract

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...

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Amphotericin B-resistant isolates from an AIDS patient were found to have defective ⌬8-7 isomerase activity (274). Furthermore, isolates selected in vitro that were cross-resistant to amphotericin B and fluconazole showed a reduced cellular accumulation of the antifungals, suggesting a possible role for a common multidrug transporter (258). Another means by which C. neoformans can acquire resistance to amphotericin B is through the formation of biofilms, which display elevated levels of resistance to the polyenes and azoles in comparison to their planktonic counterparts (370).…”

Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance

“…An amphotericin B-resistant isolate from an AIDS patient was found to have defective sterol ⌬8-7 isomerase activity (88). Isolates selected in vitro that were cross-resistant to both amphotericin B and fluconazole showed reduced cellular accumulation of the antifungals, suggesting a possible role for multidrug transporters (82). Another means by which C. neoformans can acquire resistance to amphotericin B is through the formation of biofilms, which are more resistant both to triazoles and to this polyene than planktonic cells (124).…”

Different culture media applied to the study of Cryptococcus neoformans susceptibility to amphotericin B and fluconazole

“…Amphotericin B (8,9,10,17) and fluconazole-C. neoformans-resistant strains were detected in antifungal susceptibility tests (1,13,14,15).…”