Amphotericin B resistant isolates of Cryptococcus neoformans without alteration in sterol biosynthesis.

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.

Altered P450 activity associated with direct selection for fungal azole resistance.

Azole antifungals inhibit CYP51A1-mediated sterol 14 alpha-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 substrate, 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.

Cross-resistance to polyene and azole drugs in Cryptococcus neoformans.

Fluconazole was observed to inhibit sterol 14 alpha-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.

Interaction of the lacZ beta-galactosidase of Escherichia coli with some beta-D-galactopyranoside competitive inhibitors.

1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (lacZ) beta-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg(2+) by Mn(2+) enhances both longitudinal and transverse relaxation of the methyl groups of the beta-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-beta-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn(2+) ion is located 8-9A (0.8-0.9nm) from the centroid of the trimethylammonium group and 9A (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (K(i) (o) and K(i) (+) respectively) for the isosteric ligands, beta-d-galactopyranosylbenzene and the beta-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=K(i) (+)/K(i) (o)) obtained with 2-(beta-d-galactopyranosyl)-naphthalene and the beta-d-galactopyranosylisoquinolinium ion at pH7 with Mg(2+)-enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg(2+)-enzyme can be described by Q=0.1(1+[H(+)]/4.17x10(-10))/1+[H(+)]/10(-8)). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg(2+)-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg(2+) as well as tyrosine-501.