Selective Membrane Toxicity of the Polyene Antibiotics: Studies on Natural Membranes

Four mutants strains of Aspergillus fennelliae resistant to various polyene antibiotics have been analyzed for their sterol content. The mutant strains contained A7'22 24(2 8-ergostatrien-3#-ol and A7 22-ergostadien-3,3-ol as the major sterols, whereas the wild types contained ergosterol, indicating that the mutants contain metabolic blocks for C,-C, dehydrogenation and C24-C28 reduction in the biosynthesis of ergosterol. Revertant sectors arising from the mutant colonies contained either more A7.22-ergostadien-3#-ol than the parent mutant strain or showed the reappearance of ergosterol. The revertant sectors also showed lower resistance to amphotericin B and a higher rate of sporulation than the parent mutant strain. These results confirm our previous observations that the presence of ergosterol is directly related to the drug susceptibility and the normal rate of sporulation in this species.The development of resistance to polyene antibiotics in fungi has been shown to coincide with alterations in the sterol content of the cells (1,2,(5)(6)(7)(10)(11)(12)(13)(14).In 1972 (2) identified several biosynthetic precursors of ergosterol as the major sterols in nystatin-resistant mutants of S. cerevisiae. A mutant of S. cerevisiae resistant to relatively low concentrations of nystatin was found to contain as a major sterol 5,6-dihydroergosterol, an immediate precursor of ergosterol. The mutants resistant to higher concentrations of nystatin, on the other hand, containing earlier precursors such as A7. 22, 24(28 '-ergostatrien-3fl-ol, A8, 22, 24(28)-ergostatrein -3f-ol, and fecosterol (Al8 24 (28 )-ergostadien-3#-ol.We have recently reported observations of polyene resistance and correlated sterol changes in the mutants of Aspergillus fennelliae (10). In this communication, we report the identification of the sterols accumulated in the mutants of A. fennelliae. Evidence supporting our previous hypothesis that ergosterol is one of the necessary components for the normal rate of asexual and sexual sporulation is also presented. MATERIALS AND METHODSStrains and cultural conditions. The polyeneresistant mutants, AF4-NS1, p-NS1, AF5-AB1, p-AB1, and wild-type strains of A. fennelliae, described elsewhere (10), were used in this experiment. The wild-type strains were maintained on a malt extractagar medium, and the mutants were maintained on the same medium but containing 4 tsg of amphotericin B per ml for AF5-AB1 and p-AB1 and 10 U of nystatin per ml for AF4-NS1 and p-NS1. For sterol extractions, the strains were cultured in malt extract-broth medium at 30 C on a gyrotory shaker (115 rpm) for 5 days. To isolate a reverse mutant, AF5-AB1 and p-AB1 were maintained also on drug-free malt extract-agar medium.Sterol extraction and purification. Mycelial pads grown on malt extract medium were washed once in distilled water and saponified with 30% methanolic KOH at reflux for 3 h before the sterols were extracted with hexane (10). The hexane extracts were then washed with distilled water, and the solvent was evaporated under a str...

Section: Discussionsupporting

confidence: 67%

Polyene-Resistant Mutants of Aspergillus fennelliae : Identification of Sterols

Kim

Kwon‐Chung

Milne

et al. 1974

“…1B). The imidazole-induced release from Prototheca phospholipids is dramatically increased by addition of 5% of the neutral lipid fraction of the (11,12). Several studies confirm that membrane ergosterol, the major fungal sterol, is much more effective than cholesterol, the major mammalian sterol, in conferring polyene susceptibility (3,15 (17).…”

Section: Micsmentioning

confidence: 72%

Lipid composition and sensitivity of Prototheca wickerhamii to membrane-active antimicrobial agents

Sud¹,

Feingold²

1979

The lipid composition of Prototheca wickerhamii ATCC 16529 is presented and discussed in relation to the unique susceptibility of the organism to drugs of three membrane-active antimicrobial classes: the polyenes, the polymyxins, and the imidazoles. The presence of ergosterol in the neutral lipid fraction of the membrane is likely responsible for the exquisite susceptibility to amphotericin B. The presence of a large quantity of free fatty acids in the membrane appears responsible for imidazole susceptibility. The membrane determinants of polymyxin B susceptibility are less well defined.

“…In addition, it is also widely used among pediatric patients (8)(9)(10). Conventional AMB produces renal toxicity (11)(12)(13)(14)(15), and therefore new lipidic combinations have been developed to improve the delivery of the drug and decrease its toxicity (16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

The Production of Reactive Oxygen Species Is a Universal Action Mechanism of Amphotericin B against Pathogenic Yeasts and Contributes to the Fungicidal Effect of This Drug

Mesa-Arango

Trevijano-Contador

Román

et al. 2014

168

151

e Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, and Cryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.