Amphotericin B Resistance and Membrane Fluidity in Kluyveromyces lactis Strains

Abstract

The membrane fluidity of reduced-amphotericin B (AmB)-sensitivity Kluyveromyces lactis mutant strain is higher than that of the wild-type K. lactis strain. After culture of the K. lactis and K. lactis mutant cells in the presence of subinhibitory doses of AmB (10 and 125 mg/liter, respectively), the plasma membranes of both yeast strains also showed a higher fluidity than did those of control cells. High membrane fluidity was associated with changes in the structural properties of the membranes. Culture of the K. lactis and K. lactis mutant cells in the presence of AmB induced changes in membrane lipid contents. In particular, phospholipid contents were increased in both strains treated with AmB, compared with their corresponding counterparts. As a result, the sterol/phospholipid ratio decreased. The relative proportion of monounsaturated fatty acids also increased after AmB treatment. The saturated fatty acid/monounsaturated fatty acid ratio decreased in K. lactis and K. lactis mutant cells treated with AmB but also in K. lactis mutant control cells compared to that in the K. lactis wild strain. These changes in lipid composition explain the higher fluidity, which could represent a process of metabolic resistance of the yeasts to AmB.Antifungal heptaen amphotericin B (AmB) is presently considered the "gold standard" for the intravenous chemotherapy of deep fungal infections in spite of its secondary effects (24). This antibiotic has been a therapeutic agent since 1956, but its mode of action at the molecular level has not been totally elucidated. Various interaction models with membrane sterols have been proposed. However, the most recent models are based on the formation of stable complexes not only with sterols but also with membrane phospholipids whose interactions induce pore formation (1, 6).AmB was isolated from Streptomyces nodosus cultures by Gold et al. (9). It is a polyene belonging to the macrolide family, and its molecular conformation is closed to phospholipid molecules. This antifungal agent possesses a hydrophobic part (hydrocarbon chain) and a hydrophilic part (polyhydroxyl chain); its amphipathic properties allow interactions with the membrane after diffusion through the yeast cell wall. Its interaction with membrane constituents (14,16,18) provokes the formation of pores responsible for several reactions, such as lipid oxidations and peroxidations, inhibition of membrane enzymes (3, 4), osmotic shocks, and, finally, cell death (32).Yeast resistance to antifungal agents, mainly azol derivatives, is a well-known phenomenon. Yeast cells with reduced sensitivity to AmB have been isolated from patients (24, 29). Horsburgh et al. (13) and Hakkou et al. (11) also isolated a Candida lusitaniae strain and a Kluyveromyces lactis strain, respectively, with reduced sensitivity to AmB. The reduced sensitivity of the strains either may stem from changes in the membrane chemical properties which may result from mutations or may be induced by the antifungal agent itself. The alterations of the membrane may al...

Deletion of thePDR16gene influences the plasma membrane properties of the yeastKluyveromyces lactis

“…Indeed, changes in the structural properties of the K. lactis membrane (decreased sterol/phospholipid ratio, the decreased saturated fatty acid/monounsaturated fatty acid ratio) could explain the higher membrane fluidity, which represents higher resistance of the K. lactis yeast to several drugs compared with its Klpdr16⌬ mutant. Our results thus support the observations of Mishra et al (2008), who found increased membrane fluidity in fluconazole-resistant C. albicans clinical isolates from diabetic patients, and those of Younsi et al (2000), explaining higher membrane fluidity with changes in its lipid composition, representing a process of metabolic resistance of the K. lactis yeast to amphotericin B. The precise molecular function of KlPdr16p in providing protection against azole antifungals is not known yet and requires further experimentation.…”

Membranes of Class IIa Bacteriocin-Resistant Listeria monocytogenes Cells Contain Increased Levels of Desaturated and Short-Acyl-Chain Phosphatidylglycerols

“…A less fluid membrane has also been shown to be a factor influencing the resistance of Staphylococcus aureus to the cat- (38). It is also known that the increased levels of monounsaturated fatty acids in the membrane phospholipid influence the overall decrease in the membrane molecular order (38). These findings indicate the significant roles played by unsaturated phospholipids and membrane fluidity in antibiotic or antimicrobial peptide association with membranes and consequently in resistance mechanisms.…”

“…In contrast, the larger amounts of longer and saturated PGs in the sensitive L. monocytogenes B73 and 412 strains may enhance membrane insertion by bacteriocin and thus increase sensitivity. A less fluid membrane has also been shown to be a factor influencing the resistance of Staphylococcus aureus to the cat- (38). It is also known that the increased levels of monounsaturated fatty acids in the membrane phospholipid influence the overall decrease in the membrane molecular order (38).…”

Membranes of five-fold alamethicin-resistantStaphylococcus aureus, Enterococcus faecalis andBacillus cereus show decreased interactions with alamethicin due to changes in membrane fluidity and surface charge

“…A weaker insertion ability of AMPs into unsaturated phosphatidylglycerol could point to the role of increased amounts of unsaturated fatty acids and increased membrane fluidity in resistant strains. Similarly it has been observed that Kluyveromyces lactis mutant cells with reduced amphotericin B sensitivity have a higher unsaturated fatty acid-to-saturated fatty acid ratio than do wild type K. lactic cells (Younsi et al, 2000). Hence membrane fluidity could be an important contributing factor to class IIa bacteriocins and AMPs resistance by affecting the insertion into membranes and consequently the formation and stability of the pores.…”