Membrane Sphingolipid-Ergosterol Interactions Are Important Determinants of Multidrug Resistance in Candida albicans

Abstract

In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans. We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis. While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C. albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine. Treatment of wild-type C. albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs. Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants. For example, the induction in vitro of a 12% change in the membrane fluidity of C. albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells. Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C. albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning. A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein. Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C. albicans cells.

The COP9 signalosome is involved in the regulation of lipid metabolism and of transition metals uptake in Saccharomyces cerevisiae

“…We further confirmed this finding by assessing the ability of the Δcsn5 mutant to grow in the presence of nystatin and ketoconazole. Nystatin targets ergosterol in the cell membrane, while ketoconazole interferes with ergosterol biosynthesis [31]. As expected, the results shown in Table 4 indicate that Δcsn5 cells are indeed more sensitive to ketoconazole and more resistant to nystatin compared with the wild-type, again confirming an alteration in ergosterol biosynthesis.…”

Genome-Wide Expression and Location Analyses of the Candida albicans Tac1p Regulon

“…An attractive model would be that Tac1p contributes to the raft association and proper routing of its target gene products Cdr1p, Cdr2p, and Rta3p to the plasma membrane by regulating AST2 expression. Recent studies have demonstrated that both sphingolipids and sterols are important determinants of surface localization of Cdr1p, as reduced membrane localization of Cdr1p was associated with increased susceptibility of C. albicans to ketoconazole in mutants defective in ergosterol biosynthesis (53). Interestingly, we found that Tac1p binding was enriched at the ERG1 (1.7-fold, P value of 0.0005) and ERG2 (1.6-fold, P value of 0.0011) promoters.…”

Structure and Function Analysis of CaMdr1p, a Major Facilitator Superfamily Antifungal Efflux Transporter Protein of Candida albicans : Identification of Amino Acid Residues Critical for Drug/H + Transport

“…Drug susceptibilities of yeast strains were measured by two independent methods: microdilution assay and spot assay. The MIC 80 s for the strains were determined with a broth microdilution method as described previously (11,16). For spot assay, 5-l samples of fivefold serial dilutions of each yeast culture (each with cells suspended in normal saline to an optical density at 600 nm of 0.1) were spotted onto YEPD plates in the absence (control) or in the presence of the drugs (11,16 …”

“…Current evidence suggests that Candida acquires azole resistance by employing multiple mechanisms that include (i) failure of drug accumulation mediated by extrusion pump proteins such as Cdr1p and Cdr2p, belonging to ATP binding cassette (ABC), and CaMdr1p, belonging to major facilitator superfamilies (MFS), (ii) alterations in the azole target protein Erg11p, and (iii) up regulation of the ERG11 gene (11,13,16).…”