Comparison of sterol import under aerobic and anaerobic conditions in three fungal species, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae

Abstract

Sterol import has been characterized under various conditions in three distinct fungal species, the model organism Saccharomyces cerevisiae and two human fungal pathogens Candida glabrata and Candida albicans, employing cholesterol, the sterol of higher eukaryotes, as well as its fungal equivalent, ergosterol. Import was confirmed by the detection of esterified cholesterol within the cells. Comparing the three fungal species, we observe sterol import under three different conditions. First, as previously well characterized, we observe sterol import under low oxygen levels in S. cerevisiae and C. glabrata, which is dependent on the transcription factor Upc2 and/or its orthologs or paralogs. Second, we observe sterol import under aerobic conditions exclusively in the two pathogenic fungi C. glabrata and C. albicans. Uptake emerges during post-exponential-growth phases, is independent of the characterized Upc2-pathway and is slower compared to the anaerobic uptake in S. cerevisiae and C. glabrata. Third, we observe under normoxic conditions in C. glabrata that Upc2-dependent sterol import can be induced in the presence of fetal bovine serum together with fluconazole. In summary, C. glabrata imports sterols both in aerobic and anaerobic conditions, and the limited aerobic uptake can be further stimulated by the presence of serum together with fluconazole. S. cerevisiae imports sterols only in anaerobic conditions, demonstrating aerobic sterol exclusion. Finally, C. albicans imports sterols exclusively aerobically in post-exponential-growth phases, independent of Upc2. For the first time, we provide direct evidence of sterol import into the human fungal pathogen C. albicans, which until now was believed to be incapable of active sterol import.

Fig 1

Sterol uptake in C. albicans, S. cerevisiae, and C. glabrata strains under various conditions. Cholesterol (A, B, and C) and ergosterol (A and C) uptake under normoxic and microaerophilic conditions into the strains of three fungal species. Sterol import was measured in parental strains and their UPC2-deficient derivatives (A), parental strains in the presence of fluconazole (FLC) and/or fetal bovine serum (FBS) (B), or in parental strains and their AUS1- and PDR11-deficient derivatives (C), comparing heat-killed (HK) cells with actively growing strains. The graphs in panel C represent separate experiment from those in panel A and thus are presented separately. In all cases strains were grown for 48 h under normoxic (gray bars) and microaerophilic condition (black bars) in CSM complete supplemented with uridine, oleic acid in the form of Tween 80, and the appropriate sterol (cholesterol or ergosterol) with its radiolabeled form. In the experiment in panel C, fluconazole (FLC; each strain's MIC₈₀) and/or fetal bovine serum (FBS; 10%) was added as noted. Samples were normalized to cpm/10⁸ cells. Graphs represent the average of three biological replicates with the standard errors. All differences between normoxic and microaerophilic conditions are statistically significant as determined by using the Student t test (P < 0.05), and data pairs marked with asterisks are also significant (to P < 0.1). In panel B the difference between C. albicans cholesterol uptake in the presence of FLC and FBS is not statistically significant. Heat-killed controls were only performed once due to the number of samples in the experiment. The heat killing causes significant disruption of the cell membranes, as proven by an increase in propidium iodide permeability (45).

Fig 2

Normoxic and microaerophilic growth of C. albicans, S. cerevisiae, and C. glabrata strains. A dilution series (10-fold) of cells were grown for 48 h under normoxic and microaerophilic conditions at 30°C on various agar media. CSM (CSM complete supplemented with uridine), TW (Tween 80, 0.1%), Chol (cholesterol, 20 μg/ml), and FLC (fluconazole, 256 μg/ml) were used. S. cerevisiae parental strains differ for the UPC2 mutants (W303-1a) and the AUS1/PDR11 mutant (BY4742) and thus are presented separately with the appropriate mutants. CSM supplemented with Tween 80 was performed, and it was not significantly different from CSM alone (data not shown).

Fig 3

Time course experiments of sterol uptake into C. albicans, S. cerevisiae, and C. glabrata. Cholesterol (A, B, and C) and ergosterol (D, E, and F) uptake by C. albicans (circles), S. cerevisiae (squares), and C. glabrata (triangles) under normoxic (A, C, D, and F) and microaerophilic (B and E) conditions. Parental strains are represented by solid symbols and UPC2-null mutants by open symbols. Strains were grown for 5 days in CSM complete supplemented with uridine, Tween 80, and a mix of cold and radioactively labeled cholesterol-ergosterol. Samples were analyzed every 24 h. The graphs represent average of three biological replicates with standard deviation. (G and H) Cholesterol uptake in three fungal species presented as a percentage of all available cholesterol taken up from the medium under normoxic (G) and microaerophilic (H) conditions. The graphs in panels G and H were created from data presented also in panels A and B.

Fig 4

TLC separation of radioactively labeled cholesterol and ergosterol associated with the cells of C. albicans, S. cerevisiae, and C. glabrata. Set of parental strains of the three fungal species were compared to each other under normoxic (2 and 5 days) and microaerophilic (2 days) conditions in the presence of radioactively labeled cholesterol (A) or ergosterol (B). AUS1/PDR11 mutants were compared to their parents only in the presence of radioactively labeled cholesterol (C). Total cell lipids were extracted from the cells and separated by TLC. The samples represent the amount of cells grown in equal volume of culture medium. Chol, cholesterol; CE, cholesteryl ester; Erg, ergosterol; EE, ergosteryl ester.

Fig 5

Uptake of fluorescently labeled cholesterol from agar medium. Cell cultures of C. albicans, S. cerevisiae, and C. glabrata strains spotted on CSM complete supplemented with uridine and cholesteryl-BODIPY (fluorescently labeled cholesterol) were incubated for 2 days under normoxic and microaerophilic conditions and scanned. The zone of clearance (white area) around colonies is indicative of decreased local concentration of cholesteryl-BODIPY and thus active uptake of this sterol. S. cerevisiae parental strains differ for the UPC2 mutants (W303-1a) and AUS1/PDR11 mutant (BY4742) and thus are presented both separately with the appropriate mutants.

Fig 6

MICs determined for C. albicans, S. cerevisiae, and C. glabrata. MICs determined under normoxic (A, B, C, G, H, I, J, and K) and microaerophilic (D, E, F, L, and M) conditions for C. albicans (A, D, and G), S. cerevisiae (B, E, H, J, and L), and C. glabrata (C, F, I, K, and M). Parental strains (CaBWP17, ScW303-1a/ScBY4742, and CgKUE200) are represented by black solid symbols, UPC2-null mutants (CaΔupc2/Δupc2, ScΔupc2/Δecm22, and CgΔupc2A/Δupc2B) by open symbols and AUS1/PDR11 mutants (ScΔaus1/Δpdr11 and CgΔaus1) by gray solid symbols. Circles represent medium supplemented with Tween 80, squares represent medium supplemented with Tween 80-cholesterol, and triangles represent medium supplemented with Tween 80-cholesterol-FBS. S. cerevisiae parental strains differ for the UPC2 mutants (W303-1a; graphs B, E, and H) and AUS1/PDR11 mutant (BY4742; graphs J and L) and thus are presented both in separate sections of the figure. Parental strain BY4742 is presented together with its ScΔaus1/Δpdr11 derivative (J and L). Fluconazole concentrations did not exceed 256 μg/ml, with the exception of 2-fold increase for C. glabrata, in order to prevent reaching of critical micelle concentration for this compound. Graphs represent the average of three biological replicates with standard errors.