Amphotericin B Resistance and Membrane Fluidity inKluyveromyces lactis Strains

Yeast strains and culture conditions. K. lactis (ATCC 96897) was isolated from dairy products, and the reduced-AmB-sensitivity K. lactis strain (ATCC 96896) was obtained by culture of the wild strain in the presence of subinhibitory doses of AmB (11).

The isolation of K. lactis from human sources is rarely reported, and previous reports of human infection are unavailable in the literature. An immunosuppressed heart transplant patient with pulmonary infection due to the yeast Kluyveromyces fragiliswas described by Lutwick et al. (20).

The cells were grown aerobically in Sabouraud liquid medium (2% glucose, 1% Primatone [Sigma]) at 25°C in the absence or presence of subinhibitory doses of AmB (10 and 125 mg/liter for K. lactis and K. lactis mutant, respectively).

AmB (Squibb) was solubilized in a mixture of dimethyl sulfoxide and ethanol (1:1, vol/vol).

The growth of the cells was estimated by measuring the absorbance of the culture at 620 nm on a Perkin-Elmer spectrophotometer. No difference in morphology was observed by optical microscopy.

Spheroplast preparation.After 10 h of growth, bothKluyveromyces strains were harvested at 4°C by centrifugation at 1,500 × g for 10 min and washed twice with distilled water and then twice with 1.2 M KCl. One gram of cells was suspended in 4 ml of 0.02 M phosphate buffer (25 mM Na₂HPO₄ · 12H₂O, 15 mM KH₂PO₄) (pH 7.4) with 80 mg of pronase E (Merck) (20 mg/ml) and 0.01 M dithiothreitol (Sigma). It was incubated for 60 min at 30°C with slight stirring.

After centrifugation at 1,500 × g, the cells were washed three times in an isotonic solution (0.15 M Na₂HPO₄, 0.08 M citric acid, 1.4 M KCl) (pH 5.8) and resuspended in 4 ml of the isotonic solution.

The suspension was then incubated at 30°C for 3 h with 30 mg of “lysing enzyme” from Trichoderma harzianum (Sigma) and 5 mM dithiothreitol (Sigma) with slight stirring. Once centrifuged at 1,500 × g, the spheroplasts were washed twice with 1.2 M KCl and resuspended in the isotonic solution (pH 5.8). All experiments were then performed at a temperature of 30°C.

The kinetics of spheroplast formation was monitored by enumerating the remaining intact yeast cells after dilution of the suspension with distilled water on a Malassez cell with a binocular optic microscope.

To study the influence of the ionic strength of the buffer on spheroplast stability, several dilutions of the isotonic (pH 5.8) and 1.8-ionic-strength solution were achieved. Dilutions of 10, 13, 16, 20, 25, 30, 40, and 50% of the 1.8-ionic-strength solution in distilled water corresponded, respectively, to ionic strengths of 1.6, 1.53, 1.5, 1.4, 1.3, 1.2, 1.0, and 0.8.

To study the influence of incubation time on spheroplast stability, samples were collected at intervals of 0, 20, 40, 60, 80, and 100 min in the isotonic solution (pH 5.8). The number of spheroplasts was determined, for each assay, by the difference between the total number of intact cells and the number of intact cells remaining after spheroplast lysis in a nonosmotic solution.

Spheroplast labeling with a fluorescent probe.Lipidic fluidity was assessed by steady-state fluorescence polarization using a lipophilic fluorescent probe: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH; Molecular Probes, Eugene, Oreg.), which is incorporated into the plasma membrane, remaining anchored at the lipid-water interface with the DPH moiety intercalated between the upper parts of the fatty acyl chains (26).

Stock solutions of TMA-DPH in N-N′-dimethylformamide at a concentration of 2 × 10⁻³ M were kept in the dark at 4°C.

For fluorescence polarization measurements, 1 μl of probe solution (final probe concentration, 2 × 10⁻⁶ M) was added to 1 ml of membrane suspension (spheroplast concentration, 4 × 10⁶ cells per ml) and the labeled suspension was incubated at room temperature in the dark for 5 min.

Fluorescence was measured at 25°C immediately after labeling, without further washing.

Fluorescence polarization measurements.The fluorescence anisotropy was determined at 25°C using a continuous-excitation apparatus (Fluofluidimeter; Regulest, Florange, France). The excitation wavelength was selected with a band-pass filter centered on 365 nm. The emission wavelength was 425 nm.

The intensities of the emitted light oriented parallel (I_II) and perpendicular (I_I) to the plane of the excitation beam were measured. For each sample, five determinations were done and signals from an unlabeled membrane suspension “blank” (Ib_II and Ib_I) were also determined to eliminate the influence of the light scattered by the membrane suspension. The fluorescence anisotropy, <r> (1/membrane fluidity), was calculated using the following relationship:<r>=[(IfII−IbII)−(IfI−IbI)]/[(IfII−IbII)+2(IfI−IbI)] The parameter <r> is inversely related to lipid fluidity.

Plasma membrane isolation and purification.By following the protocol of Rose and Veasey (30), the spheroplasts were suspended in a pH 7.2 Tris-mannitol buffer (50 mM Tris and 0.3 M mannitol adjusted to pH 7.2 with HCl). After 10 min at room temperature, the suspension was incubated for 20 min in an ice bath to make the membranes more rigid. Lysis was then carried out by gentle stirring at low speed with an Ika-Werk homogenizer to mechanically favor osmotic lysis. To ensure the isolation of the plasma membranes, the crushed material was checked with a binocular optic microscope to confirm the absence of cytoplasmic organelles.

The spheroplast lysate was suspended in 3 ml of a 62% (p/vol) sucrose solution then supplemented with a discontinuous gradient of sucrose constituted of 2 ml of 7, 6, 5, 4, 3, 2, and 1% (p/vol) sucrose in mannitol buffer. After centrifugation of this suspension at 100,000 × g, the pellet contained the rough membrane fraction. This preparation was suspended in 4 ml of mannitol buffer in a 40-ml Ultraclear centrifugation tube (Beckman). Then a discontinuous gradient of sucrose was prepared by successively introducing 5 ml of 62 and 50% (p/vol) sucrose and 3 ml of 45, 40, 35, 30, 20, and 10% (p/vol) sucrose in mannitol buffer. After centrifugation at 24,000 × g for 90 min at 4°C, the eight fractions were collected and submitted to a protein concentration determination by the method of Lowry et al. (19). The fractions containing the plasma membranes were also submitted to lipid concentration determination.

Extraction and analysis of membrane constituents.The membrane lipids were extracted by use of a chloroform-methanol (2:1, vol/vol) mixture for 18 h with gentle stirring at 37°C under nitrogen by following the method of Folch et al. (7), with a slight modification (18 h instead of 3 min).

After filtration on Whatman no. 2 filter paper, a washing with the Folch solvent (chloroform-methanol-water, 200:100:75, vol/vol/vol), and decantation, the chloroform extract was evaporated and dried in a vacuum. The dry extract contained the membrane total lipids and was submitted to saponification in 30% (p/vol) KOH-methanol for 4 h at 100°C under reflux. After cooling, the mixture was filtered on Whatman filter paper. After the addition of 30 ml of distilled water, the filtrate was submitted to three successive extractions with petroleum ether.

The petroleum ether extract containing the sterols was washed with water, dried, evaporated to dryness in a vacuum, and then acetylated with a pyridine-acetic anhydride (1:1, vol/vol) mixture, purified, and finally identified according to the method of Belrhiti et al. (2). The total sterol content was analyzed by the method of Zak (33), with a slight modification (ergosterol was used as the standard instead of cholesterol). The different acetylated sterols were determined by gas-liquid chromatography using a PE 1701 (15 by 0.25 m; Perkin-Elmer) capillary column at 260°C. The nitrogen flow rate was 20 ml/min, and the injector and detector temperatures were 250 and 300°C, respectively.

The aqueous filtrate containing the fatty acid salts was acidified. The free fatty acids liberated were methylated according to the protocol of Belrhiti et al. (2) and identified by gas-liquid chromatography using a PE 225 (30 m by 0.25 mm; Perkin-Elmer) capillary column at 220°C. The nitrogen flow rate was 20 ml/min; the injector and detector temperatures were 250 and 300°C, respectively. Saturated (SFA) (C₁₀ to C₁₈), monounsaturated (MUFA) (C_14:1, C_16:1, and C_18:1), and polyunsaturated (C_18:2 and C_18:3) fatty acids in methylester form were purchased from Sigma and used as standards.

From the dry extract containing the membrane total lipids, the amount of phosphorus was estimated by the method of MacClare (21) and the phospholipid amount in the sample was estimated by multiplying the phosphorus level of each extract by 25, which is the mean of the phosphorus level in yeast membrane phospholipids (23).