Modifications of pH and K+ gradients in Candida albicans blastospores induced by amphotericin B. A 31P NMR and K+ atomic absorption study.

Candida albicans blastospores harvested from 8 h (exponential) or 48 h (stationary) cultures were incubated with increasing doses of amphotericin B (AmB). The time course of H+ influx and K+ efflux was monitored by in vivo 31P NMR and K+ atomic absorption respectively. AmB was shown to be more active on exponential phase cells than on stationary phase cells. For both growth phases, K+ leakage occurred before pH acidification. In light of these results, together with iodoacetate experiments, it seems difficult to assert that K+ leakage is a secondary effect resulting from an increase in the permeability to protons, as formerly proposed. In addition, no H+ over K+ selectivity of pores formed by AmB could be detected. Finally, some unexpected results were afforded by 31P NMR experiments: a broadening of Pi signals was detected on exponential phase cell spectra when the blastospores were incubated with 10(-3) and 10(-4) M AmB reflecting a transient heterogeneity of the intracellular pH within the cell population. For stationary phase blastospores, two subpopulations (IIa and IIb) were detected; population IIb, with a more acidic pHi, was much more sensitive to AmB action.

Na+ and K+ transport by 4-chlorophenylurethane-monensin in Enterococcus hirae de-energized and energized cells studied by 23Na-NMR and K+ atomic absorption.

Na+ and K+ movements induced by 4-chlorophenylurethane-monensin, which presents an inverted ion selectivity (K+ > Na+) in model systems compared with monensin, were followed on Enterococcus hirae cells by 23Na-NMR and K+ atomic absorption. For de-energized cells, the urethane derivative is much more selective for K+ than monensin, but only at low concentrations (10(-3)-10(-4) mM). For higher concentrations, as previously shown for monensin, the sodium and potassium movements are driven by the ion gradients present. On energized cells, both K+ and Na+ gradients were highly perturbed, and this can be related to the higher toxicity in mice and bacteria for this derivative.

Conditions modulating the ionic selectivity of transport by monensin examined on Enterococcus hirae (Streptococcus faecalis) by 23Na-NMR and K+ atomic absorption.

Factors likely to modulate the ionic selectivity of monensin were examined on Enterococcus hirae (Streptococcus faecalis) in two states previously characterized: the resting (de-energized) cell and the active (energized) cell. Internal and external Na+ were followed by corresponding 23Na-NMR resonances K+ concentrations were measured by atomic absorption. For a given cellular population of de-energized cells, the apparent transport rates and the final cationic concentrations reached at the steady state were decreasing with the ionophore dose. Monensin was selective for sodium only at low concentrations, in the range 1 mM-10(-4) mM the transport was depending on the effective cationic gradients. Comparison of the activity curves for two cell populations (7.10(9) and 7.10(10) cells/ml) showed the importance of the ratios of monensin/mg phospholipid and also of the ratios of external/internal volumes. On energized cells, except for low monensin concentrations, the main effect was a K(+)-induced efflux and not a Na+ influx. Two factors were modulating the resulting selectivity of this ionophore: the response of the intrinsic bacterial carriers and the generation of the gradients (mainly the external pH) which were favourable to a K+/Na+ transport. Once again the results obtained for two cell populations could be compared, the determining factors were the ratio external/internal volume and the generation of the pH gradient.

Intracellular pH of Candida albicans blastospores as measured by laser microspectrofluorimetry and 31P-NMR.

The intracellular pH (pHi) of Candida albicans blastospores harvested from 8 h or 48 h cultures was determined under identical experimental conditions by two different techniques: 31P-NMR and laser microspectrofluorimetry. Time dependence of pHi was monitored by 31P-NMR on the whole cell population. Microspectrofluorimetry, after loading of the cells with SNARF-1, enabled the determination of pHi in isolated cells and its distribution among the cell population. By this method, the vacuolar pH could not be distinguished from the cytoplasmic pH in C. albicans blastospores, but alkalization of pHi was observed at the beginning of germ tubes. The absolute values of pHi determined by 31P-NMR were slightly different from those obtained by laser microspectrofluorimetry. However, the pH distributions in the cell population were converging. For blastospores in exponential phase a gaussian distribution of pHi was observed with both methods, the cells maintained a steady pHi value when the external pH was varied from 5.5 to 8.5. For cells in stationary phase two pools were identified: the combination of the two techniques demonstrated the presence of two different subpopulations. One of these population (with lower pH) was able to commute to the other one with time as shown by 31P-NMR kinetics. This information is reported here for the first time in C. albicans.

Effects of amphotericin B on glucose metabolism in Candida albicans blastospores evidenced by 13C NMR.

Candida albicans blastospores harvested from 8- (exponential phase) or 48-h (stationary phase) cultures were incubated with 60 x 10(-3)M [1-(13)C]glucose with or without 10(-4)M amphotericin B (AmB). The utilization of [1-(13)C]glucose was monitored by in vivo 13C NMR under anaerobiosis. With exponential phase cells, in the presence of AmB, the consumption of glucose and the production of ethanol, trehalose, and glycerol continuously decreased with time, and after 25 min, the metabolism was blocked. On stationary phase cells AmB had almost no effect on glucose metabolism. Comparison with previous experiments evidenced that AmB induced first K+ leakage, then acidification, and finally a stop of the metabolism. In parallel, in vitro 13C NMR spectra were performed on supernatants and cell-free extracts of yeast suspension incubated under the same conditions. For both exponential and stationary phase cells, AmB induced an increase in the membrane permeability to glycerol; no change was observed for the other metabolites.

Phosphate-dependent sodium transport in S. faecalis investigated by 23Na and 31P NMR.

Na+ movements in S. faecalis were studied by 23Na NMR. They proved to be dependent on phosphate concentration in the buffer during the de-energization step. K+ and H+ were also studied respectively by potentiometry and 31P NMR and were shown not to be implicated. For de-energized cells the internal phosphate concentration, on the contrary, was directly linked to the external phosphate contained in the buffer. The experiments showed a Na+/Pi dependence in this prokaryote so far known only in eukaryotes.