Amphotericin B kills unicellular leishmanias by forming aqueous pores permeable to small cations and anions.

The polyene antibiotic amphotericin B (AmB) is known to form two types of ionic channels across sterol-containing liposomes, depending on its concentration and time after mixing (Cohen, 1992). In the present study, it is shown that AmB only kills unicellular Leishmania promastigotes (LPs) when aqueous pores permeable to small cations and anions are formed. Changes of membrane potential across ergosterol-containing liposomes and LPs were followed by fluorescence changes of 3,3' dipropylthiadicarbocyanine (DiSC3(5)). In KCl-loaded liposomes suspended in an iso-osmotic sucrose solution, low AmB concentrations ( NO3 > Cl > I > Br > acetate (SO2-4 being impermeable). Cell killing by AmB was followed by fluorescence changes of the DNA-binding compound ethidium bromide (EB). At low concentrations (/=0.1 microM), a salt influx via the aqueous pores formed by the antibiotic was followed by osmotic changes leading to cell lysis. This last stage is supported by electron microscopy observations of the changes of parasite morphology immediately upon addition of AmB, which indicated that the typical elongated promastigote cell forms became rounded and the flagella swells and round up. The present work is the first demonstration of the in vitro sensitivity of Leishmania promastigotes to osmotic lysis by AmB.

Concentration and time dependence of amphotericin B-induced permeability changes across plasma membrane vesicles from Leishmania sp.

The main purpose of this paper is to show that the polyene antibiotic amphotericin B may form two types of channels in plasma membrane vesicles from Leishmania sp.: ionic and aqueous. The experimental design was to follow the light scattering changes of a suspension of membrane vesicles under an osmotic shock. The results show that a low concentration of amphotericin B(0.2 to 0.8 microM) led to an enhancement of urea and salt permeability without affecting the total osmotic response of Leishmania vesicles. Such an increment of solute permeability induced by low concentrations of amphotericin B was 100% blocked by tetraethylammonium. Low concentrations of amphotericin B were also able to induce an enhancement of glucose permeability but only after Leishmania membrane vesicles were incubated for 15 min with the antibiotic, previous to mixing. On the other hand, high amphotericin B concentrations (greater than 0.8 microM) induced a decrease in the total extent of shrinkage of membrane vesicles immediately after its mixing with urea solutions. At this high concentration of amphotericin B the blocking of tetraethylammonium was reduced by 50%. These results support the authors' previous conclusion (1) that in ergosterol-containing membranes, amphotericin B may form two different types of channels differing in internal diameter.

The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp.

An osmotic method has been used to study the effect of the polyene antibiotics amphotericin B, nystatin and candicidin on the water permeability of plasma membranes prepared from Leishmania sp. The effect of amphotericin B on the permeability of Leishmania membranes to a salt such as potassium nitrate was also investigated. A non-linear and saturable enhancement of water and salt permeability was measured with increasing polyene concentrations, which could be adjusted to Hill cooperativity equation. The antibiotic concentrations that induce at 30 degrees C half-maximal effects on the water permeability of Leishmania vesicles were 0.021 microM for candicidin, 0.21 microM for amphotericin B and 1.4 microM for nystatin. At 30 degrees C, the concentration of amphotericin B required to induce half of the maximal effect on the permeability of Leishmania vesicles to potassium nitrate was 1.8 microM. The temperature dependence for amphotericin B, nystatin and candicidin enhancement of the water permeability of Leishmania vesicles was determined by using Q10 data at 20 and 30 degrees C. The estimated activation energies at increasing polyene concentrations display the same general pattern for all three polyene antibiotics investigated, that is, a maximal positive value at about the polyene concentrations required for half-maximal effect. The significance of these results for understanding the mechanisms of action of polyene antibiotics on natural membranes is discussed.