Efflux of amino acids from cells treated with LTX109.

<p>Exponentially growing yeast cells were washed, resuspended in water, and challenged with 70 µg/ml LTX109 (black bars) or water (grey bars) for 16 minutes. Amino acids (one letter code) in the extracellular medium were subsequently measured by HPLC. Each data point is the average of three individual measurements ± standard deviation.</p

Transport of H⁺, K⁺ and a fluorescent dye by cells treated with LTX109.

<p>(A) Glucose-induced acidification of medium by yeast cells. Exponentially growing <i>S. cerevisiae</i> was washed and suspended in sterile water and exposed to 100 µg/ml LTX109 (squares) or water (circles) before glucose addition at time zero. Medium pH was measured and H⁺ concentration calculated from pH = −log [H⁺]. Each data point is the average of three individual measurements with standard deviations as error bars. (B) Potassium release from yeast cells. Exponentially growing yeast cells were washed, resuspended in water, and challenged with 100 µg/ml LTX109 (squares) or water (circles) at time zero. Potassium release was measured using flame atomic absorption spectrometry in binary increasing intervals. Each data point is the average of three individual measurements ± standard deviation. (C) Nomarski (left) and fluorescent (right) microscopy of SYTOX Green-stained yeast cells. Exponential growing cells were exposed to 100 µg/ml LTX109 and SYTOX Green uptake was monitored. Cells treated with SYTOX Green and 0 µg/ml LTX109 served as control. SYTOX green uptake upon LTX109 treatment was observed in three independent experiments.</p

Fungicidal properties of LTX109 and amphotericin B.

<p>Time-kill kinetics of exponentially growing yeast cells exposed to water (circles) or five times the MIC of LTX109 (40 µg/ml) (squares) or amphotericin B (10 µg/ml) (triangles). Viability was examined every half hour as CFUs. Each data point is the average of three individual measurements ± standard deviation.</p

<i>S. cerevisiae</i> genes that confer LTX109 resistance upon deletion.

<p>n, number of mutants identified.</p

Activity of LTX109 against yeast biofilm.

<p>Confocal Laser Scanning Microscopy of <i>S. cerevisiae</i> (Σ1278<i>b</i>) biofilm. Cells were grown in Lab-Tek™ Chamber Slide™ System; Permanox - (NUNC, Denmark) in 1 ml synthetic complete medium After 12 hours, the cells were exposed to 0 µg/ml LTX109 (control) or 70 µg/ml LTX109 for another 5 hours. The biofilm cells were then stained with Syto9 (green) and propidium iodide (red) LIVE/DEAD stain before confocal laser scanning microscopy. Images are 3D reconstructions of biofilm made from 2 µm thick images in stacks of 20 individual images. CLSM was perform with a Zeiss LSM510 microscope using a 63x/0.95NA a water immersion lens. Life dead staining of biofilm treated with LTX109 was repeated in four independent experiments. White bar is 30 µm.</p