The synthetic amphipathic peptidomimetic LTX109 is a potent fungicide that disturbs plasma membrane integrity in a sphingolipid dependent manner

Abstract

The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.

Figure 1. Fungicidal properties of LTX109 and amphotericin B.

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.

Figure 2. Transport of H⁺, K⁺ and a fluorescent dye by cells treated with LTX109.

(A) Glucose-induced acidification of medium by yeast cells. Exponentially growing S. cerevisiae 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.

Figure 3. Efflux of amino acids from cells treated with LTX109.

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.

Figure 4. Mutants in sphingolipid biosynthesis are resistant to LTX109.

(A) Spot test of wild type (WT) S. cerevisiae and eight deletion mutants identified by screening a deletion collection for LTX109 resistance. Exponentially growing yeast was resuspended in YPD to 10⁷ cells/ml and serially diluted 1∶10. Aliquots (6 µl) were spotted on solid YPD plates containing 70 µg/ml LTX109 (left panel), and without LTX109 (right panel). (B) S. cerevisiae sphingolipid biosynthetic pathway.

Figure 5. Activity of LTX109 against yeast biofilm.

Confocal Laser Scanning Microscopy of S. cerevisiae (Σ1278b) 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.