The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner.

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

Formation of extrachromosomal circular DNA from long terminal repeats of retrotransposons in <i>Saccharomyces cerevisiae</i>

Extrachromosomal circular DNA (eccDNA) derived from chromosomal Ty retrotransposons in yeast can be generated in multiple ways. Ty eccDNA can arise from the circularization of extrachromosomal linear DNA during the transpositional life cycle of retrotransposons, or from circularization of genomic Ty DNA. Circularization may happen through nonhomologous end-joining (NHEJ) of long terminal repeats (LTRs) flanking Ty elements, by Ty autointegration, or by LTR–LTR recombination. By performing an in-depth investigation of sequence reads stemming from Ty eccDNAs obtained from populations of Saccharomyces cerevisiae S288c, we find that eccDNAs predominantly correspond to full-length Ty1 elements. Analyses of sequence junctions reveal no signs of NHEJ or autointegration events. We detect recombination junctions that are consistent with yeast Ty eccDNAs being generated through recombination events within the genome. This opens the possibility that retrotransposable elements could move around in the genome without an RNA intermediate directly through DNA circularization

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in <i>Saccharomyces cerevisiae</i>

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.ISSN:1362-4962ISSN:0301-561

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

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

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

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

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

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