PMC

Analysis of sensitivity to peptides and to SDS of specific S. cerevisiae deletion mutants. (A), (B) and (C) show results of three independent experiments, with specific genes as indicated in the figure. See the text for additional details on the selected genes. Other details as in Figure 4.

Differential interaction of S. cerevisiae deletion mutants with FITC-PAF26. Representative fluorescence micrographs of the parental BY4741 and S. cerevisiae deletion strains Δssd1, Δecm33, and Δarg1, as indicated at the left. Optical and image acquisition settings were the same for each fluorophore and thus differences in fluorescence intensity among strains reflect real differences. Others details as in Figure 6B.

Distribution of differentially expressed genes after peptide treatment. A z-test for two independent conditions was conducted for each peptide treatment compared to the control treatment. Effective p-values were <3.3E-03 and <3.7E-03 for PAF26 and melittin, respectively. Diagram shows genes induced (up) or repressed (down) by peptides. The small circles on the upper part refer to 15 genes induced by PAF26 and repressed by melittin and 7 genes induced by melittin and repressed by PAF26.

Differential interaction of S. cerevisiae deletion mutants with FITC-PAF26. Flow cytometry measurements of FITC-PAF26 binding to S. cerevisiae deletion mutants shown below as compared with the parental strain BY4741. Graph shows the percentage of fluorescence bound to cells after exposure of 20,000 cells to either 5 (upper panel) or 30 μM (lower panel) FITC-PAF26. Mean and SD from two replicas in each of two independent experiments are shown for each strain.

Analysis of sensitivity to peptides and to CW disturbing compounds of S. cerevisiae deletion mutants in CW-related genes. Data on sensitivity of the single gene deletion strains Δsed1, Δssd1, Δpir2, Δdse2, Δecm33, and the corresponding parental strain BY4741 are shown. (A) and (B) show results after treatment of serial 5-fold dilutions of exponentially growing cells with each peptide for 2 hours (Panel A) or 24 hours (Panel B) and subsequent plating onto YPD peptide-free plates. (C) and (D) show growth of serial dilutions of the same deletion strains on YPD plates containing SDS (Panel C) or CFW (Panel D).

Antifungal activity of peptides PAF26 and melittin to S. cerevisiae FY1679. (A) Dose response curve of cell killing activity. Cells were exposed to different concentrations of peptides for 24 h. Cell survival (measured as CFU/mL) was determined by dilution and plating. (B) Time course of cell population growth was followed in the presence of 5 μM of peptide. No significant differences were found between each of the peptides and the control treatment. In both (A) and (B) panels, grey circles and white triangles indicate PAF26 and melittin samples, respectively; in (B), white squares show controls in the absence of peptide.

Quantitative real time PCR analysis of gene expression changes after peptide treatment. All the panels show the mean relative expression ± SD (y-axis) of each individual gene upon each peptide treatment as compared to the control treatment with no peptide. (A) and (B) graphs are end-point analyses of expression of the indicated genes (x-axis) after 3 h of peptide treatment; grey bars indicate 5 μM PAF26, black bars 25 μM PAF26, and white bars 5 μM melittin. Note the different expression scales in panels (A) and (B). (C) Graph shows time-course changes of expression of ARG1 following treatment with either 5 μM PAF26 or 5 μM melittin. In all the panels, the genes ALG9, TAF10 and UBC6 were simultaneously used as constitutive references (see Methods for details).

Fluorescence microscopy of S. cerevisiae exposed to FITC-PAF26. (A) Internalization of FITC-PAF26 into S. cerevisiae FY1679 demonstrated by confocal fluorescence microscopy. Cells were exposed to 30 μM FITC-PAF26 for 30 min. Bright-field (A1) and fluorescence (A2) micrographs of the same field are shown. (B) Interaction of FITC-PAF26 with S. cerevisiae BY4741 visualized by fluorescence microscopy: DIC bright field image, as well as FITC, propidium iodide (PI), and calcofluor white (CFW) signals of the same field are shown. Cells were incubated with 30 μM FITC-PAF26 at 30°C for 2 h, and then at 20°C with 2 μM PI and 25 μM CFW for 5 min. Open arrowheads indicate peptide internalization (compare location of the CW outer signal of CFW with the internal signal of PI and the FITC fluorescence resulting from FITC-PAF26). Solid arrowhead indicates the lower FITC signal in the vacuole compared to the cytosol.