Deletion strains in genes involved in cell wall construction such as SSD1 or ECM33 showed a correlation with the higher sensitivity to PAF26 in that a proportion of cells higher than in the parental strain were labeled by the peptide and showed intense staining by PI. However, the resistant Δarg1, Δnop16 or Δipt1 mutants did not show #Poziotinib clinical trial randurls[1|1|,|CHEM1|]# a noticeable difference of peptide labeling as compared with the parental strain

(Figure 8) and in some experiments, such as the one shown in the corresponding panel of Figure 7 (Δarg1), a higher proportion of cells were labeled with the peptide. This latter result indicates that the higher resistance of these strains is not due to lack of interaction and/or internalization of the peptide. Figure 7 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. Figure 8 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 click here Fenbendazole 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. Discussion and Conclusions We have carried out a functional genomic approach on yeast to gain insight into the mechanism of two AMP that presumably have different modes of antifungal killing. Analogous reports have addressed the mode of action of distinct antifungal agents [35–38, 61, 62],

including other AMP [30, 32, 33]. These latter studies on AMP used inhibitory concentrations and found an array of multifactorial effects, but could not distinguish those processes primary related to peptide mechanism from those secondarily derived from cell death. Since we have observed biological changes of P. digitatum after exposure to sub-inhibitory (sub-micromolar) concentrations of PAF26 that include peptide internalization [46], we decided to use non-inhibitory concentrations of AMP in the gene expression experiments (5 μM, Figure 1) in an attempt to unveil primary effects of the peptides. Also, by choosing two peptides with differentiated interactions with fungal cells, we could isolate processes both common and specific of each one. The transcriptomic data demonstrates specific and statistically significant changes under these conditions that our fungicidal assays demonstrate that are involved in sensitivity to peptides.