Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides.

There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 µM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories.

The Penicillium digitatum protein O-mannosyltransferase Pmt2 is required for cell wall integrity, conidiogenesis, virulence and sensitivity to the antifungal peptide PAF26.

The activity of protein O-mannosyltransferases (Pmts) affects the morphogenesis and virulence of fungal pathogens. Recently, PMT genes have been shown to determine the sensitivity of Saccharomyces cerevisiae to the antifungal peptide PAF26. This study reports the identification and characterization of the three Pdpmt genes in the citrus post-harvest pathogen Penicillium digitatum. The Pdpmt genes are expressed during fungal growth and fruit infection, with the highest induction for Pdpmt2. Pdpmt2 complemented the growth defect of the S. cerevisiae Δpmt2 strain. The Pdpmt2 gene mutation in P. digitatum caused pleiotropic effects, including a reduction in fungal growth and virulence, whereas its constitutive expression had no phenotypic effect. The Pdpmt2 null mutants also showed a distinctive colourless phenotype with a strong reduction in the number of conidia, which was associated with severe alterations in the development of conidiophores. Additional effects of the Pdpmt2 mutation were hyphal morphological alterations, increased sensitivity to cell wall-interfering compounds and a blockage of invasive growth. In contrast, the Pdpmt2 mutation increased tolerance to oxidative stress and to the antifungal activity of PAF26. These data confirm the role of protein O-glycosylation in the PAF26-mediated antifungal mechanism present in distantly related fungal species. Important to future crop protection strategies, this study demonstrates that a mutation rendering fungi more resistant to an antifungal peptide results in severe deleterious effects on fungal growth and virulence.

The myosin motor domain-containing chitin synthase PdChsVII is required for development, cell wall integrity and virulence in the citrus postharvest pathogen Penicillium digitatum.

Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit.

Genes involved in protein glycosylation determine the activity and cell internalization of the antifungal peptide PAF26 in Saccharomyces cerevisiae.

We have previously characterized the synthetic hexapeptide PAF26 as a cell-penetrating and non-lytic antifungal peptide that is active against Saccharomyces cerevisiae and filamentous fungi. Numerous cell wall (CW) proteins are glycosylated in fungi and many of these play important roles in fungal pathogenesis. In this study, we screened a collection of S. cerevisiae deletion mutants for protein glycosylation genes whose deletion altered the sensitivity to PAF26. Increased tolerance to PAF26 was observed in mutants with the following disrupted genes: PMT1-6, EOS1, ALG5, MNN1, MNN4 and MNN5. Significantly, genes coding for protein O-mannosyltransferase 2 (Pmt2p), which is responsible for the addition of the first mannosyl residue of O-linked carbohydrates, and for Eos1p, an enzyme involved in N-linked glycosylation of proteins, showed resistance to PAF26 and defects in CW integrity. Microscopic studies on the S. cerevisiae Δeos1 deletion mutant demonstrated a blockage of peptide internalization by cells. Protoplasts lacking CWs interacted with the peptide, but were more resistant to peptide killing than cells possessing CWs due to a blockage in PAF26 internalization. Interestingly, protoplasts obtained from Δeos1 behaved similarly to those of the parental strain. Collectively, these observations demonstrate that the CW is a positive factor that determines the internalization of the PAF26, and that Eos1p exerts its activity through the glycosylation of specific protein(s) involved in peptide internalization.

Two functional motifs define the interaction, internalization and toxicity of the cell-penetrating antifungal peptide PAF26 on fungal cells.

The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW) is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW) and PAF96 (RKKAAA), were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i) its interaction with the cell envelope; (ii) its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii) its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the cytoplasm, which coincides with cell death. Peptide containment within vacuoles preserves fungal cells from peptide toxicity.

Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus.

BACKGROUND: Penicillium digitatum is a fungal necrotroph causing a common citrus postharvest disease known as green mold. In order to gain insight into the genetic bases of its virulence mechanisms and its high degree of host-specificity, the genomes of two P. digitatum strains that differ in their antifungal resistance traits have been sequenced and compared with those of 28 other Pezizomycotina. RESULTS: The two sequenced genomes are highly similar, but important differences between them include the presence of a unique gene cluster in the resistant strain, and mutations previously shown to confer fungicide resistance. The two strains, which were isolated in Spain, and another isolated in China have identical mitochondrial genome sequences suggesting a recent worldwide expansion of the species. Comparison with the closely-related but non-phytopathogenic P. chrysogenum reveals a much smaller gene content in P. digitatum, consistent with a more specialized lifestyle. We show that large regions of the P. chrysogenum genome, including entire supercontigs, are absent from P. digitatum, and that this is the result of large gene family expansions rather than acquisition through horizontal gene transfer. Our analysis of the P. digitatum genome is indicative of heterothallic sexual reproduction and reveals the molecular basis for the inability of this species to assimilate nitrate or produce the metabolites patulin and penicillin. Finally, we identify the predicted secretome, which provides a first approximation to the protein repertoire used during invasive growth. CONCLUSIONS: The complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.

Identification and characterization of chitin synthase genes in the postharvest citrus fruit pathogen Penicillium digitatum.

In this study, we carried out the isolation and characterization of chitin synthase genes (CHS) of the main citrus fruit postharvest pathogen Penicillium digitatum. Using distinct sets of degenerate primers designed from conserved regions of CHS genes of yeast and filamentous fungi, PCR methods, and a DNA genomic library, five putative CHS genes (PdigCHSI, PdigCHSII, PdigCHSIII, PdigCHSV, and PdigCHSVII) were identified, isolated, sequenced, and characterized. Phylogenetic analyses, sequence identity, and domain conservation support the annotation as CHS. A very high sequence identity and strong synteny were found with corresponding regions from the genome of Penicillium chrysogenum. Gene expression of P. digitatum CHS genes during mycelium axenic growth, under oxidative and osmotic stress conditions, and during infection of citrus fruits was confirmed and quantified using quantitative RT-PCR (qRT-PCR). PdigCHSIII had the highest expression among the five genes by one order of magnitude, while PdigCHSII had the lowest. However, PdigCHSII was strongly induced coincident with conidial production, suggesting a role in conidiogenesis. The expression of PdigCHSI, PdigCHSIII, PdigCHSV, and PdigCHSVII was upregulated during infection of citrus fruit. PdigCHSV and PdigCHSVII coexpressed in most of the experiments carried out, and they are separated by a 1.77 kb intergenic region and arranged in opposite directions.