The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.

Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses.

The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca(2+) ([Ca(2+)]c) dynamics and a distinct Ca(2+) signature that involves Ca(2+) influx from the external medium and internal Ca(2+) stores. We investigated the molecular basis of this Ca(2+) response by using [Ca(2+)]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca(2+) signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca(2+) entry does not occur through the hitherto described high- and low-affinity Ca(2+) uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca(2+) release from internal stores following IP3 formation combines with the extracellular Ca(2+) influx.

MsDef1 and MtDef4 from Medicago spp. are small cysteine-rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ-core motif (GXCX(3-9) C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ-core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ-core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane/cell wall. Using N. crassa expressing the Ca(2+) reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca(2+) homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca(2+) were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ-core motifs play important roles in their different modes of antifungal action.

Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

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.

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.

Recent evidence indicates that antimicrobial peptides can kill microbes in more complex ways than just by membrane permeabilization. In this study, the mechanism of internalization of the de novo designed cationic hexapeptide PAF26 has been characterized in detail using Neurospora crassa. Live-cell imaging of fluorescently labelled PAF26, organelle probes and mutants indicate that the peptide is endocytically internalized at low fungicidal concentrations (2.0-5 µM). At these concentrations, PAF26 initially accumulated in vacuoles that expanded, and then was actively transported into the cytoplasm, which coincided with cell death. Deletion mutants of the endocytic proteins RVS-161, RVS-167 and RAB-5 exhibited reduced rates of PAF26 internalization and fungicidal activity. Pharmacological experiments with live-cell probes showed that PAF26 internalization and antifungal action at low fungicidal concentrations was energy-dependent, primarily actin-mediated, and disrupted intracellular calcium homeostasis. PAF26 antifungal activity at low concentrations was shown to rely on its endocytic internalization. PAF26 also induced plasma membrane depolarization which, however, was independent of peptide internalization and killing of fungal cells. At high fungicidal concentrations (20 µM), PAF26 internalization was energy-independent, suggesting the involvement of passive peptide translocation. Our results provide new mechanistic insights into the mode-of-action of small cationic antimicrobial peptides that should facilitate improvements in their design.

Mycelial interconnectedness achieved by hyphal fusion has been hypothesized to facilitate the distribution and sharing of nutrients between different parts of a mycelium, especially when nutrients are heterogeneously distributed in the environment. However, the link between environmental nutrient availability and hyphal fusion is little understood. Here, we report that amino acids and extracellular pH regulate conidial anastomosis tube (CAT) fusion during colony initiation in Neurospora crassa. Quantitative analyses revealed that low extracellular pH and certain amino acids, particularly tryptophan, inhibit CAT fusion. Conidial germination was also inhibited by tryptophan but this inhibition was mitigated by the presence of other amino acids. This provides evidence for tryptophan having a role as a signal molecule that regulates CAT fusion. Tryptophan acts intracellularly because two amino acid permease mutants (Δmtr and Δaap-20) exhibited resistance against tryptophan-mediated inhibition of CAT fusion. Tryptophan and low pH did not significantly affect vegetative hyphal fusion in mature colonies, indicating that the latter is regulated in a different manner to CAT fusion.

<p style="text-align: justify;"><strong>Aims</strong>: Developing, by classical genetic methods, new wine yeast strains showing improved release of mannoproteins during wine fermentation, as well as suitable selection procedures for this purpose. These strains would be useful to improve quality characters associated to wine mannoprotein content.</p><p style="text-align: justify;"><strong>Methods and results</strong>: UV mutagenesis was used for genetic improvement of the industrial wine yeast strain ADY1. Cell wall-related phenotypes were used as primary selection criteria; an additional screening procedure was developed based on the detection of the released mannoproteins by hybridization with peroxidase-labeled Concanavalin A. Mannoprotein overproduction was assessed in laboratory media as well as in grapevine juice. One mutant strain, renamed HPS, was selected using these criteria. HPS showed increased mannoprotein release in different culture media, including natural must. Moreover, white wines fermented with this improved strain were less susceptible to protein haze than equivalent wines fermented with the original ADY1 strain. Red wines fermented with the mutant strain were also polysaccharide-enriched as compared to the original one.</p><p style="text-align: justify;"><strong>Conclusion</strong>: No clear correlation between a specific cell wall-related phenotype, or a combination of them, and improved release of polysaccharides by yeast random mutants could be established, and not all strains identified by in vitro assays as mannoprotein overproducing mutants were found positive for mannoprotein release in industrial conditions. Nevertheless, UV mutagenesis, combined with Concanavalin a detection, seems to be a viable way to improve mannoprotein release by industrial wine yeast strains.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This study is one of the few recent reports on genetic improvement of wine yeast strains by non-recombinant genetic tools. It shows that mannoprotein release can be genetically improved and, for the first time, describes a successful selection procedure for such a complex character. These strains are potentially useful for the improvement of mannoprotein-related characters of white and red wines.</p>

BACKGROUND: the mechanism of action of antimicrobial peptides (AMP) was initially correlated with peptide membrane permeation properties. However, recent evidences indicate that action of a number of AMP is more complex and involves specific interactions at cell envelopes or with intracellular targets. In this study, a genomic approach was undertaken on the model yeast Saccharomyces cerevisiae to characterize the antifungal effect of two unrelated AMP. RESULTS: Two differentiated peptides were used: the synthetic cell-penetrating PAF26 and the natural cytolytic melittin. Transcriptomic analyses demonstrated distinctive gene expression changes for each peptide. Quantitative RT-PCR confirmed differential expression of selected genes. Gene Ontology (GO) annotation of differential gene lists showed that the unique significant terms shared by treatment with both peptides were related to the cell wall (CW). Assays with mutants lacking CW-related genes including those of MAPK signaling pathways revealed genes having influence on sensitivity to peptides. Fluorescence microscopy and flow cytometry demonstrated PAF26 interaction with cells and internalization that correlated with cell killing in sensitive CW-defective mutants such as Δecm33 or Δssd1. GO annotation also showed differential responses between peptides, which included ribosomal biogenesis, ARG genes from the metabolism of amino groups (specifically induced by PAF26), or the reaction to unfolded protein stress. Susceptibility of deletion mutants confirmed the involvement of these processes. Specifically, mutants lacking ARG genes from the metabolism of arginine pathway were markedly more resistant to PAF26 and had a functional CW. In the deletant in the arginosuccinate synthetase (ARG1) gene, PAF26 interaction occurred normally, thus uncoupling peptide interaction from cell killing. The previously described involvement of the glycosphingolipid gene IPT1 was extended to the peptides studied here. CONCLUSIONS: Reinforcement of CW is a general response common after exposure to distinct AMP, and likely contributes to shield cells from peptide interaction. However, a weakened CW is not necessarily indicative of a higher sensitivity to AMP. Additional processes modulate susceptibility to specific peptides, exemplified in the involvement of the metabolism of amino groups in the case of PAF26. The relevance of the response to unfolded protein stress or the sphingolipid biosynthesis, previously reported for other unrelated AMP, was also independently confirmed.

Peptides and small proteins exhibiting antimicrobial activity have been isolated from many organisms ranging from insects to humans, including plants. Their role in defense is established, and their use in agriculture was already being proposed shortly after their discovery. However, some natural peptides have undesirable properties that complicate their application. Advances in peptide synthesis and high-throughput activity screening have made possible the de novo and rational design of novel peptides with improved properties. This review summarizes findings in the identification and design of short antimicrobial peptides with activity against plant pathogens, and will discuss alternatives for their heterologous production suited to plant disease control. Recent studies suggest that peptide antimicrobial action is not due solely to microbe permeation as previously described, but that more subtle factors might account for the specificity and absence of toxicity of some peptides. The elucidation of the mode of action and interaction with microbes will assist the improvement of peptide design with a view to targeting specific problems in agriculture and providing new tools for plant protection.

Short antimicrobial peptides represent an alternative to fight pathogen infections. PAF26 is a hexapeptide identified previously by a combinatorial approach against the fungus Penicillium digitatum and shows antimicrobial properties towards certain phytopathogenic fungi. In this work, PAF26 was used as lead compound and its properties were compared with two series of derivatives, obtained by either systematic alanine substitution or N-terminal amino acid addition. The alanine scan approach underlined the optimized sequence of PAF26 in terms of potency and permeation capability, and also the higher contribution of the cationic residues to these properties. The N-terminal addition of amino acids resulted in new heptapeptides with variations in their antimicrobial characteristics, and very low cytolysis to human red blood cells. Positive (Arg or Lys) and aromatic (Phe or Trp) residue addition increased broad spectrum activity of PAF26. Noteworthy, addition of selected residues had specific effects on the properties of derivatives of PAF26.

The objective of this study was to investigate and compare the in vitro efficacy and in vivo potential of eight distinct short antimicrobial peptides to control the postharvest green mold disease of oranges caused by the fungus Penicillium digitatum. The L-amino acid versions of the four peptides PAF26, PAF38, PAF40, and BM0, previously obtained by combinatorial approaches, were examined. The study included two antibacterial peptides formerly identified by rational design, BP15 and BP76, and it is demonstrated that they also have in vitro antifungal properties. The natural antimicrobial peptides melittin and indolicidin were also selected for comparison, due to their well-known properties and modes of action. In vitro and in vivo results indicated differential behaviors among peptides, regarding the inhibitory potency in growth media, selectivity against distinct microorganisms, fungicidal activity towards nongerminated conidia of P. digitatum, and efficacy in fruit inoculation assays. Interestingly, a high in vitro inhibitory activity did not necessarily associate with an effective control of fruit infection by P. digitatum. The short tryptophan-rich cationic peptides PAF26, PAF38, PAF40, and BM0 were lethal to conidia of P. digitatum, and this property is correlated with better protection in the decay control test.

AIM: to evaluate the activity against fungal phytopathogens of two synthetic peptides derived from the protein bovine lactoferricin: the antibacterial active core of six amino acid residues (LfcinB(20-25)) and an extension of 15 amino acids (LfcinB(17-31)). METHODS AND RESULTS: in vitro activity against fungal pathogens was determined and compared with that against model micro-organisms. Activity was demonstrated against fungi of agronomic relevance. Distinct antimicrobial properties in vitro were found for the two peptides. LfcinB(17-31) had growth inhibitory activity higher than LfcinB(20-25). However, LfcinB(17-31) was not fungicidal to quiescent conidia of Penicillium digitatum at the concentrations assayed, while LfcinB(20-25) killed conidia more efficiently. Microscopical observations showed that the mycelium of P. digitatum treated with LfcinB(17-31) developed alterations of growth, sporulation and chitin deposition, and permeation of hyphal cells. In experimental inoculations of mandarins, both peptides showed limited protective effect against the disease caused by P. digitatum. CONCLUSIONS: LfcinB(20-25) and LfcinB(17-31) peptides were shown to have antimicrobial activity against plant pathogenic filamentous fungi, with distinct properties and mode of action. SIGNIFICANCE AND IMPACT OF THE STUDY: LfcinB(20-25) and LfcinB(17-31) peptides offer novel alternatives to develop resistant plants by molecular breeding.

The small antimicrobial peptide PAF26 (Ac-RKKWFW-NH(2)) has been identified by a combinatorial approach and shows preferential activity toward filamentous fungi. In this work, we investigated the mode of action and inhibitory effects of PAF26 on the fungus Penicillium digitatum. The dye Sytox Green was used to demonstrate that PAF26 induced cell permeation. However, microscopic observations showed that sub-MIC concentrations of PAF26 produced both alterations of hyphal morphology (such as altered polar growth and branching) and chitin deposition in areas of no detectable permeation. Analysis of dose-response curves of inhibition and permeation suggested that growth inhibition is not solely a consequence of permeation. In order to shed light on the mode of PAF26 action, its antifungal properties were compared with those of melittin, a well-known pore-forming peptide that kills through cytolysis. While the 50% inhibitory concentrations and MICs of the two peptides against P. digitatum mycelium were comparable, they differed markedly in their fungicidal activities toward conidia and their hemolytic activities toward human red blood cells. Kinetic studies showed that melittin quickly induced Penicillium cell permeation, while PAF26-induced Sytox Green uptake was significantly slower and less efficient. Therefore, the ultimate growth inhibition and morphological alterations induced by PAF26 for P. digitatum are not likely a result of conventional pore formation. Fluorescently labeled PAF26 was used to demonstrate its specific in vivo interaction and translocation inside germ tubes and hyphal cells, at concentrations as low as 0.3 muM (20 times below the MIC), at which no inhibitory, morphological, or permeation effects were observed. Interestingly, internalized PAF26 could bind to cellular RNAs, since in vitro nonspecific RNA binding activity of PAF26 was demonstrated by electrophoretic mobility shift assays. We propose that PAF26 is a short, de novo-designed penetratin-type peptide that has multiple detrimental effects on target fungi, which ultimately result in permeation and killing.