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.
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.
SummaryRecent 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 mM). 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 mM), PAF26 internalization was energyindependent, 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.
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 M (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.
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