The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis.

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

The Plant Defensin NaD1 Enters the Cytoplasm of Candida Albicans via Endocytosis.

Antimicrobial peptides are widespread in nature and are produced by many organisms as a first line of defence against pathogens. These peptides have a broad range of biological activities, such as antibacterial or antifungal activities and act with varied mechanisms of action. A large number of the peptides are amphipathic α-helices which act by disrupting plasma membranes and allowing leakage of intracellular contents. However, some peptides have more complex mechanisms of action that require internalisation into the target organisms' cytoplasm. The method by which these peptides enter the cytoplasm varies, with some requiring the energy dependent processes of endocytosis or polyamine transport and others entering via passive transport. Here we describe the mechanism that the antimicrobial peptide, the plant defensin NaD1, uses to transverse the fungal membrane and gain access to the fungal cytoplasm. By inhibiting ATP synthesis and using an inhibitor of actin polymerisation, we show that NaD1 is internalised into C. albicans yeast cells by the energy-dependent process of endocytosis.

Synergistic Activity between Two Antifungal Proteins, the Plant Defensin NaD1 and the Bovine Pancreatic Trypsin Inhibitor.

Defensins are a large family of small, cationic, cysteine-rich proteins that are part of the defense arsenal that plants use for protection against potentially damaging fungal infections. The plant defensin NaD1 from Nicotiana alata is a potent antifungal protein that inhibits growth and kills a variety of fungal pathogens that affect both plant and animal (human) hosts. Some serine protease inhibitors have also been reported to be antifungal molecules, while others have no inhibitory activity against fungi. Here we describe the synergistic activity of the plant defensin NaD1 with a selection of serine protease inhibitors against the plant pathogens Fusarium graminearum and Colletotrichum graminicola and the animal pathogen Candida albicans. The synergistic activity was not related to the protease inhibitory activity of these molecules but may arise from activation of fungal stress response pathways. The bovine pancreatic trypsin inhibitor (BPTI) displayed the most synergy with NaD1. BPTI also acted synergistically with several other antifungal molecules. The observation that NaD1 acts synergistically with protease inhibitors provides the foundation for the design of transgenic plants with improved resistance to fungal disease. It also supports the possibility of naturally occurring accessory factors that function to enhance the activity of innate immunity peptides in biological systems. IMPORTANCE This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them individually. This study identified synergy between an antifungal peptide and a group of peptides that do not affect fungal growth in vitro. This provides the foundation for generation of transgenic plants with increased resistance to fungal disease and identification of antifungal accessory factors that enhance the activity of innate immune molecules but do not have an antifungal effect on their own.

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-ß motif which consists of an α helix and a triple-stranded ß-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake.

Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg(2+) levels. Populations treated with BPTI had fewer cells in S-phase of the cell cycle and a corresponding increase of cells in G(0)/G(1) and G(2) phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.

Activation of stress signalling pathways enhances tolerance of fungi to chemical fungicides and antifungal proteins.

Fungal disease is an increasing problem in both agriculture and human health. Treatment of human fungal disease involves the use of chemical fungicides, which generally target the integrity of the fungal plasma membrane or cell wall. Chemical fungicides used for the treatment of plant disease, have more diverse mechanisms of action including inhibition of sterol biosynthesis, microtubule assembly and the mitochondrial respiratory chain. However, these treatments have limitations, including toxicity and the emergence of resistance. This has led to increased interest in the use of antimicrobial peptides for the treatment of fungal disease in both plants and humans. Antimicrobial peptides are a diverse group of molecules with differing mechanisms of action, many of which remain poorly understood. Furthermore, it is becoming increasingly apparent that stress response pathways are involved in the tolerance of fungi to both chemical fungicides and antimicrobial peptides. These signalling pathways such as the cell wall integrity and high-osmolarity glycerol pathway are triggered by stimuli, such as cell wall instability, changes in osmolarity and production of reactive oxygen species. Here we review stress signalling induced by treatment of fungi with chemical fungicides and antifungal peptides. Study of these pathways gives insight into how these molecules exert their antifungal effect and also into the mechanisms used by fungi to tolerate sub-lethal treatment by these molecules. Inactivation of stress response pathways represents a potential method of increasing the efficacy of antifungal molecules.

Identification and mechanism of action of the plant defensin NaD1 as a new member of the antifungal drug arsenal against Candida albicans.

In recent decades, pathogenic fungi have become a serious threat to human health, leading to major efforts aimed at characterizing new agents for improved treatments. Promising in this context are antimicrobial peptides produced by animals and plants as part of innate immune systems. Here, we describe an antifungal defensin, NaD1, with activity against the major human pathogen Candida albicans, characterize the mechanism of killing, and identify protection strategies used by the fungus to survive defensin treatment. The mechanism involves interaction between NaD1 and the fungal cell surface followed by membrane permeabilization, entry into the cytoplasm, hyperproduction of reactive oxygen species, and killing induced by oxidative damage. By screening C. albicans mutant libraries, we identified that the high-osmolarity glycerol (HOG) pathway has a unique role in protection against NaD1, while several other stress-responsive pathways are dispensable. The involvement of the HOG pathway is consistent with induction of oxidative stress by NaD1. The HOG pathway has been reported to have a major role in protection of fungi against osmotic stress, but our data indicate that osmotic stress does not contribute significantly to the adverse effects of NaD1 on C. albicans. Our data, together with previous studies with human beta-defensins and salivary histatin 5, indicate that inhibition of the HOG pathway holds promise as a broad strategy for increasing the activity of antimicrobial peptides against C. albicans.