A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value

As fungal infections are becoming more prevalent in the medical or agricultural fields, novel and more efficient antifungal agents are badly needed. Within the scope of developing new strategies for the management of fungal infections, antifungal compounds that target essential fungal cell wall components are highly preferable. Ideally, newly developed antimycotics should also combine major aspects such as sustainability, high efficacy, limited toxicity and low costs of production. A naturally derived molecule that possesses all the desired characteristics is the antifungal protein (AFP) secreted by the filamentous ascomycete Aspergillus giganteus. AFP is a small, basic and cysteine-rich peptide that exerts extremely potent antifungal activity against human- and plant-pathogenic fungi without affecting the viability of bacteria, yeast, plant and mammalian cells. This review summarises the current knowledge of the structure, mode of action and expression of AFP, and highlights similarities and differences concerning these issues between AFP and its related proteins from other Ascomycetes. Furthermore, the potential use of AFP in the combat against fungal contaminations and infections will be discussed.     

The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells

The antifungal protein AFP is a small, cystein-rich protein secreted by the imperfect ascomycete Aspergillus giganteus. The protein efficiently inhibits the growth of filamentous fungi, including a variety of serious human and plant pathogens mainly of the genera Aspergillus and Fusarium, whereas AFP does not affect the growth of yeast and bacteria. This restricted susceptibility range makes it very attractive for medical or biotechnological use to combat fungal infection and contamination. We, therefore, analyzed whether AFP affects the growth or function of a number of mammalian cells. Here we show that the protein neither provokes any cytotoxic effects on human endothelial cells isolated from the umbilical vein nor activates the immune system. Moreover, potassium currents of neurons and astrocytes do not change in the presence of AFP and neither excitatory processes nor the intracellular calcium homeostasis of cultured skeletal muscle myotubes are affected by AFP. Our data, therefore, suggest that AFP is indeed a promising candidate for the therapeutic or biotechnological use as a potential antifungal agent.     

Filamentous fungi found in Atta sexdens rubropilosa colonies after treatment with different toxic bait formulations

Leaf‐cutting ants maintain a symbiotic relationship with basidiomycetous fungi cultivated as food. Here, we profiled the non‐symbiotic filamentous fungi in laboratory nests of Atta sexdens rubropilosa submitted to treatments with different toxic bait formulations (using the insecticide sulfluramide as the active ingredient). After treatment, several filamentous fungi were found in different nest compartments. Culture‐dependent techniques recovered a total of 93 fungal isolates comprising 10 genera, 11 species and four unidentified fungi. The genus Penicillium was prevalent in both control and insecticide treatments. Overall, the majority of fungal isolates obtained in this study are commonly found in soil. Escovopsis spp., the specialized parasite of the ant‐fungus mutualism was only recorded in the fungus gardens of nests submitted to the toxic treatments. Moreover, no correlation was found regarding the presence of fungi in the different nest compartments (chi‐square, P > 0.4182). This study reveals that Escovopsis spp. is not the only fungus to overgrow the fungus garden of debilitated nests, thus adding more evidence on the possible negative impacts of such alien fungi. As suggested by previous studies, fast‐growing filamentous fungi likely overgrow the fungus garden in such conditions.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi

The antifungal protein AFP from Aspergillus giganteus is highly effective in restricting the growth of major human- and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of Fusarium oxysporum and Aspergillus oryzae showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an Aspergillus niger reporter strain in which the cell wall integrity pathway was strongly induced by AFP.     

Biotechnologically relevant enzymes and proteins

The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.     

Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.     

Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins

Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of β-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.     

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus

Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.     

The effect of Galleria mellonella apolipophorin III on yeasts and filamentous fungi

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal β-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.     

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) producing ROS affects growth and viability of filamentous fungi

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) causes intracellular superoxide production and oxidative stress and enhances the susceptibility of Saccharomyces cerevisiae, Candida albicans, and C.?glabrata cells to cycloheximide, 5-fluorocytosine, and azole antimycotic drugs. Here, we demonstrate the antifungal activity of CTBT against 14 tested filamentous fungi. CTBT prevented spore germination and mycelial proliferation of Aspergillus niger and the pathogenic Aspergillus fumigatus. The action of CTBT is fungicidal. CTBT increased the formation of reactive oxygen species in fungal mycelium as detected by 2',7'-dichlorodihydrofluorescein diacetate and reduced the radial growth of colonies in a dose-dependent manner. Co-application of CTBT and itraconazole led to complete inhibition of fungal growth at dosages lower than the chemicals alone. Antifungal and chemosensitizing activities of CTBT in filamentous fungi may be useful in combination treatments of infections caused by drug-resistant fungal pathogens.     

真菌鞘糖脂的生物学功能及应用研究进展

丝状真菌作为一类重要的微生物，被广泛应用于发酵食品、工业酶和次生代谢物等工业生产中。真菌鞘糖脂主要由鞘氨醇、脂肪酸链和特殊的极性基团组成，根据极性基团的不同，分为中性鞘糖脂和酸性鞘糖脂两大类。鞘糖脂不仅参与真菌生长、细胞分化、增殖、细胞凋亡、逆境胁迫等重要生理活动，中性鞘糖脂还可作为功能性医药用品、化妆品和保健食品的重要活性组分。本文论述了真菌鞘糖脂的主要种类、结构、生物合成途径和及其参与丝状真菌生长、分化和响应逆境胁迫的生物学功能；探讨了真菌中性鞘糖脂作为抗菌肽的靶点和酸性鞘糖脂在开发抗真菌药物中的应用；同时还综述了中性鞘糖脂作为化妆品的保湿成分或保健食品的功能成分，在改善皮肤屏障功能和预防特应性皮炎中的重要作用的相关研究进展，尤其是来源于曲霉的中性鞘糖脂，可显著增强皮肤屏障功能，并可作为益生元预防肠道损伤；另外还探讨了曲霉尤其是米曲霉作为开发中性鞘糖脂生物资源的优势。     

Characterization of the<Emphasis Type="Italic"> Penicillium chrysogenum</Emphasis> antifungal protein PAF

The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF (Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.     

Fungi isolated from Sclerotia ofSclerotium cepivorum and from soil and their effects upon the pathogen

Summary Data are presented on the antagonistic effects of the fungi isolated from sclerotia ofSclerotium cepivorum and from nonrhizosphere soil taken from around the roots of infected onions upon mycelial growth and sclerotial germination ofS. cepivorum. Most of the isolated fungi especiallyPenicillium species were antagonistic to mycelial growth. Sclerotial germination was slightly inhibited by diffusates of these fungal isolates. Testing the antifungal effect of someAllium extracts against the fungal isolates by the inhibition zone method showed that garlic extract has the greatest antifungal effects and onion extract is the least potent. However, spore germination tests indicated that onion extract completely inhibits the spore germination of all test fungi. The role of host-plant extracts in stimulating sclerotial germination is discussed.     

Survival strategies of yeast and filamentous fungi against the antifungal protein AFP

The activities of signaling pathways are critical for fungi to survive antifungal attack and to maintain cell integrity. However, little is known about how fungi respond to antifungals, particularly if these interact with multiple cellular targets. The antifungal protein AFP is a very potent inhibitor of chitin synthesis and membrane integrity in filamentous fungi and has so far not been reported to interfere with the viability of yeast strains. With the hypothesis that the susceptibility of fungi toward AFP is not merely dependent on the presence of an AFP-specific target at the cell surface but relies also on the cell's capacity to counteract AFP, we used a genetic approach to decipher defense strategies of the naturally AFP-resistant strain Saccharomyces cerevisiae. The screening of selected strains from the yeast genomic deletion collection for AFP-sensitive phenotypes revealed that a concerted action of calcium signaling, TOR signaling, cAMP-protein kinase A signaling, and cell wall integrity signaling is likely to safeguard S. cerevisiae against AFP. Our studies uncovered that the yeast cell wall gets fortified with chitin to defend against AFP and that this response is largely dependent on calcium/Crz1p signaling. Most importantly, we observed that stimulation of chitin synthesis is characteristic for AFP-resistant fungi but not for AFP-sensitive fungi, suggesting that this response is a successful strategy to protect against AFP. We finally propose the adoption of the damage-response framework of microbial pathogenesis for the interactions of antimicrobial proteins and microorganisms in order to comprehensively understand the outcome of an antifungal attack.     

Influence of fungi on growth and survival of Onychiurus armatus (Collembola) in a metal polluted soil

Summary The influence of food quantity and quality on growth and survival of Onychiurus armatus (Tullb.) in metal polluted environments has been investigated in laboratory experiments. The Collembola was reared on five species of fungi isolated from a metal polluted soil close to a brass mill in SE Sweden.Survival of O. armatus was improved when fungal biomass was continuously added in a polluted mor (1,300 ppm Zn and 200 ppm Cu), and when specimens were fed metal polluted fungi for 1, 3 and 7 days a week, only those that were starved had increased mortality. Allometric growth, on the other hand, was significantly reduced when Collembola was given surplus of metal polluted fungi, whereas growth losses caused by metals were offset by protein rich food. Hence, sufficient food quantities alone could overcome mortality losses but not growth retardation in a metal polluted environment.Feeding preference of O. armatus was not determined by the protein content of the fungi although this was beneficial for growth. Metals changed the relative palatability of fungal species, but one of the metal tolerant species, Paecilomyces farinosus, which was also protein rich, remained reasonably attractive for O. armatus also when it was metal polluted. The mechanisms by which growth and survival of O. armatus were promoted by a combination of protein and Zn/Cu rich fungi seemed to be crucial in understanding the fate of a population of this species in a metal polluted soil.     

Growth and reproduction of fungal feeding Collembola as affected by fungal species,melanin and mixed diets

Fungal feeding soil invertebrates feed on a wide spectrum of fungal species suggesting that mixed diets increase fitness. We investigated relationships between food preferences for seven saprophytic fungal species/forms and fitness parameters (mortality, growth, time to reproduction, reproduction, egg size) in two Collembola species, Folsomia candida and Protaphorura armata. The fungal species/forms studied included the wild type and a melanin-deficient form of Aspergillus fumigatus to investigate the role of melanin in collembolan nutrition. Also, three mixed diets consisting of a preferred fungal species (Cladosporium cladosporioides) and species of intermediate or low food quality were investigated. Both Collembola species preferred similar fungal species/forms as food. Food preference generally matched fitness parameters, i.e. growth and reproduction of Collembola was at a maximum when feeding on preferred fungi. This was not the case for A. fumigatus. The wild type and the melanin-deficient form ranked among the least preferred fungi. Growth and reproduction of Collembola were low when feeding on the wild type but high when feeding on the melanin-deficient form indicating that the Collembola misjudged the food quality of the latter in the preference tests. The results show for the first time that genes driving melanin syntheses (pksP) strongly affect the food quality of fungi for fungal feeding invertebrates. Feeding on mixed diets generally increased growth and reproduction of Collembola except when the diets included toxic species (Penicillium sp.). The results support the nutrient balance hypothesis and also show that the detection of toxic species in the diet is important. They indicate that the widespread generalist feeding mode of Collembola maximizes fitness if toxic fungal species are avoided. The fitness parameters growth, reproduction and time until onset of reproduction were correlated closely but egg volume, which also varied with fungal diet, correlated poorly with the other fitness parameters. Variation in egg size with fungal diet shows that the diet of Collembola may have transgenerational effects.     

Fruit,flies and filamentous fungi – experimental analysis of animal–microbe competition using Drosophila melanogaster and Aspergillus mould as a model system

In addition to their fundamental role in nutrient recycling, saprobiotic microorganisms may be considered as typical consumers of food‐limited ephemeral resource patches. As such, they may be engaged in inter‐specific competition with saprophagous animals feeding from the same resource. Bacteria and filamentous fungi are known to synthesise secondary metabolites, some of which are toxic and have been proposed to deter or harm animals. The microorganisms may, however, also be negatively affected if saprophagous animals do not avoid microbe‐laden resources but feed in the presence of microbial competitors. We hypothesised that filamentous fungi compete with saprophagous insects, whereby secondary metabolites provide a chemical shield against the insect competitors. For testing this, we developed a new ecological model system representing a case of animal–microbe competition between saprobiotic organisms, comprising Drosophila melanogaster and species of the fungus Aspergillus (A. nidulans, A. fumigatus, A. flavus). Infestation of Drosophila breeding substrate with proliferating fungal colonies caused graduated larval mortality that strongly depended on mould species and colony age. Confrontation with conidiospores only, did not result in significant changes in larval survival, suggesting that insect death may not be ascribed to pathogenic effects. When confronted with colonies of transgenic fungi that lack the ability to express the global secondary metabolite regulator LaeA (ΔlaeA), larval mortality was significantly reduced compared to the impact of the wild type strains. Yet, also in the ΔlaeA strains, inter‐specific variation in the influence on insect growth occurred. Competition with Drosophila larvae impaired fungal growth, however, wild type colonies of A. nidulans and A. flavus recovered more rapidly from insect competition than the corresponding ΔlaeA mutants (not in A. fumigatus). Our findings provide genetic evidence that toxic secondary metabolites synthesised by saprotrophic fungi may serve as a means to combat insect competitors. Variation in the ability of LaeA to control expression of various secondary metabolite gene clusters might explain the observed species‐specific variation in Drosophila–Aspergillus competition.     

Studies on the synergistic effect of amphotericin B and 5-fluorocytosine on the growth rate of single hyphae ofAspergillus fumigatus by a biocell-tracer system

The biocell-tracer system is a microscopical system to measure the growth rate of a single fungal hypha. The synergistic effect of amphotericin B (AMPH) and 5-fluorocytosine (5-FC) on the growth of hyphae ofAspergillus fumigatus was studied by using this system. Although neither 2µg/ml of AMPH nor 250µg/ml of 5-FC alone showed any effect on the hyphal growth, their combination at these concentrations showed a distinct inhibitory activity. The biocell-tracer system is useful for antifungal activity testing in filamentous fungi.     

Culturable Fungi of Stored ‘Golden Delicious’ Apple Fruits: A One-Season Comparison Study of Organic and Integrated Production Systems in Switzerland

The effects of organic and integrated production systems on the culturable fungal microflora of stored apple fruits from five matched pairs of certified organic and integrated ‘Golden Delicious’ farms were studied at five representative production sites in Switzerland. Isolated fungi were identified morphologically. Colonization frequency (percentage of apples colonized), abundance (colony numbers), and diversity (taxon richness) were assessed for each orchard. The standard quality of the stored fruits was comparable for both organic and integrated apples and complied with national food hygiene standards. Yeasts (six taxa) and the yeast-like fungus Aureobasidium pullulans were the dominant epiphytes, filamentous fungi (21 taxa) the dominant endophytes. The most common fungi occurred at all sites and belonged to the “white” and “pink” yeasts, yeast-like A. pullulans, filamentous fungi Cladosporium spp., Alternaria spp., and sterile filamentous fungi. Canonical correspondence analysis of the total fungal community revealed a clear differentiation among production systems and sites. Compared to integrated apples, organic apples had significantly higher frequencies of filamentous fungi, abundance of total fungi, and taxon diversity. The effects of the production system on the fungal microflora are most likely due to the different plant protection strategies. The incidence of potential mycotoxin producers such as Penicillium and Alternaria species was not different between production systems. We suggest that higher fungal diversity may generally be associated with organic production and may increase the level of beneficial and antagonistically acting species known for their potential to suppress apple pathogens, which may be an advantage to organic apples, e.g., in respect to natural disease control.