The infectious propagules of Aspergillus fumigatus are coated with antimicrobial peptides.

Fungal spores are unique cells that mediate dispersal and survival in the environment. For pathogenic fungi encountering a susceptible host, these specialised structures may serve as infectious particles. The main causative agent of the opportunistic disease aspergillosis, Aspergillus fumigatus, produces asexual spores, the conidia, that become dissipated by air flows or water currents but also serve as propagules to infect a susceptible host. We demonstrate that the defX gene of this mould encodes putative antimicrobial peptides resembling cysteine-stabilised (CS)αß defensins that are expressed in a specific spatial and temporal manner in the course of asexual spore formation. Localisation studies on strains expressing a fluorescent proxy or tagged defX alleles expose that these antimicrobial peptides are secreted to coat the conidial surface. Deletion mutants reveal that the spore-associated defX gene products delay the growth of Gram-positive Staphylococcus aureus and demonstrate that the defX gene and presumably its encoded spore-associated defensins confer a growth advantage to the fungal opponent over bacterial competitors. These findings have implications with respect to the ecological niche of A. fumigatus that serves as a 'virulence school' for this human pathogenic mould; further relevance is given for the infectious process resulting in aspergillosis, considering competition with the host microbiome or co-infecting microorganisms to break colonisation resistance at host surfaces.

Surface display of HFBI and DewA hydrophobins on Saccharomyces cerevisiae modifies tolerance to several adverse conditions and biocatalytic performance.

Hydrophobins are relatively small proteins produced naturally by filamentous fungi with interesting biotechnological and biomedical applications given their self-assembly capacity, efficient adherence to natural and artificial surfaces, and to introduce modifications on the hydrophobicity/hydrophilicity of surfaces. In this work we demonstrate the efficient expression on the S. cerevisiae cell surface of class II HFBI of Trichoderma reesei and class I DewA of Aspergillus nidulans, a hydrophobin not previously exposed, using the Yeast Surface Display a-agglutinin (Aga1-Aga2) system. We show that the resulting modifications affect surface properties, and also yeast cells' resistance to several adverse conditions. The fact that viability of the engineered strains increases under heat and osmotic stress is particularly interesting. Besides, improved biocatalytic activity toward the reduction of ketone 1-phenoxypropan-2-one takes place in the reactions carried out at both 30 °C and 40 °C, within a concentration range between 0.65 and 2.5 mg/mL. These results suggest interesting potential applications for hydrophobin-exposing yeasts. KEY POINTS : • Class I hydrophobin DewA can be efficiently exposed on S. cerevisiae cell surfaces. • Yeast exposure of HFBI and DewA increases osmotic and heat resistance. • Engineered strains show modified biocatalytic behavior.

Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins.

Fungal hydrophobins are small amphiphilic proteins that can be used for coatings on hydrophilic and hydrophobic surfaces. Through the formation of monolayers, they change the hydrophobicity of a given surface. Especially, the class I hydrophobins are interesting for biotechnology, because their layers are stable at high temperatures and can only be removed with strong solvents. These proteins self-assemble into monolayers under physiological conditions and undergo conformational changes that stabilize the layer structure. Several studies have demonstrated how the fusion of hydrophobins with short peptides allows the specific modification of the properties of a given surface or have increased the protein production levels through controlled localization of hydrophobin molecules inside the cell. Here, we fused the Aspergillus nidulans laccase LccC to the class I hydrophobins DewA and DewB and used the fusion proteins to functionalize surfaces with immobilized enzymes. In contrast to previous studies with enzymes fused to class II hydrophobins, the DewA-LccC fusion protein is secreted into the culture medium. The crude culture supernatant was directly used for coatings of glass and polystyrene without additional purification steps. The highest laccase surface activity was achieved after protein immobilization on modified hydrophilic polystyrene at pH 7. This study presents an easy-to-use alternative to classical enzyme immobilization techniques and can be applied not only for laccases but also for other biotechnologically relevant enzymes. IMPORTANCE: Although fusion with small peptides to modify hydrophobin properties has already been performed in several studies, fusion with an enzyme presents a more challenging task. Both protein partners need to remain in active form so that the hydrophobins can interact with one another and form layers, and so the enzyme (e.g., laccase) will remain active at the same time. Also, because of the amphiphilic nature of hydrophobins, their production and purification remain challenging so far and often include steps that would irreversibly disrupt most enzymes. In our study, we present the first functional fusion proteins of class I hydrophobins from A. nidulans with a laccase. The resulting fusion enzyme is directly secreted into the culture medium by the fungus and can be used for the functionalization of hard surfaces.

Fungal hydrophobins render stones impermeable for water but keep them permeable for vapor.

The conservation of architectural heritage is a big challenge in times with increasing air pollution with aggressive gases. A second major threat to buildings is the combination of water and air contaminants which may be used by microorganisms for their metabolism. Hence, myriads of different bacteria and fungi populate stone surfaces and penetrate into the fine pores and cracks. Whereas epoxid-based paintings (or other paintings) may protect the coated surfaces from water and aggressive gases, these chemicals seal the stone surface and prevent also the evaporation of vapor from the inside of the buildings. Here, we tested a natural, fungal protein-based coating method. Fungi use small, amphiphilic proteins to turn their surfaces hydrophobic. We found that Aspergillus nidulans hydrophobin DewA and Trichoderma reesei HFBI confer hydrophobicity to stones but keep their pores open. The effect resembles "Gore-tex" fabric material.

Comparative analysis of surface coating properties of five hydrophobins from Aspergillus nidulans and Trichoderma reseei.

Fungal hydrophobins are small amphiphilic proteins that self-assemble into monolayers on hydrophobic:hydrophilic interfaces and can be used for surface coatings. Because e.g. Aspergillus nidulans contains six different hydrophobins, it is likely that they have different properties and are used for different "applications" in the fungus. We established a method for recombinant production of different class hydrophobins in Escherichia coli. We produced DewA, DewC, DewD, DewE from A. nidulans and HFBI from Trichoderma reesei and compared surface coating properties of these hydrophobins. All tested proteins formed coatings on glass, strongly increasing the hydrophobicity of the surface, and showed emulsion-stabilizing properties. But whereas the typical class I hydrophobin DewA formed the most stable coating on glass, the intermediate class hydrophobins DewE and DewD were more effective in stabilization of oil:water emulsions. This work gives insights into correlations between structural characteristics of hydrophobins and their behaviour as surface binding agents. It could help with the clarification of their biological functions and lead to novel biotechnological applications.

Improving the performance of a biofuel cell cathode with laccase-containing culture supernatant from Pycnoporus sanguineus.

Laccases are multicopper oxidoreductases that can be used in biofuel cells to improve cathode performance by cathodic oxygen reduction. Here we present a laccase from the ligninolytic white-rot fungus Pycnoporus sanguineus that, in contrast to the Trametes versicolor laccase, can be produced in the absence of inducers in a standard culture medium. After 7days of cultivation the activity of this laccase in culture supernatant reached 2.5U/ml, which is high enough for direct application of the supernatant in biofuel cells. The highest current density of 115.0±3.5µA/cm(2) at 400mV vs. SCE was obtained at pH 5 with a buckypaper cathode with a laccase-containing culture supernatant. The enzyme also showed electrocatalytic activity at pH 6 and 7. These results not only present a new cost-efficient laccase for improving cathode performance, but also show that new laccases with different catalytic properties can be suitable for biofuel cells.