Multifunctional hydrophobin-like protein (HFB-NJ1): A versatile solution for wastewater treatment.

As wastewater contains a variety of contaminating bacteria and oily residues, there is an urgent need for environmentally safe bactericidal agents and surfactants which can be applied for wastewater treatment. The present study emphasizes on the potential of hydrophobin-like protein (HFB-NJ1) extracted from sporulating mycelia of Aspergillus sp. NJ1 for wastewater treatment. The purified HFB-NJ1, depicted the presence of one single protein band of molecular size approximately 11-12 kDa on silver-stained SDS-PAGE gel. HFB-NJ1 also presented properties such as surface modification of glass and stable emulsification of sunflower oil. HFB-NJ1 depicted exceptional antibacterial activity against bacterial pathogens such as Bacillus subtilis and Pseudomonas aeruginosa at low MIC of 0.5 µg/mL and 0.75 µg/mL respectively. Additionally, HFB-NJ1 depicted enhanced emulsification of various vegetable and petroleum-based oils (E24 > 80%). HFB-NJ1 effectively reduced gold ions, producing nanospheres with a size of 15.33 nm - a recognized antimicrobial agent. This study underscores the multifunctional attributes of HFB-NJ1, highlighting its efficacy in removing pathogenic bacteria, emulsifying organic compounds from wastewater, and demonstrating a reduction ability for nanoparticle synthesis.

Simple Method to Assess Foam Structure and Stability using Hydrophobin and BSA as Model Systems.

The properties and arrangement of surface-active molecules at air-water interfaces influence foam stability and bubble shape. Such multiscale-relationships necessitate a well-conducted analysis of mesoscopic foam properties. We introduce a novel automated and precise method to characterize bubble growth, size distribution and shape based on image analysis and using the machine learning algorithm Cellpose. Studying the temporal evolution of bubble size and shape facilitates conclusions on foam stability. The addition of two sets of masks, for tiny bubbles and large bubbles, provides for a high precision of analysis. A python script for analysis of the evolution of bubble diameter, circularity and dispersity is provided in the Supporting Information. Using foams stabilized by bovine serum albumin (BSA), hydrophobin (HP), and blends thereof, we show how this technique can be used to precisely characterize foam structures. Foams stabilized by HP show a significantly increased foam stability and rounder bubble shape than BSA-stabilized foams. These differences are induced by the different molecular structure of the two proteins. Our study shows that the proposed method provides an efficient way to analyze relevant foam properties in detail and at low cost, with higher precision than conventional methods of image analysis.

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris.

Fungal disease of mushroomCordyceps militaris (CM) caused byCalcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.

Function of a hydrophobin in growth and development, nitrogen regulation, and abiotic stress resistance of Ganoderma lucidum.

Fungal hydrophobins have many important physiological functions, such as maintaining hydrophobicity and affecting virulence, growth, and development. In Ganoderma lucidum, the molecular regulation mechanisms of hydrophobins in mushroom are unclear. In this study, we investigated a hydrophobin protein 1 (Hyd1) in G. lucidum, which belongs to the fungal Class I hydrophobins. The hyd1 gene was highly expressed during the formation of primordia, and expression was the lowest in fruiting bodies. Through the construction of hyd1 silenced strains, we found that primordia formation was not initiated in these strains. This finding indicated that Hyd1 played an important role in the development of G. lucidum. Second, AreA, a key transcription factor in nitrogen metabolism, negatively regulated the expression of hyd1. In an areA-silenced strain, the expression of hyd1 increased by ∼14-fold compared with that of the wild-type (WT) strain. Electrophoretic mobility shift assays (EMSA) indicated binding of AreA to the promoter of hyd1. Additionally, expression of hyd1 was determined in the presence of different nitrogen sources. Compared with that in the ammonia nitrogen source, the expression of hyd1 in nitrate nitrogen source significantly increased. Finally, we found that hyd1 plays important roles not only in nitrogen regulation but also in the resistance to other abiotic stresses. After silencing of hyd1, the resistance to heat, cell wall, and salt stresses decreased. Our findings reveal the important roles of Hyd1 in the development and resistance to abiotic stresses in G. lucidum and provide insights into the nitrogen regulation mechanism of hydrophobins in higher basidiomycetes.

Involvement of ionic interactions in self-assembly and resultant rodlet formation of class I hydrophobin RolA from Aspergillus oryzae.

Hydrophobins are small amphiphilic proteins that are conserved in filamentous fungi. They localized on the conidial surface to make it hydrophobic, which contributes to conidial dispersal in the air, and helps fungi to infect plants and mammals and degrade polymers. Hydrophobins self-assemble and undergo structural transition from the amorphous state to the rodlet (rod-like multimeric structure) state. However, it remains unclear whether the amorphous or rodlet state is biologically functional and what external factors regulate state transition. In this study, we analyzed the self-assembly of hydrophobin RolA of Aspergillus oryzae in detail and identified factors regulating this process. Using atomic force microscopy, we observed RolA rodlet formation over time, and determined "rodlet elongation rate" and "rodlet formation frequency." Changes in these kinetic parameters in response to pH and salt concentration suggest that RolA rodlet formation is regulated by the strength of ionic interactions between RolA molecules.

Features of disruption mutants of genes encoding for hydrophobin Vmh2 and Vmh3 in mycelial formation and resistance to environmental stress in Pleurotus ostreatus.

Hydrophobins, which are small-secreted proteins with both hydrophobic and hydrophilic parts, can self-assemble into an amphiphilic film at the air-water interface, helping the fungus to form aerial hyphae. In the agaricomycete Pleurotus ostreatus, more than 20 putative hydrophobin genes have been predicted. Of these, two hydrophobin genes, vmh2 and vmh3, are predominantly expressed in the vegetative mycelium. In this study, we focused on the functions of Vmh2 and Vmh3 in vegetative mycelia. Based on the observation of the mycelial cross-section by transmission electron microscopy and the disappearance time of water droplets on the mycelial surface, Vmh2 and Vmh3 were considered essential for the maintenance of the surface hydrophobicity of the mycelium. The Δvmh3 and Δvmh2Δvmh3 strains exhibited relatively slower aerial mycelia formation on a liquid medium, and no significant alteration was observed in Δvmh2 strains. Only the Δvmh3 and Δvmh2Δvmh3 strains grew slower than the wild-type strain under stress conditions involving SDS and H2O2 on agar plates. This study revealed possible distinct roles for these hydrophobins in stress resistance. These results suggest that Agaricomycetes, including P. ostreatus, have evolved to possess multiple different hydrophobins as a means of adapting to various environments.

Physiological function of hydrophobin Vmh3 in lignin degradation by white-rot fungus Pleurotus ostreatus.

Hydrophobins are small-secreted proteins comprising both hydrophobic and hydrophilic parts, that can self-assemble into an amphiphilic film at the air-liquid interface. More than 20 hydrophobin genes have been estimated in the white-rot fungus Pleurotus ostreatus. In our previous studies, three hydrophobin genes were shown to be predominantly expressed under ligninolytic conditions, and only vmh3 was downregulated in both the delignification-deficient mutant Δgat1 and Δhir1 strains. Here, we focused on the function of the hydrophobin Vmh3 to clarify its physiological role in lignin degradation. When the hyphae were observed by transmission electron microscopy, deletion of vmh3 resulted in the disappearance of black aggregates at the interface between the cell wall and outer environment. Deletion of vmh3 resulted in reduced hydrophobicity when 0.2% sodium dodecyl sulfate was dropped onto the mycelial surface. These results suggest that Vmh3 functions on the cell surface and plays a major role in mycelial hydrophobization. Furthermore, the Δvmh3 strain showed a marked delay in lignin degradation on beech wood sawdust medium, while the production of lignin-modifying enzymes was not reduced. This study demonstrated, for the first time, the possible effect of hydrophobin on lignin degradation by a white-rot fungus.

Hydrophobin Gene Cmhyd4 Negatively Regulates Fruiting Body Development in Edible Fungi Cordyceps militaris.

A deep understanding of the mechanism of fruiting body development is important for mushroom breeding and cultivation. Hydrophobins, small proteins exclusively secreted by fungi, have been proven to regulate the fruiting body development in many macro fungi. In this study, the hydrophobin gene Cmhyd4 was revealed to negatively regulate the fruiting body development in Cordyceps militaris, a famous edible and medicinal mushroom. Neither the overexpression nor the deletion of Cmhyd4 affected the mycelial growth rate, the hydrophobicity of the mycelia and conidia, or the conidial virulence on silkworm pupae. There was also no difference between the micromorphology of the hyphae and conidia in WT and ΔCmhyd4 strains observed by SEM. However, the ΔCmhyd4 strain showed thicker aerial mycelia in darkness and quicker growth rates under abiotic stress than the WT strain. The deletion of Cmhyd4 could promote conidia production and increase the contents of carotenoid and adenosine. The biological efficiency of the fruiting body was remarkably increased in the ΔCmhyd4 strain compared with the WT strain by improving the fruiting body density, not the height. It was indicated that Cmhyd4 played a negative role in fruiting body development. These results revealed that the diverse negative roles and regulatory effects of Cmhyd4 were totally different from those of Cmhyd1 in C. militaris and provided insights into the developmental regulatory mechanism of C. militaris and candidate genes for C. militaris strain breeding.

Curcumin-Encapsulated Fusion Protein-Based Nanocarrier Demonstrated Highly Efficient Epidermal Growth Factor Receptor-Targeted Treatment of Colorectal Cancer.

Curcumin, a polyphenol derived from turmeric, has multiple biological functions, such as anti-inflammatory, antioxidant, antibacterial and, above all, antitumor activity. Colorectal cancer is a common malignancy of the gastrointestinal tract with an extremely high mortality rate. However, the low bioavailability and poor targeting properties of curcumin generally limit its clinical application. In the present study, we designed a fusion protein GE11-HGFI as a nanodrug delivery system. The protein was connected by flexible linkers, inheriting the self-assembly properties of hydrophobin HGFI and the targeting ability of GE11. The data show that the encapsulation of curcumin by fusion protein GE11-HGFI can form uniform and stable nanoparticles with a size of only 80 nm. In addition, the nanocarrier had high encapsulation efficiency for curcumin and made it to release sustainably. Notably, the drug-loaded nanosystem selectively targeted colorectal cancer cells with high epidermal growth factor receptor expression, resulting in high aggregated concentrations of curcumin at tumor sites, thus showing a significant anticancer effect. These results suggest that the nanocarrier fusion protein has the potential to be a novel strategy for enhancing molecular bioactivity and drug targeting in cancer therapy.

Tomato Xylem Sap Hydrophobins Vdh4 and Vdh5 Are Important for Late Stages of Verticillium dahliae Plant Infection.

Verticillium dahliae causes economic losses to a wide range of crops as a vascular fungal pathogen. This filamentous ascomycete spends long periods of its life cycle in the plant xylem, a unique environment that requires adaptive processes. Specifically, fungal proteins produced in the xylem sap of the plant host may play important roles in colonizing the plant vasculature and in inducing disease symptoms. RNA sequencing revealed over 1500 fungal transcripts that are significantly more abundant in cells grown in tomato xylem sap compared with pectin-rich medium. Of the 85 genes that are strongly induced in the xylem sap, four genes encode the hydrophobins Vdh1, Vdh2, Vdh4 and Vdh5. Vdh4 and Vhd5 are structurally distinct from each other and from the three other hydrophobins (Vdh1-3) annotated in V. dahliae JR2. Their functions in the life cycle and virulence of V. dahliae were explored using genetics, cell biology and plant infection experiments. Our data revealed that Vdh4 and Vdh5 are dispensable for V. dahliae development and stress response, while both contribute to full disease development in tomato plants by acting at later colonization stages. We conclude that Vdh4 and Vdh5 are functionally specialized fungal hydrophobins that support pathogenicity against plants.

Characterization of the structure and self-assembly of two distinct class IB hydrophobins.

Hydrophobins are small proteins secreted by fungi that accumulate at interfaces, modify surface hydrophobicity, and self-assemble into large amyloid-like structures. These unusual properties make hydrophobins an attractive target for commercial applications as emulsifiers and surface modifying agents. Hydrophobins have diverse sequences and tertiary structures, complicating attempts to characterize how they function. Here we describe the atomic resolution structure of the unusual hydrophobin SLH4 (86 aa, 8.4 kDa) and compare its function to another hydrophobin, SC16 (99 aa, 10.2 kDa). Despite containing only one charged residue, SLH4 has a similar structure to SC16 yet has strikingly different rodlet morphology, propensity to self-assemble, and preferred assembly conditions. Secondary structure analysis of both SC16 and SLH4 suggest that during rodlet formation residues in the first intercysteine loop undergo conformational changes. This work outlines a representative structure for class IB hydrophobins and illustrates how hydrophobin surface properties govern self-assembly, which provides context to rationally select hydrophobins for applications as surface modifiers. KEY POINTS: • The atomic-resolution structure of the hydrophobin SLH4 was determined using nuclear magnetic resonance spectroscopy. • The structure of SLH4 outlines a representative structure for class IB hydrophobins. • The assembly characteristics of SLH4 and SC16 are distinct, outlining how surface properties of hydrophobins influence their function.

The Hydrophobin Gene Family Confers a Fitness Trade-off between Spore Dispersal and Host Colonization in Penicillium expansum.

Hydrophobins are small amphipathic surface proteins found exclusively in fungi. In filamentous ascomycetes, one conserved role of a subset of hydrophobins is their requirement for spore dispersal. Other contributions of these proteins to fungal biology are less clear and vary across genera. To determine the functions of hydrophobins in the biology and virulence of this fungus, we created seven single mutants and a septuple-deletion mutant (Δsep) of the entire putative P. expansum hydrophobin gene family. One spore hydrophobin, HfbA, shared 72.56% sequence identity to the Aspergillus fumigatus spore hydrophobin RodA and was required for efficient spore dispersion in P. expansum. The Δsep mutant was likewise reduced in spore dispersal, hypothesized to be due to the aberrant shape and clumping of the Δsep conidia and conidiophores. Additionally, the Δsep mutant presented several differences in physiological traits, including decreased survival in extreme cold temperatures and increased production of several toxic secondary metabolites. Most striking was the unexpected fitness advantage that the Δsep strain displayed in competitive passaging with the wild-type strain on host apple where the mutant significantly increased in percentage of the colonizing population. This work uncovers potential ecological trade-offs of hydrophobin presence in filamentous fungi. IMPORTANCE Hydrophobins are amphipathic secreted proteins uniquely found in filamentous fungi. These proteins self-assemble and constitute the outer most layer of fungal surfaces thus mediating multiple aspects of fungal interactions with their environments. Hydrophobins facilitate spore dispersal, yet a full understanding of the function and need for multiple hydrophobins in fungal species remains elusive. To address the role of this protein family in Penicillium expansum, the causative agent of blue mold disease in pome fruit, all seven putative hydrophobin genes were deleted and the mutant assessed for numerous physiological traits and virulence on fruit. Despite showing a decrease in spore dispersal, the septuple-deletion mutant was more fit than the wild type in competitive pathogenicity tests on apple. Our findings suggest this gene family illustrates a functional trade-off between dispersal and host colonization in P. expansum.

A novel hydrophobin encoded by hgfII from Grifola frondosa exhibiting excellent self-assembly ability.

Hydrophobins are small proteins from filamentous fungi, which have remarkable self-assembly properties of great potential, e.g., as drug carriers and as anti-bacterial agents, but different hydrophobins, with improved properties, are needed. HGFI (a hydrophobin from Grifola frondosa) is a class I hydrophobin, which can self-assemble into rodlet structures with a length range 100-150 nm. In this study, we identified a new hydrophobin gene (hgfII) from the mycelium of G. frondosa with a much higher transcriptional level than hgfI. Heterologous expression of hgfII was accomplished in the Pichia pastoris. X-ray photoelectron spectroscopy and water contact angle assay measurements revealed that HGFII can self-assemble into a protein film at the air-solid interface, with circular dichroism and thioflavin T fluorescence studies showing that this effect was accompanied by a decrease in α-helix content and an increase in ß-sheet content. Using atomic force microscopy, it was shown that HGFII self-assembled into rodlet-like structures with a diameter of 15-30 nm, showing that it was a class I hydrophobin, with self-assembly behavior different from HGFI. The surface hydrophobicity of HGFII was stronger than that of HGFI, meanwhile, in emulsification trials, HGFII displayed better dispersive capacity to the soybean oil than HGFI, producing a more stable and durable emulsion.

The Colletotrichum siamense Hydrophobin CsHydr1 Interacts with the Lipid Droplet-Coating Protein CsCap20 and Regulates Lipid Metabolism and Virulence.

Previous studies of the lipid droplet-coating protein Cap20 in Colletotrichum show that it plays a key role in appressorium development and virulence. In this study, the hydrophobin CsHydr1, which contains a signal peptide of 19 amino acids and a hydrophobic domain (HYDRO), was shown to interact with CsCap20 in Colletotrichum siamense. The CsHydr1 deletion mutant showed slightly enhanced mycelial growth, small conidia, slow spore germination and appressoria formation, cell wall integrity and virulence. Like CsCAP20, CsHydr1 is also localized on the lipid droplet surface of C. siamense. However, when CsCap20 was absent, some CsHydr1 was observed in other parts. Quantitative lipid determination showed that the absence of either CsHydr1 or CsCap20 reduced the content of lipids in mycelia and conidia, while the effect of CsCap20 was more obvious; these results suggest that an interaction protein CsHydr1 of CsCap20 is localized on the lipid droplet surface and involved in lipid metabolism, which affects appressorium formation and virulence in C. siamense.

Aspergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation.

Hydrophobins are small amphipathic proteins conserved in filamentous fungi. In this review, the properties and functions of Aspergillus hydrophobins are comprehensively discussed on the basis of recent findings. Multiple Aspergillus hydrophobins have been identified and categorized in conventional class I and two non-conventional classes. Some Aspergillus hydrophobins can be purified in a water phase without organic solvents. Class I hydrophobins of Aspergilli self-assemble to form amphipathic membranes. At the air-liquid interface, RolA of Aspergillus oryzae self-assembles via four stages, and its self-assembled films consist of two layers, a rodlet membrane facing air and rod-like structures facing liquid. The self-assembly depends mainly on hydrophobin conformation and solution pH. Cys4-Cys5 and Cys7-Cys8 loops, disulfide bonds, and conserved Cys residues of RodA-like hydrophobins are necessary for self-assembly at the interface and for adsorption to solid surfaces. AfRodA helps Aspergillus fumigatus to evade recognition by the host immune system. RodA-like hydrophobins recruit cutinases to promote the hydrolysis of aliphatic polyesters. This mechanism appears to be conserved in Aspergillus and other filamentous fungi, and may be beneficial for their growth. Aspergilli produce various small secreted proteins (SSPs) including hydrophobins, hydrophobic surface-binding proteins, and effector proteins. Aspergilli may use a wide variety of SSPs to decompose solid polymers.

Isolation and Characterization of a Novel Hydrophobin, Sa-HFB1, with Antifungal Activity from an Alkaliphilic Fungus, Sodiomyces alkalinus.

The adaptations that alkaliphilic microorganisms have developed due to their extreme habitats promote the production of active natural compounds with the potential to control microorganisms, causing infections associated with healthcare. The primary purpose of this study was to isolate and identify a hydrophobin, Sa-HFB1, from an alkaliphilic fungus, Sodiomyces alkalinus. A potential antifungal effect against pathogenic and opportunistic fungi strains was determined. The MICs of Sa-HFB1 against opportunistic and clinical fungi ranged from 1 to 8 µg/mL and confirmed its higher activity against both non- and clinical isolates. The highest level of antifungal activity (MIC 1 µg/mL) was demonstrated for the clinical isolate Cryptococcus neoformans 297 m. The hydrophobin Sa-HFB1 may be partly responsible for the reported antifungal activity of S. alkalinus, and may serve as a potential source of lead compounds, meaning that it can be developed as an antifungal drug candidate.

Identification and characterization of a hydrophobin Vmh3 from Pleurotus ostreatus.

Hydrophobins (HPs) are relatively small surface-active proteins of fungal origin. Being an industrially important protein, isolation of new molecules from GRAS (Generally Regarded as Safe) strains like mushrooms is the need of the time. In the present work, hydrophobin Vmh3-1 is isolated, purified, and identified from a culture broth and vegetative mycelia of Pleurotus ostreatus grown in a Potato dextrose broth (PDB) in static culture conditions. Purified proteins from the broth and the cell wall showed bands of 11 kDa and 17 kDa when analyzed on SDS-PAGE. Hydrophobin Vmh3-1 was identified in purified protein samples by the Orbitrap-HR-LC-MS/MS analysis with a maximum of 66% sequence coverage. The amphipathic nature of the protein was revealed by an increase in the water contact angle (WCA) of the hydrophilic surface of glass by 87% as well as a decrease in the WCA of the hydrophobic surface of Teflon by 19%. The emulsification property was tested with food-grade oils and Hexane. A maximum activity (EI 24) of 87.64% was recorded for Sunflower oil. In CD (Circular dichroism) spectra, Vmh3-1 showed the typical spectra of hydrophobin with a dominance of ß-sheets (51%) in the secondary structure and a minimum percentage of the α-helix (2%). The protein did not show a self-aggregating property on vigorous shaking making it suitable for numerous industrial applications. The identification of Vmh3-1 with detailed amino acid sequencing and the characterization of the protein to evaluate its potential in surface modifications for various industrial applications is demonstrated herein for the first time.

Identification and Functional Analysis of a Novel Hydrophobic Protein VdHP1 from Verticillium dahliae.

Verticillium dahliae could cause destructive vascular wilt disease on hundreds of plant species around the world, including cotton. In this study, we characterized the function of a hydrophobin gene VdHP1 in pathogen development and pathogenicity. Results showed that VdHP1 could induce cell death and activate plant immune responses. The VdHP1 deletion mutants (ΔVdHP1) and the complement mutants (C-ΔVdHP1) were obtained by the homologous recombination method. The VdHP1 deletion mutants exhibited increased hydrophilicity, inhibited microsclerotial formation, and reduced spore smoothness. In addition, the deletion mutants were more sensitive to NaCl, while relatively insensitive to KCl and sorbitol. Mutants also had greater resistance to Congo red, UV radiation, and high temperature, which suggested that ΔVdHP1 strains have stronger resistance to abiotic stress in general. Different carbon source assays showed that the utilization ability of skim milk, cellulose, and starch was greatly enhanced in ΔVdHP1, compared with that of WT and complemented strains. Furthermore, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The pathogenicity test found that the crude toxin content, colonization, and dispersal of ΔVdHP1 was significantly increased compared with the WT and complementary strains. In addition, cotton seedlings showed more severe wilting symptoms after inoculation with ΔVdHP1 strains. These results suggested that the hydrophobin VdHP1 negatively regulated the virulence of V. dahliae, and played an important role in development, adaptability, and pathogenicity in V. dahliae, which maybe provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence. IMPORTANCE Verticillium dahliae is a soilborne fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, it was illustrated that the hydrophobin VdHP1 could induce cell death and activate plant immune responses. VdHP1 affected the hydrophobicity of V. dahliae, and negatively regulated the strains resistant to stress, and the utilization ability of different carbon sources. In addition, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The VdHP1 gene negatively regulated the total virulence, colonization, and dispersal of V. dahliae, with enhanced pathogenicity of mutant strains in this gene. These results suggested that the hydrophobin VdHP1 played an importance in development, adaptability, and pathogenicity in V. dahliae, and would provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence.

Adsorption Kinetics and Self-Assembled Structures of Aspergillus oryzae Hydrophobin RolA on Hydrophobic and Charged Solid Surfaces.

Hydrophobins are small secreted amphipathic proteins ubiquitous among filamentous fungi. Hydrophobin RolA produced by Aspergillus oryzae attaches to solid surfaces, recruits polyesterase CutL1, and thus promotes hydrolysis of polyesters. Because the N-terminal region of RolA is involved in the interaction with CutL1, the orientation of RolA on the solid surface is important. However, the kinetic properties of RolA adsorption to solid surfaces with various chemical properties remain unclear, and RolA structures assembled after the attachment to surfaces are unknown. Using a quartz crystal microbalance (QCM), we analyzed the kinetic properties of RolA adsorption to the surfaces of QCM electrodes that had been chemically modified to become hydrophobic or charged. We also observed the assembled RolA structures on the surfaces by atomic force microscopy and performed molecular dynamics (MD) simulations of RolA adsorption to self-assembled monolayer (SAM)-modified surfaces. The RolA-surface interaction was considerably affected by the zeta potential of RolA, which was affected by pH. The interactions of RolA with the surface seemed to be involved in the self-assembly of RolA. Three types of self-assembled structures of RolA were observed: spherical, rod-like, and mesh-like. The kinetics of RolA adsorption and the structures formed depended on the amount of RolA adsorbed, chemical properties of the electrode surface, and the pH of the buffer. Adsorption of RolA to solid surfaces seemed to depend mainly on its hydrophobic interaction with the surfaces; this was supported by MD simulations, which suggested that hydrophobic Cys-Cys loops of RolA attached to all SAM-modified surfaces at all pH values. IMPORTANCE The adsorption kinetics of hydrophobins to solid surfaces and self-assembled structures formed by hydrophobin molecules have been studied mostly independently. In this report, we combined the kinetic analysis of hydrophobin RolA adsorption onto solid surfaces and observation of RolA self-assembly on these surfaces. Since RolA, whose isoelectric point is close to pH 4.0, showed higher affinity to the solid surfaces at pH 4.0 than at pH 7.0 or 10.0, the affinity of RolA to these surfaces depends mainly on hydrophobic interactions. Our combined analyses suggest that not only the adsorbed amount of RolA but also the chemical properties of the solid surfaces and the zeta potential of RolA affect the self-assembled RolA structures formed on these surfaces.

The Gß-like Protein AfCpcB Affects Sexual Development, Response to Oxidative Stress and Phagocytosis by Alveolar Macrophages in Aspergillus fumigatus.

G-protein signaling is important for signal transduction, allowing various stimuli that are external to a cell to affect its internal molecules. In Aspergillus fumigatus, the roles of Gß-like protein CpcB on growth, asexual development, drug sensitivity, and virulence in a mouse model have been previously reported. To gain a deeper insight into Aspergillus fumigatus sexual development, the ΔAfcpcB strain was generated using the supermater AFB62 strain and crossed with AFIR928. This cross yields a decreased number of cleistothecia, including few ascospores. The sexual reproductive organ-specific transcriptional analysis using RNAs from the cleistothecia (sexual fruiting bodies) indicated that the CpcB is essential for the completion of sexual development by regulating the transcription of sexual genes, such as veA, steA, and vosA. The ΔAfcpcB strain revealed increased resistance to oxidative stress by regulating genes for catalase, peroxiredoxin, and ergosterol biosynthesis. The ΔAfcpcB strain showed decreased uptake by alveolar macrophages in vitro, decreased sensitivity to Congo red, decreased expression of cell wall genes, and increased expression of the hydrophobin genes. Taken together, these findings indicate that AfCpcB plays important roles in sexual development, phagocytosis by alveolar macrophages, biosynthesis of the cell wall, and oxidative stress response.