Hydrophobin appointed as EAS. The insoluble nature

Hydrophobin proteins have attracted a great deal of attention over the past decade,due to their unique surface active properties for medical, biotechnology and applicatioin food science. These small amphipathic proteins are produce and secreted byflamentous fungi during its growth and development cycles to assist with the formatioof aerial hyphea, prevent wetting of the aerial spores to allow for efcient dispersal,protect the spores from the external environment and enhance attachment to surfacesprior to infections. At the hydrophobic:hydrophilic interface, the surface activehydrophobin proteins self assemble to form into a biocompatible amphipathic flm.Due to the diverse function provided by these hydrophobin proteins, researchershave been exploiting the properties of these proteins for advancements in surfacemodifcation, developments of biosensors, producing stable emulsions and surfacefunctionalisation. Understanding the structure and properties of these hydrophobinproteins may provide insights for the development and design of novel nanomaterials
The fbrillar structures coating the surface of fungi spores were frst observed withtransmission electron microscopy in the 1960’s 1, 2, were not termed “hydrophobin”till 1991 by Wessels et al 3. The rodlet coating found on the surface of fungi sporesof Neurospora crassa was discovered to function as water repelling flm in 1978 byDempsey and Beever4. When comparing wild type spores with mutant sporeswhich did not form the rodlet coating, due to a gene knock out of Neurosporacrassa, the lack of the rodlet coating rendered the mutant spores easily wettable.The missing gene was designated “eas” due to the “easily” wettable phenotypeand the protein was appointed as EAS. The insoluble nature of the rodlet flmmade attempts at solublising the protein with commonly used denaturing bu?erchallenging 4, 5. It was not until 1995, Templeton et al were able to solublisedthe rodlet flms with 100% tri?uroacetic acid and extract a 7 kDa protein foundto be the EAS gene product 6. The term “hydrophobin” was coined by during
the discovery of characteristic proteins, SC1, SC3 and SC4, produced during theformation of aerial hyphae and fruiting bodies of Schizophyllum commune 3. Theproteins have a relatively high numbers of hydrophobic residues and contained eightcysteines organised in a conserved pattern. These characteristics are found in otherhydrophobin proteins produced by di?erent flamentous fungi 6–12.
Fungi are essential part of the ecosystem with multiple roles in nature. As dominatedecomposer of organic materials in nature, fungi recycle nutrients back into theirhabitat or lives symbiotically with other plants or animals. Generally found in moist,dark environment, and without the ability to photosynthesise, flamentous fungicells forms long tubular flaments called hyphae, that spreads to form mycelium, toabsorb food and reproduce. During asexual reproduction, new aerial morphologies,such as conidiophores and fruiting bodies, are produced to disperse spores intothe air and germinate in new locations. Hydrophobin proteins are produced andsecreted during these growth and development cycles and function to assist with theformation of aerial hyphea, prevent wetting of the aerial spores to allow for efcientdispersal, protect the spores from the external environment and enhance attachmentto surfaces prior to infections.
A fungus can produce multiple types of hydrophobin protein, each able fulfldi?erent roles and act as synergist. In Schizophyllum commune four hydrophophbins,Sc1, Sc3, Sc4 and Sc6 can be found on the surface of the fungi, contributing to thehydrophobic nature of the fungi. During the formation of aerial hyphae, Sc3 isupregulated to lower the surface tension of the growth medium allowing the hyphaeto break through the water interface. As the aerial hyphae breaches the interface,hydrophobins coats the aerial hyphae preventing the structure from wetting, whichassists with dispersal 3. In the fruiting bodies, Sc1, Sc4 and Sc6 can be found on theas structural supports, in particular Sc4 assembles as hydrophobic flm lining the gaschannels to prevent water logging. Other hydrophobins such as Aspergillus nidulansproduces six di?erent hydrophobins; RodA, DewA, DewB, DewC, DewD, DewE,which all contributes to the spore hydrophobicity, yet the expression level and timeof expression varies 15. The phytopathogenic fungus Magnaporthe grisea producesthe hydrophobin MPG1, and MHP1 which are critical for attachment and infection.At the fungal and host interface, MPG1 can self-assemble into hydrophobin rodletstructures which is able to retain enzyme cutinase at the interface. Yet MHP1 cansuppress rodlet assembly suggesting a dynamic mechanism allowing the spore tocontrol and localise cutinase activities 16. The gene which encodes the hydrophobinEAS is activated by blue light and the circadian clock near morning and afternoonperiod. Interestingly, this coincide with the time of greatest di?erent in air pressure,which provides the greatest potential for the dispersal of spores 17. Hydrophobinsare upregulated for survival during nutrient, carbon or nitrogen starvation to formprotective coating around the spores or hyphae to prevent the loss of excess nutrients18.All hydrophobin proteins provide di?erent functionalities for the survival and growthduring the fungi lifecycle, with active roles during reproduction, development andgermination (Figure 1.2).Distinctions between hydrophobins were observed in the early studies, determinedby the hydrophathy profles, and solubility of the hydrophobin layers. Hydrophobinswere divided into two di?erent classes; Class I and Class II 19–21.
Class I hydrophobins are able to form an insoluble amphiphilic monolayer consistingof cross ?-sheet structures called rodlets. Monolayers from Class I hydrophobins arephysically and chemically stabled in strong alkaline solutions (3 M NaOH) andmany acidic conditions (3 M HCL) 22. The monolayers can only be disrupted andsolublised by strong acids such as tri?uoroacetic acid (TFA) or formic acid, however,once monomeric, hydrophobins are still able to self-assemble back into a rodlet flmunder the correct conditions 23. The rodlets are 10 nm wide and assemble laterallyto form flms between 2.5 nm to 3 nm thick 16, 22, 24 Class I hydrophobin proteinstend to be larger than Class II, containing around 50-150 residues.