review article on hydrophobins

This is an amended version of an article that was published in Mycologist 14 (4), 153-159, 2000, reproduced with permission of the British Mycological Society

Hydrophobins, unique fungal proteins

J.G.H. WESSELS

Department of Plant Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

Hydrophobins are proteins that appear unique to mycelial fungi. They play essential roles in the emergence of aerial structures, gas exchange in fruiting bodies and lichens, and in pathogenesis. These roles are all based on the remarkable properties of these proteins as observed in vitro.

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Structure and occurrence of hydrophobins

Hydrophobins are proteins that appear unique to fungi and do not occur in other organisms. In fact they may occur exclusively in mycelial fungi being apparently absent in those fungi that can grow as yeast only such as baker's yeast (Saccharomyces cerevisiae). Hydrophobins are relatively small secreted proteins (100-150 amino acids), all typified by the presence of 8 cysteine residues at conserved places along the chain of amino acids. Two classes of hydrophobins have been distinguished, Class I and Class II (Wessels, 1994). Figure 1 shows representatives of the two classes, SC3 from Schizophyllum commune and cerato-ulmin from Ophiostoma ulmi, the causative agent of Dutch elm disease. Members of the Class I and Class II hydrophobins share many properties but the latter have not been recorded in basidiomycetes, they form less stable complexes, and less is known about their functions. Only Class I hydrophobins will be considered further in this brief overview.

Figure 1. Polar (blue) and apolar (red) amino acids and cysteine residues (black) in SC3 (a ClassI hydrophobin) and cerato-ulmin (CU, a Class II hydrophobin). Plus and minus signs indicate charged amino acids.

We know that in Class I hydrophobins all cysteine residues are paired by means of disulphide bridges but which cysteine residues are actually paired has not been established. In Figure 1 the disulphide bridges in SC3 have been tentatively drawn as determined for the Class II hydrophobin cerato-ulmin (Yaguchi et al., 1993). Apart from the cysteine residues the positions of polar and apolar amino acids (Fig. 1) are well preserved, so that the pattern of hydrophobicity along the amino acid chain (the hydropathy plot) is characteristic for these hydrophobins. Otherwise the amino acid sequences are quite diverse, even among different hydrophobins from the same organism. Figure 2 shows Class I hydrophobins from different fungi (for references see Wessels, 1997; Wösten and Wessels, 1997) grouped in a dendrogram that shows their relatedness.

Figure 2. Dendrogram of Class I hydrophobin amino acid sequences. The highly variable amino-terminal sequence before the first cysteine residue has been omitted. Proteins indicated with a black bullet have been physically isolated. References in Wessels, 1997 and Wösten and Wessels, 1997. XEH1 and XPH1 are from Scherrer et al. (2000).

Because of the diversity in amino acid sequences it is not surprising that hydrophobin genes have only rarely been detected on the basis of DNA homology by cross-hybridisation. Most of these genes have been detected as being very active at certain stages of development, often producing enormous amounts of mRNAs. For instance, the SC1, SC3, SC4 and SC6 hydrophobin genes of Schizophyllum commune, the first that have been cloned, were isolated on the basis of the abundant mRNAs produced by these genes at the time of formation of aerial hyphae and fruiting bodies (Mulder and Wessels, 1988). Another successful strategy has been isolation of Class I hydrophobins on the basis of their distinguished property of self-assembly into extremely insoluble complexes (see below). This is followed by determination of a small part of the amino acid sequence at the amino-terminal end of the protein and working backward towards the sequence of the gene. The sequence of the cloned gene then predicts the whole amino acid sequence. Although Fig. 2 lists no zygomycetes, Class I hydrophobins have at least been suggested to occur in Mucor mucedo (de Vries et al. 1993). The dendrogram (Fig. 2) suggests that Class I hydrophobins were 'invented' by fungi at least before the split occurred between ascomycetes and basidiomycetes which occurred in the early Devonian (approximately 400 million years ago). They may even be more ancient if hydrophobins do occur in zygomycetes, which preceded the septate fungi (Taylor, 1990). Unfortunately the occurrence of hydrophobins in chytridiomycetes is unknown.

Molecular properties of hydrophobins

De Vries et al. (1993) estimated that at least 10% of all proteins produced at the time of emergent growth in S. commune were hydrophobins and they are also abundant in other fungi. If hydrophobins are such characteristic fungal proteins that are produced in large quantities, then why were they not discovered earlier? At least for the class I hydrophobins the answer lies in a remarkable property of these proteins, not known for any other protein. When confronted with the interface between a hydrophobic and hydrophilic environment, the protein monomers assemble into an SDS-insoluble amphipathic membrane. The monomers undergo a conformational change such that the side of the membrane facing the hydrophobic phase becomes extremely hydrophobic (similar to Teflon) while the other side is hydrophilic. For instance, as shown in the cartoon of Fig. 3A, hydrophobin monomers in aqueous solution assemble where the water meets the air and form a membrane with its hydrophobic side facing the air. This side of the protein membrane features a typical rodlet pattern (Fig. 3C), The hydrophobin assemblage is very stable and even withstands boiling in a detergent solution, such as 2% sodium dodecyl sulphate (SDS). It can, however, be dissociated into monomers with 100% trifluoroacetic acid (TFA) or formic acid (FA) and, after removal of the acid, the soluble monomers can again be assembled. This also demonstrates resilience of hydrophobins to harsh chemical treatments. Since nearly all hydrophobins in a fungus occur in the assembled state, they do not show up in the SDS extracts that are usually made for resolution of component proteins. It was only after cloning the genes that a search for the products of these genes led to the discovery of hydrophobins. However, once we had discovered this insolubility of the hydrophobin assemblages in hot SDS solutions it was easy to isolate these hydrophobins from any fungus. All other proteins were simply dissolved in hot SDS after which hydrophobins in a practically pure state could be extracted from the residue with TFA.

Figure 3. Assembly of SC3 hydrophobin at the water-air interface (A) and at the surface of a water-teflon interface (B). The hydrophobic surface of the membrane facing the air shows fascicles of parallel rodlets measuring 10 nm in width (bar represents 100 nm).

Figure 3B illustrates that immersion of a hydrophobic object (such as a sheet of Teflon) into an aqueous hydrophobin solution leads to the coating of the hydrophobic surface with an assembled amphipathic hydrophobin layer. After removal of the object from the solution its surface has now become hydrophilic and hot detergents cannot remove the responsible coating. Hydrophobins thus can make a hydrophilic surface hydrophobic and a hydrophobic surface hydrophilic by assembling into an extremely thin coating of 10 nanometres (one-millionth of a millimetre).

Our research on the nature of the stable conformational changes accompanying assembly of these hydrophobins shows that these changes involve extensive b -sheet formation (de Vocht et al. 1998). These conformational changes are not unlike those occurring in amyloid fibril formation as in Alzheimer and Creuzfeldt-Jacob disease (to be published). However, instead of leading to dysfunction and debilitating the organism, hydrophobins seem to have evolved to undergo this conformational change in order to fulfil a number of essential functions in the life of fungi. It is also noteworthy that the disulphide bridges in the hydrophobin monomer prevent the protein from aggregating spontaneously in the absence of a hydrophilic-hydrophobic interface (de Vocht et al. 2000). Clearly, precocious intracellular assembly could be harmful to the fungus.

Formation of aerial structures

As is clear from the properties of the isolated hydrophobins, these proteins can change the nature of a surface. When we first saw the rodlet pattern of assembled SC3 hydrophobin (Fig. 3C), we were immediately reminded of similar rodlets we had seen on the surface of aerial hyphae of S. commune and generally known to occur on fungal spores. In Neurospora crassa a mutation was known, eas, resulting in easily wettable conidia without rodlets. Indeed, two groups (Bell Pedersen et al., 1992; Lauter et al., 1992) independently showed that this mutant produced insufficient amounts of a hydrophobin. Stringer et al. (1991) isolated a gene expressed during conidiation of Aspergillus nidulans that, by comparison to the genes isolated from S. commune, was unmistakably a hydrophobin gene. Upon disruption of this gene, the mutant produced wet conidia without rodlets. Probably the hydrophobins on the conidia have an important function in keeping the spores dry and to prevent clumping, allowing dispersal of the spores by wind.

Figure 4. Proposed model for the role of hydrophobins in the emergence of aerial hyphae. Symbols as in Fig. 3.

We found the assembled SC3 hydrophobin as a hydrophobic rodlet layer on the surface of aerial hyphae of S. commune, explaining their dry unwettable surface and their tendency to grow into the air. However, SC3 was also produced by the submerged hyphae and, in the absence of an interface, the hydrophobin accumulated in large quantities as monomers into the culture medium. It was not until we discovered that the hydrophobin tremendously lowered the water surface tension by assembling at the medium-air interface (see Fig. 3A), that we grasped the reason for this. Apparently, hyphae cannot easily overcome the high surface tension of water to breach the surface and to grow into the air. Indeed, when we disrupted the SC3 gene we found that hyphae growing in liquid medium were no longer able to grow into the air. This could be remedied by adding SC3 hydrophobin to the medium of the mutant although the aerial hyphae now had a wettable surface. This led to the model depicted in Fig. 4 (Wösten et al., 1999). It would seem that without the hydrophobin at the surface of the medium, submerged hyphae approaching the surface are deflected by the high water surface tension and continue to grow along the path of least resistance. However, with the hydrophobin assembled at the medium surface and the water surface tension reduced to less than 50% of normal a hypha would be able to extend the surface of the water by continuously secreting hydrophobins at its growing tip. The hydrophobin secreted by the aerial hypha now assembles on the hyphal surface conferring hydrophobicity to the wall. In this model the polysaccharide wall underlying the hydrophobin coating is considered to be an extension of the aqueous phase. An SC3 mutant sends hyphae into the air when provided with SC3 in the medium and these hyphae must break through the assembled hydrophobin layer but we are still ignorant of any mechanical resistance that this layer poses to the emerging hypha. When growing in a (semi) solid agar medium the phenotype of the SC3 mutant is less pronounced. Probably the anchorage in the substrate allows hyphae to exert more force to breach the surface and to grow into the air. However, even under these conditions fewer aerial hyphae were formed and fewer fruiting bodies arose on a dikaryon with disrupted SC3 genes.

Keeping gas channels open in fruiting bodies and lichens

S. commune contains at least four different hydrophobin genes. Of these, the SC3 gene is active in monokaryons and dikaryons but the SC1, SC4 and SC6 genes are active in dikaryons only, at the time of fruiting body formation. At least the role of the SC4 hydrophobin has now been clarified. The tissue of the fruiting body is traversed by numerous air channels, which probably have a role in providing efficient gas exchange during respiration in this massive tissue. By capillary action such channels would easily fill with water, were it not for the presence of assembled SC4 providing a hydrophobic lining (Fig. 5a). This hydrophobic coating is expected to keep all liquid water out of these channels. Disrupting both SC4 genes present in the dikaryon proved this role (van Wetter et al., 2000). The air channels now filled with water when the fruiting bodies were submerged in water. Also in Agaricus bisporus we found a hydrophobin assemblage lining the air channels in the mushroom, probably serving the same function. With regard to formation of these hydrophobic linings it should be noted that hydrophobin monomers secreted into the extracellular matrix of the fruiting bodies would automatically assemble at the wall of the channels when they meet the gas phase, as they do on the surface of aerial hyphae.

Fig.5a. A hydrophobic rodlet layer assembles at the walls of air channels in the large fruiting bodies of basidiomycetes preventing these to fill with water under wet conditions

Lichens, which are exposed to regular cycles of drying and wetting, are metabolically active only under wet conditions. It is thus of great functional significance that gas spaces in the lichen remain open under wet conditions. Honegger (1991) described the occurrence of a rodlet layer lining medullar gas spaces in several lichens. Hydrophobins that upon assembly in vitro formed the same type of rodlets as seen in the lichen have now been isolated from Xanthoria ectaneoides and X. parietina and the fungal hydrophobin genes were cloned (Scherrer et al., 2000). The deduced sequences were similar to those of other ascomycete hydrophobins of Class I (Fig. 2). We assume that the fungus secretes the hydrophobin monomers, which diffuse into the apoplast that surrounds both mycobiont and photobiont. When reaching the interface with the gas space the fungal hydrophobin then assembles over the surface of both partners (Fig. 5). According to Honegger (1991) this hydrophobic lining of the gas spaces (the rodlet layer is often covered by other hydrophobic substances) allows efficient apoplastic transport of water and solutes between the symbionts as well as permitting optimal gas exchange during wet periods. Perhaps these hydrophobins are the key to the existence of this symbiotic relationship.

Figure 5. A hydrophobic rodlet layer of assembled hydrophobin (red line) lines the medullar gas spaces of a lichen, covering both the mycobiont and the photobiont

Fossil lichens are known from the Early Devonian (Taylor et al., 1995), about 400 million years ago, but they probably occurred much earlier. They may even have been the first terrestrial eukaryotic organisms (Taylor, 1990). If so, then the 'invention' of hydrophobins may have been a decisive event at this crucial point in the history of life.

Adherence to hydrophobic substrates

Monokaryotic and dikaryotic hyphae of S. commune tightly adhere to hydrophobic surfaces by means of the SC3 hydrophobin that assembles between the hydrophilic wall and the hydrophobic substrate. This was shown by localisation of the hydrophobin and by the effect of SC3 gene disruption (Wösten et al., 1994). The amphipathic hydrophobin membrane effectively glues two incompatible materials together (Fig. 6A). Another possibility is that secreted hydrophobin first assembles on the hydrophobic surface making this surface hydrophilic, after which a water-based glue can easily secure the hypha to the primed surface (Fig. 6B). Such mechanisms may be of great importance for fungi when they interact with the hydrophobic surfaces of plants and animals as a prelude to a beneficial or parasitic relationship. The role of a hydrophobin in a pathogenic interaction has been shown for Magnaporthe grisea infecting rice plants (Talbot et al., 1993). Without firm attachment of the germ tube to the leaf by means of the hydrophobin MPG1 no appressoria were formed and no infection ensued. But also in ectomycorrhizal interactions hydrophobins probably play, an as yet unknown, role (Martin et al., 1999).

Figure 6. A hypha adheres to a hydrophobic surface by means of a hydrophobin while growing in air (A) or in an aqueous medium (B). Symbols as in Fig. 3.

Prospects

It is likely that other roles of hydrophobins will be discovered. However, what is now known already suggests that the 'invention' of hydrophobins may have been central to the evolution of the Kingdom Fungi. Also their roles in the interactions of fungi with plants, algae and cyanobacteria, as in mycorrhiza and lichens, is intriguing because these suggest a crucial role in the emergence and maintenance of terrestrial life. Perhaps these ancient proteins can now be put to use in biotechnological applications in which we want to change the nature of a surface. Natura artis magistra.

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