6.8 On the far side

By definition, a wall has an outer side; an interface between that which is contained and that which is excluded. Consequently there are molecules which can be described as being on the outside of the fungal wall. Some of these we have already mentioned; such as the mating proteins called agglutinins which are responsible for ‘agglutination’ (cell-to-cell adhesion) between cells of different mating type in Saccharomyces cerevisiae as an early process in mating (discussed in Chapter 8; CLICK HERE to view the page). The interaction takes place between two GPI-anchored cell surface glycoproteins anchored in the outer face of the plasma membranes of the two mating cells. These are representative of a growing class of cell adhesion proteins, generally called adhesins, which are critical to a wide range of fungal cell interactions with the outside world (Dranginis et al., 2007).

Adhesins are glycoproteins that are secreted to the wall, then anchored with their interactive binding domains elevated beyond the wall surface; they have several domains with discrete functions and the need to project beyond the rest of the wall determines the order of their structural domains. The characteristic order (from the outside inwards) is:

  • as glycoproteins their polypeptides carry amino-terminal secretion signals;
  • then there are the interactive binding domains which are highly specific for their ligands (targets);
  • there may be central threonine-rich glycosylated domains that make possible cell-cell interactions with other cells of the same species (or of the same mycelium), so-called homotypic interactions;
  • N- and O-glycosylated stalks serve to elevate these binding domains above the wall surface;
  • carboxy-terminal regions mediate covalent cross-linking to the wall matrix and possibly tethering to the plasma membrane through modified GPI anchors (Dranginis et al., 2007).

These structural motifs are common but not universal, although we currently have an incomplete knowledge of what appears to be a very large and diverse range of glycoproteins. Adhesins are known to mediate homotypic interactions in mating (as in budding yeast) as well as in multicellular differentiation, an example being glycoproteins known as galectins which are specific to the fruit body of Coprinopsis.

Adhesins also enable adhesion to surfaces. Fibrils extending from the wall into the suspending fluid are commonly found where fungi attach to surfaces. The fibrils are highly specific to a complementary molecular structure on the surface. This system of recognition and communication between fungus and partner is widespread in pathogenic and mutualistic interactions, enabling adherence to the surfaces of host organisms (animal and plant). But they also enable the formation of biofilms, both on natural surfaces and on catheters and other medical devices.

A hint of other capabilities of these molecules is provided by an adhesin secreted by Blastomyces dermatiditis. B. dermatitidis grows as a filamentous mould at room temperature in soils contaminated with animal excreta (like many pastures); spores inhaled by man and his animals convert into large invasive yeasts in the lungs, and cause a potentially fatal blastomycosis. The adhesin produced by the fungus has a domain structured like an animal epidermal growth factor that binds to host cells and downregulates the host’s immune response, consequently potentiating the infection (Dranginis et al., 2007).

The surface interactions described so far take place at least in aqueous films and in some cases in a fluid medium, either natural or artificial. In the latter case the boundary between ‘wall’ and ‘medium’ may not always be clearly demarcated (as is indicated in Fig 2A) because the zone beyond the wall contains successively diminishing concentrations of polysaccharides and glycoproteins.

However, many hyphal surfaces are dry and even hydrophobic (that is, non-wettable). Particular examples are the surfaces of the innumerable air-dispersed spores found in fungi, their sporophores, as well as all sorts of aerial mycelium and aerial structures like fruit bodies and stromata. Less obvious examples are cavities within organised tissues that are kept fluid-free by hydrophobic coatings to enable gas exchange (for example see Lugones et al., 1999; Pareek, Allaway & Ashford, 2006).

The outer surface of the hyphal or spore walls in these cases is usually found to be composed of a layer of 10 nm wide rodlets composed of proteins which modify the biophysical properties of the wall surface; the most commonly encountered family of such proteins are called hydrophobins (Wessels, 1996; Wösten & de Vocht, 2000; Sunde et al., 2008; Cox & Hooley, 2009). Hydrophobins belong to a large, diverse group of related proteins found widely in the fungi; when expressed to the maximum they may constitute up to 10% of total wall protein. Each molecule consists of a hydrophobic domain and a hydrophilic domain; that is, they are amphipathic (a term you may have met in relation to the phospholipids that make up biological membranes, which also have a hydrophilic group at one end and a hydrophobic group at the other). Their amino-terminal part determines the hydrophilic side of the assemblage. The amphipathic structure of hydrophobins provides the molecules with an extraordinary potential array of functions:

  • enable hyphae to break through the air/water interface of fluid habitats;
  • provide the hydrophobicity required by hyphae and spores in contact with air;
  • participate in morphogenetic signalling, initiating conidiation and fruit body formation;
  • have important roles in tissue formation, particularly in controlling fluid and air spaces;
  • promote adhesion between the hydrophilic cell wall of the fungus and the hydrophobic surfaces of plants and insects, and so potentiate infection and aid penetration of the host surface;
  • interestingly, aerial conidia of Aspergillus, Penicillium and Cladosporium that have surface layers of hydrophobin do not activate the immune system; hydrophobin rodlets are said to ‘immunologically silence airborne moulds’, which means that although fungal spores are ubiquitous in the air we breathe they neither continuously activate host immunity nor induce inflammatory responses after inhalation (Aimanianda et al., 2009);
  • mediate symbiotic interactions with plant roots (mycorrhizas) and algae (lichens).

The molecules that do all this are relatively small proteins, usually around 100 amino acids, that have extensive homologies and characteristically contain signal sequences for secretion, and eight cysteine residues conserved in the same position. These eight conserved cysteine residues form four disulphide bridges and they prevent self-assembly of the hydrophobin in the absence of a hydrophilic–hydrophobic interface. Hydrophobins are unique to mycelial fungi but are expressed by both Ascomycota and Basidiomycota. Each fungus has genes for more than one, often more than ten different hydrophobins, and the genes are usually expressed at different times. In Schizophyllum commune the hydrophobin found in the vegetative hyphal wall differs from that expressed in the hyphal walls of the fruit body (Wessels, 1996).

Hydrophobins are excreted from the hyphal tip; if the hypha is in an aqueous environment, the hydrophobins pass into solution. But the protein molecules are able to self-assemble into covering films at the water/air (= hydrophilic/hydrophobic) interface and when a hypha emerges from the solution, the polypeptide polymerises on the surface of the hyphal wall, forming an array of parallel rodlets. Each hydrophobin molecule is bound to the fungal wall by its hydrophilic end; the hydrophobic domain being exposed to the outside world (Linder et al., 2005; Cox & Hooley, 2009). A difference in solubility of these assemblages divides hydrophobins into two groups: class I hydrophobins form highly insoluble membranes dissolved only by trifluoroacetic acid and formic acid, while assemblies of class II hydrophobins dissolve readily in ethanol or sodium dodecyl sulfate (SDS).

The hydrophobin assembly on the hypha reduces water movement through the wall, protecting from desiccation, but the exposed hydrophobic surface enables bonding to other hydrophobic surfaces. This happens because hydrophobes are not electrically polarised and the lowest energy-state for two hydrophobes is for them to bond together to exclude electrically polarised water molecules. A fungal wall coated with hydrophobins will be able to use this hydrophobic interaction to bond to other aerial hyphae, leading to the formation of multicellular hyphal structures. A hydrophobin-coated spore could also attach immediately and firmly to the hydrophobic (for example, waxy) surface of its plant or insect host; giving time for formation of appressoria or other penetration structures.

Within fungal tissues, and that includes the tissues of lichen thalli, hydrophobins provide control over the movement of water and gases within the tissue because the exposed hydrophobic domains prevent water logging in airspaces, and allow the fungus to control which channels through the tissue are used for movement of water and aqueous nutrients, and which are kept free of fluid and used for movement of gasses (Wösten & de Vocht, 2000; Sunde et al., 2008). The remarkable ability of hydrophobins to change the nature of a surface (they turn hydrophobic surfaces hydrophilic and hydrophilic surfaces hydrophobic) make hydrophobins interesting candidates for use in commercial and medical applications (Cox & Hooley, 2009; Cox, Aldred & Russell, 2009).

Another fungal wall protein that deserves specific mention is the glycoprotein known as glomalin, which is produced abundantly in the walls of hyphae and spores of arbuscular mycorrhizal fungi in soil and in roots. What makes this protein deserving of mention is the amount of it that is produced. It permeates soil organic matter, and can accounts for around 30% of the carbon in soil. In the soil, glomalin forms clumps of soil granules called aggregates that add structure to soil. It is such a major component of soil organic matter that the Agricultural Research Service of the United States Department of Agriculture, which is not normally sensationalist, published an article in the  September 2002 issue of its Agricultural Research magazine entitled Glomalin: hiding place for a third of the world’s stored soil carbon, which explains that: ‘Glomalin gives soil its tilth - that subtle texture that enables experienced farmers and gardeners to judge great soil by feeling the smooth granules as they flow through their fingers’ (CLICK HERE to view the page [requires Internet connection]).

Glomalin is produced only by members of the Glomeromycota, fungi that form arbuscular mycorrhizas. When first discovered it was clearly present in such quantity that it must make a massive contribution to the aggregation of soil particles. So it was first thought that it must be secreted or otherwise released into the soil by arbuscular mycorrhizal fungi specifically to control soil structure; a mechanism that has been called habitat engineering. The view being that increased soil aggregation (= improved tilth) would benefit the host plant, and thereby the associated mycorrhizal fungus, and so justify the energetic ‘cost’ of producing the glomalin. There is some experimental support for this idea though there is also evidence that glomalin is not secreted, but is covalently bound into the hyphal wall matrix where it protects the hypha. It may be that the characteristics that enable glomalin to protect the hyphal wall also allow the protein to promote soil aggregation (Driver, Holben & Rillig, 2005; Purin & Rillig, 2007).

Updated December 17, 2016