Fungal melanin detection by the use of copper sulfide-silver.

Silver-staining procedures were investigated for their effectiveness in identifying cell wall-based fungal melanins in live and fixed plastic embedded samples, particularly 1,8-dihydroxynaphthalene (DHN) based polyketide melanins. We developed a simple and reliable melanin-staining technique based on a silver accumulation method originally published for histological demonstration of heavy metal sulfides in mammalian tissues. Copper is bound to fungal melanin followed by formation of the copper sulfide at melanin sites in fungal cell walls, which then are amplified into vivid black stains using a silver enhancement step. The method demonstrates patterns of melanization in a range of fungal hyphae and is suitable for light and electron microscopy. Albino mutant fungi and normally nonmelanized fungi do not stain with the sulfide-silver technique. Mammalian melanocytes also were labeled by the technique, indicating its universality as a melanin probe.

Localization of fungal fimbriae by immunocytochemistry in pathogenic and nonpathogenic isolates of Venturia inaequalis.

Pathogenic and nonpathogenic isolates of Venturia inaequalis were grown in liquid culture. Hyphae were treated with two types of fimbrial antiserum (AU- and AV-1) and examined by immunofluorescent microscopy, in order to establish the distribution of fimbrial epitopes in whole cell mounts. The AV-1 antiserum was specific for the glycoprotein subunits while the AU-antiserum was specific for the protein moieties present on the fimbriae of Mycobotryum violaceum. The use of fimbrial antiserum with immunocytochemistry and transmission electron microscopy demonstrated a clear distinction between pathogenic and nonpathogenic isolates of V. inaequalis, based on the appearance of the fungal cell wall and the distribution of fimbrial epitopes labeled with AV-1 antiserum and immunogold complex. In actively growing hyphae of the pathogenic isolate, characterized by distinct cellular organelles, small vacuoles, and lipid bodies, fimbrial epitopes were concentrated in the fungal cell wall and were present minimally on the outer surface. In contrast, actively growing hyphae of the nonpathogenic isolate of V. inaequalis had extensive fine hair-like protrusions in the fungal cell wall which labeled with the AV-1 antiserum and immunogold. The distribution of fimbrial epitopes in V. inaequalis was highly dependent on the developmental growth stage of the fungal mycelium. Aging mycelia in both the pathogenic and nonpathogenic isolates of V. inaequalis were characterized by a large central vacuole and no label. In the pathogenic and nonpathogenic isolates of V. inaequalis grown in vitro, the distribution of fimbrial glycoprotein epitopes provided a more complex profile than that seen in M. violaceum.

Fungal fimbriae are composed of collagen.

Fungal fimbriae are surface appendages that were first described on the haploid cells of the smut fungus, Microbotryum violaceum. They are long (1-20 microm), narrow (7 nm) flexuous structures that have been implicated in cellular functions such as mating and pathogenesis. Since the initial description, numerous fungi from all five phyla have been shown to produce fimbriae on their extracellular surfaces. The present study analyses the protein component of M.violaceum fimbriae. The N-terminus and three internal amino acid sequences were determined. All four show a strong similarity to sequences which are characteristic of the collagen gene family. Enzymatic digests and immunochemical analyses support this finding. Based on these results, it is suggested that the proteinaceous subunits of fimbriae should be termed fungal collagens. Previously, collagen has been found only among members of the kingdom Animalia where it is the principal component of the animal extracellular matrix and is the most abundant animal protein. The unexpected finding of collagen in the members of the Mycota suggests that it may have evolved from a common ancestor that existed before the divergence of fungi and animals. Further, native fungal fimbriae can function as a mammalian extracellular matrix component. They can act as a substratum which permits animal cells to adhere, spread, and proliferate in a manner similar to animal collagens. The implications of this finding to both phylogeny and pathology are discussed.

Evidence that fimbriae of the smut fungus Microbotryum violaceum contain RNA.

The cells of the fungus Microbotryum violaceum produce many long, fine surface hairs that are similar in size and morphology to bacterial pili or fimbriae. These fungal fimbriae are assembled from 74 kDa glycoprotein subunits. We now present evidence that these fimbriae also have a RNA component. Isopycnic centrifugation of fimbriae in caesium chloride produced one band at a density intermediate to that of protein and nucleic acid. The absorbance spectrum of the intact fimbriae was consistent with that of a nucleoprotein. After extraneous RNAs were enzymically removed from the purified fimbrial preparation, disruption of the fibrils resulted in the release of not only the 74 kDa glycoprotein subunits, but also a 30 base single-stranded RNA species. To our knowledge, this is the first example of extracellular RNA as a component of a surface appendage.

Somatic nuclear division in the sporidia of Ustilago violacea. III. Ultrastructural observations.

The paper provides detailed ultrastructural observations on nuclear division in the smut fungus Ustilago violacea and is based on previous light-microscopic work outlining the division in living and stained cells. The division as in many other Basidiomycetes is not intranuclear, but occurs within a partially disrupted membrane. The division takes place after migration of most of the nucleus into the bud cell, after limited breakdown of the nuclear membrane, and after the formation of a spindle between two spindle-pole bodies (SPB). The remaining part of the nucleus containing the nucleolus is left behind in the parent cell and degenerates there. The SPB, as in other Basidiomycetes, is a dome-shaped relatively structureless body, quite distinct from the flat plaques of many Ascomycetes and the elaborat centrioles of Phycomycetes. The SPB divides shortly before migration into the daughter cell and invariably is located at the apex of the migrating nucleus. Nuclear division is completed when the two masses of chromatin clustered about each of the SPB's are separated as the spindle elongates. One daughter neculeus reforms in the bud and the other is reformed in the mother cell. Cells fixed and stained by conventional light-microscopic methods were examined in the light of the electron-microscopic observations to determine whether these procedures induce artefacts. Aceto-orcein and Giemsa when used cold were found to produce relatively artefact-free preparations. However, previous results in which the cells were warmed gently in these stains are now seen to contain artefacts in the form of contracted chromatinic granules often arranged in chains. These artefacts may provide useful information but clearly they must be interpreted cautiously until the nature of the changes induced by heating are known.

Somatic nuclear division in the sporidia of Ustilago violacea. IV. Microtubules and the spindle-pole body.

In unbudded cells of the anther smut fungus Ustilago violacea there is a dome-shaped spindle-pole body (SPB) consisting of a core 0.1 mum in diameter surrounded by a ribosome-free region 0.3-0.4 mum in diameter lying in a pocket of the nuclear membrane. After budding the nucleus moves towards the bud and begins to rotate rapidly. At about this stage the SPB divides into two parallel bars each about 0.1-0.15 mum in diameter and 0.3 mum long, separated by a distance of about 0.3 mum. Microtubules associated with the nuclear membrane but not with the SPB are present at the time of nuclear rotation. These microtubules disappear when rotation stops. Microtubules attached to the SPB are found during migration of the chromatinic portion of the nucleus into the bud cell. These microtubules disappear when migration stops and the nuclear membrand begins to break down. The twin SPB bars appear to move into the nucleus through a break in the membrane and begin to move apart forming a spindle about 1 mum long. Chromosomal microtubules (one per kinetochore) were found in several serial sections, and in addition there appeared to be several continuous microtubules present. The separation of the two chromatinic masses appeared to result from elongation of the continuous microtubules to about 3 mum long. Cytoplasmic microtubules and spindle microtubules were both found attached to the SPB as it elongated and one nucleus returned to the mother cell. The paper concludes with a discussion of the SPB as a multifuncitonal control center affecting nuclear migration, spindle formation, membrane breakdown and synthesis, karyogamy, conjugation, budding, chromosomal movement, replication, and disjunction.

Fungal fimbriae. II. Their role in conjugation in Ustilago violacea.

During conjugation in the anther smut fungus Ustilago violacea cells of opposite mating type first pair tightly and then develop a conjugation tube or bridge between them. The cells of both mating types are covered in long fine hairs or fimbriae, some of which appear to end in knobs. Experiments involving enzyme treatments of the cell surface indicate that these fimbriae do not play an essential role in cell pairing, instead pairing seems to be initiated when one or both mating types produce amorphous masses of alpha-amylase-sensitive material. Electron micrographs, enzyme and inhibitor studies, and experiments using restrictive temperatures suggest, however, that fimbriae may be essential for the later stages of conjugation i.e. development of the conjugation tube. If so, it is suggested that they may permit the exchange of macromolecules between the conjugating cells, initiating localized wall-softening and wall-breakdown.

Fungal fimbriae. III. The effect on flocculation in Saccharomyces.

Flocculent strains of Saccharomyces cerevisiae and S. carlsbergensis (S. uvarum) produced many short (0.5 mum) hairs (fimbriae) on the outer cell walls. Non-flocculent strains produce few fimbriae. Cells that are flocculent in wort but not in defined medium produce fimbriae only in the former medium. Cells treated with pronase lose both their fimbriae and the ability to flocculate. Cells treated with alpha-amylase retain some fimbriae but lose the ability to flocculate. It is suggested that the fimbriae may be the surface mannan-protein complexes known to be involved in flocculation and also in protein secretion. Haploid cells of both mating types also produced fimbriae, some of which apparently have a terminal knob.

Fungal fimbriae. I. Structure, origin, and synthesis.

Fine hair-like appendages on the cell walls of the another smut Ustilago violacea are described. These hairs are termed fimbriae because of their close similarity to the fimbriae (pili) found on certain Gram-negative bacteria. Cells of U. violacea may carry more than 200 fimbriae varying in length from about 0.5 mum to over 10 mum, and having a diameter of about 60-70 A. Some fimbriae produce knobs similar to those found on bacterial sex fimbriae. Log-phase cells are the most densely fimbriated, while stationary phase cells are devoid of fimbriae. The cells can be defimbriated by sonication, high-speed agitation, or centrifugation through a 40% sucrose solution. The fimbriae can regenerate in these defimbriated cells in about 1 h. This regeneration is inhibited by both cycloheximide and rifampin, but not by chloramphenicol and therefore appears to depend on de novo protein synthesis on cytoplasmic ribosomes. Similar long fimbriae are found on U. maydis and Leucosporidium (Candida) scottii. Short fimbriae, about 0.5 mum long, were found on all the other species of yeast-like fungi examined (Rhodotorula, Saccharomyces, Schizosaccharomyces, Hansenula, Lipomyces, Nadsonia, and Torulopsis spp.).