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PubMed2005

Hyphal differentiation in the exploring mycelium of Aspergillus niger.

Mycelial fungi play a central role in element cycling in nature by degrading dead organic material such as wood. Fungal colonization of a substrate starts with the invasion of exploring hyphae. These hyphae secrete enzymes that convert the organic material into small molecules that can be taken up by the fungus to serve as nutrients. Using green fluorescent protein (GFP) as a reporter, we show for the first time that exploring hyphae of Aspergillus niger differentiate with respect to enzyme secretion; some strongly express the glucoamylase gene glaA, while others hardly express it at all. When a cytoplasmic GFP was used, 27% of the exploring hyphae of a 5-day-old colony belonged to the low expressing hyphae. By fusing GFP to glucoamylase and by introducing an ER retention signal, this number increased to 50%. This difference is due to cytoplasmic streaming of the reporter in the former case, as was shown by using a photo-activatable GFP. Our findings indicate that a fungal mycelium is highly differentiated, especially when taking into account that hyphae in the exploration zone were exposed to the same nutritional conditions.

Aspergillus niger↗
PubMed2005

Asp f6, an Aspergillus allergen specifically recognized by IgE from patients with allergic bronchopulmonary aspergillosis, is differentially expressed during germination.

BACKGROUND: Aspergillus fumigatus is a pathogenic mould causing allergic and invasive respiratory diseases. Allergic bronchopulmonary Aspergillosis (ABPA) is a severe pulmonary complication resulting from hypersensitivity to A. fumigatus proteins. Aspergillus allergen Asp f6 is recognized by IgE from ABPA patients, but not from sensitized individuals, a fact that can be used to differentiate between these two groups of allergic patients. METHODS: Proteins from hyphae, resting and germinating conidia of A. fumigatus were compared by SDS-PAGE. Protein identification was performed using MALDI-TOF mass spectrometry. Recombinant A. fumigatus allergens were used to isolate specific monoclonal antibodies (mab) from a hybridoma bank generated against Aspergillus proteins. RESULTS: A hyphae-specific 23 kDa A. fumigatus protein was identified as the allergen Asp f6/manganese-dependent superoxide dismutase (MnSOD). Differential expression of MnSOD was confirmed by immunoblot using a specific mab. In contrast, Asp f8 another intracellular, but not ABPA-specific allergen, was detected in hyphae and conidia. CONCLUSIONS: Aspergillus fumigatus is able to colonize its environment by the formation of hyphae. Hyphae are found in the lung of ABPA patients, but not in patients suffering from atopic asthma. Our finding that Asp f6 is specifically expressed in hyphae might explain why an IgE response to Asp f6 is specific for ABPA patients.

Allergens↗
PubMed2003

Asexual development is increased in Neurospora crassa cat-3-null mutant strains.

We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3(RIP) colonies were sensitive to H(2)O(2), indicating that wild-type (Wt) resistance to external H(2)O(2) was due to CAT-3. cat-3(RIP) colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3(RIP) mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3(RIP) mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

Animals↗
PubMed1976

[Microscopic studies of Streptomyces hygroscopicus growth kinetics].

Growth kinetics, branch formation, and cytological properties of mycelial growth of Streptomyces hygroscopicus on solid media were investigated by phase-contrast microscopy using a microculture method. Measurements were made on taken photographs of the growing hyphae from the beginning of spore germination up to maximal 18 hours. The specific growth rate of the germ tube was much higher than the specific growth rate of the mycelium. The doubling time of the total length of the mycelial hyphae and the doubling time of branch formation was quite the same for the period investigated. After a short time of outgrowth each individual hypha grows at a constant rate, i.e. the length increases linearly, but the growth kinetics of the whole mycelium becomes exponentially by branching. It seems, that nucleoids only divide in that part of a hypha between the tip and the nearest branch. A cell unit (1.4-1.9 mum on different media) could be calculated by the length of a hypha and the number of nucleoids. A hypha is growing with the cell unit at the tip or with the polar cap only. No interkalary growth in length could be found. Branches were formed only up to about 100 mum from the tip on complex medium and up to about 50 mum from the tip on mineral salt medium. A simple method of mycelial growth has been developed. Some properties showing the connection between Streptomycetes and prokaryotic organisms on one hand and hyphal growing fungi on the other hand are discussed.

Culture Media↗
PubMed1997

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press

Journal Article↗
PubMed1988

Unusual structures of Sporothrix schenckii.

A series of environmental and clinical isolates of Sporothrix schenckii being studied in our laboratories under standard conditions of temperature, humidity and media, displayed, in some of the isolates, large dematiaceous hyphae. The large hyphae could be seen to produce the fine hyphae associated with S. schenckii on which typical microconidia developed. Typical microconidia also developed occasionally on the large hyphae, and strands of the unusually large hyphae also were seen to form arthroconidia. Some strains formed large, thin-walled cells on thin and/or thick hyphae resembling the 'balloon cells' seen in mature colonies of Trichophyton tonsurans. All strains which have demonstrated these various and unusual structures histopathologically produced typical sporotrichosis in laboratory animals. When reisolated from these animals, the cultures again exhibited the unusual structures, as well as those typical of S. schenckii.

Air Microbiology↗
PubMed1997

Flow sensing in mycelial fungi.

Hyphae of a variety of mycelial fungi were shown to reorient their direction of growth to grow upstream in a continuous flowing current of liquid medium. Among those tested fungi, Aspergillus niger hyphae was most polarized towards the upstream. Hyphae of A. niger which had been attached to poly-L-lysine coated plates on the base of a continuous-culture flow cell were exposed to a current of medium of flow rates in the range of 0-6400 microns s-1 for 1-4 h. Hyphal tips quickly reoriented towards the upstream of the flowing medium and became aligned parallel to the flow axis as they grew larger. At flow rates above 1600 microns s-1, however, the percentage of polarization of hyphae reached a constant level. With other fungi, germ tube emergence was not polarized but hyphae and branches grew towards the upstream. There was no detectable difference in the pH and oxygen concentration of the flowing medium between inlet and outlet of the flow cell. This result suggests that hyphae of mycelial fungi sense transient flow of liquid medium over their surface and reorient their direction to grow upstream.

Aspergillus niger↗
PubMed2003

Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil.

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.

Agriculture↗
PubMed2005

Colonization of cantaloupe roots by Monosporascus cannonballus.

Penetration of Monosporascus cannonballus into and growth within cantaloupe roots was studied using light and electron microcopy. Germ tubes penetrated the epidermis, and hyphae grew, without branching, almost directly to the xylem. The hyphae traversed the endodermis into protoxylem cells, and then grew extensively within the lumen of metaxylem vessels. Eventually, the hyphae grew back out into the cortical cells. A relatively low percentage of cells within both the cortex and xylem of lesions contained hyphae. The hyphae were generally localized within the lesion and could rarely be isolated more than 2 mm away from the margin of the lesion. Regardless of tissue type, hyphae were predominately intracellular. M. cannonballus appeared to be most similar to vascular wilt pathogens in its mode of parasitism, but does not spread via the vascular system to above-ground plant tissues.

California↗
PubMed2005

N-cadherin mediates endocytosis of Candida albicans by endothelial cells.

Candida albicans is the most common cause of fungal bloodstream infections. To invade the deep tissues, blood-borne organisms must cross the endothelial cell lining of the vasculature. We have found previously that C. albicans hyphae, but not blastospores, invade endothelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the endothelial cell receptor that mediates the endocytosis of C. albicans. We determined that endocytosis of C. albicans was not mediated by bridging molecules in the serum and that it was partially dependent on the presence of extracellular calcium. Using an affinity purification procedure, we discovered that endothelial cell N-cadherin bound to C. albicans hyphae but not blastospores. N-cadherin also co-localized with C. albicans hyphae that were being endocytosed by endothelial cells. Chinese hamster ovary (CHO) cells expressing human N-cadherin endocytosed significantly more C. albicans hyphae than did CHO cells expressing either human VE-cadherin or no human cadherins. The expression of N-cadherin by the CHO cells resulted in enhanced endocytosis of hyphae, but not blastospores, indicating the selectivity of the N-cadherin-mediated endocytosis. Down-regulation of endothelial cell N-cadherin expression with small interfering RNA significantly inhibited the endocytosis of C. albicans hyphae. Therefore, a novel function of N-cadherin is that it serves as an endothelial cell receptor, which mediates the endocytosis of C. albicans.

Animals↗
PubMed1999

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote.

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.

Cell Membrane↗
PubMed1985

A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT.

A rapid colorimetric assay for viability of fungi that uses the tetrazolium salt MTT is described. All live species of a wide range of medically important fungi that were tested reduced MTT, with resultant intracellular formation within several hours of clearly visible purple crystals of MTT-formazan. The assay proved particularly useful in measuring viability of individual hyphae; greater than 99% of live Aspergillus fumigatus and Rhizopus oryzae hyphae stained purple when incubated with MTT compared with 0% of hyphae killed by any of a variety of methods. The MTT method for measuring fungal viability closely correlated with a slide-culture technique when killing of A. fumigatus hyphae by graded concentrations of hydrogen peroxide was compared. MTT-formazan was easily extracted from the fungi by alcohol, a procedure allowing spectrophotometric quantification of fungal killing in both cell-free and cellular systems. A linear relation between inocula and MTT reduction of several fungi, including Candida albicans blastospores and A. fumigatus hyphae, was demonstrated. Human leukocytes killed 80.3% of C. albicans blastospores as measured by the MTT assay, compared with 70.5% as measured by dilutions and pour plates. Killing of A. fumigatus hyphae by human leukocytes was also quantitated by using the MTT assay.

Fungi↗
PubMed2004

Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis.

We show that two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis, that have been chemically fixed with a combination of methylglyoxal and formaldehyde and stained with Alexa phalloidin. In approximately one half of the hyphae examined, an F-actin depleted zone within the apical F-actin cap was observed. The remaining hyphae had a continuous apical cap. In live, growing hyphae two types of cytoplasmic organization were observed at the tips, one in which a clear zone was present which may correlate with the F-actin depleted zone, and one where no such clear zone existed which may represent the continuous cap. We suggest that the F-actin depleted zone may be a structural component of the actin network in a subpopulation of oomycete hyphae and may be comparable to similar F-actin depleted zones at the apices of other tip growing cells such as pollen tubes and root hairs. This observation has implications with regard to models of hyphal extension. Hyphae fixed with formaldehyde alone showed continuous apical F-actin caps. Our ability to resolve the F-actin depleted zone likely reflects the cross-linking capabilities of methylglyoxal. The methylglyoxal-formaldehyde combination fixative gave more stained hyphae, brighter staining and more complete staining of F-actin compared to formaldehyde alone.

Achlya↗
PubMed1978

Resistance of selected saprobic and zoopathogenic fungi to cycloheximide.

Spore germination was used as an assay to measure the sensitivities of selected fungi (Achlya bisexualis, Cladosporium sp., Trichophyton mentagrophytes and Microsporum gypseum) to cyclohexamide and to determine their abilities to adapt to the drug. Two patterns of response were noted. The saprobes, A. bisexualis and Cladosporium sp., demonstrated acquired resistance. Spores from hyphae previously exposed to cycloheximide either germinated in the presence of concentrations of the drug that completely inhibited spores from unexposed hyphae (Achlya), or germinates with a shorter lag and to a greater extent in the presence of the antibiotic than did spores from unexposed hyphae (Cladosporium). Hyphae of Achlya adapted at concentrations of cycloheximide in which spores did not germinate and hyphae of Cladosporium adapted more rapidly than spores. Achlya adapted to only 12 muM-cycloheximide whereas Cladosporium acquired resistance to 18 mM-cycloheximide. These fungi lost this acquired resistance after a single transfer to media lacking cycloheximide. The zoopathogens, T. mentagrophytes and M. gypseum, had a contrasting response, indicating constitutive resistance. Conidia from unexposed hyphae showed 90 to 100% germination on media containing up to 18 mM-cycloheximide; prior exposure to the drug did not affect their response.

Adaptation, Physiological↗
PubMed1998

Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans.

The role of actin in apical growth and enzyme secretion in the filamentous fungus Aspergillus nidulans was studied by treating the hyphae with cytochalasin A (CA), which inhibits actin polymerization. Indirect immunofluorescence microscopy revealed actin at the tips of main hyphae and branches, and at the site of developing septa. CA inhibited the growth of the fungus and changed the growth pattern of hyphal tips from cylindrical tubes to spherical beads. The regions with swellings showed no actin fluorescence, and neither was actin seen in association with septa. After 4 h exposure, hyphae were able to resume the normal tip growth pattern in the presence of CA for a short period of time and new cylindrical hyphae, with actin fluorescence at the apex, emerged from the swollen tips. Later, the tips of the hyphae swelled again, which led to a beaded appearance. We also studied the effect of CA on the secretion of alpha- and beta-galactosidase. alpha-Galactosidase is secreted into the culture medium, whereas beta-galactosidase remains in the mycelium, with part of its activity bound to the cell wall. When A. nidulans mycelium was incubated in the presence of CA, a reduction in the secretion of alpha-galactosidase into the culture medium and a decrease in the alpha- and beta-galactosidase activities bound to the cell wall was detected. However, the CA dose used for the hyphae did not modify the secretion of the enzymes from protoplasts. Results described here provide evidence that a polymerized actin cytoskeleton is required for normal apical growth, hyphal tip shape and polarized enzyme secretion in A. nidulans. Cytochalasin-induced disruptions of the actin cytoskeleton could result in the alterations of apical growth and inhibition of enzyme secretion observed by blocking secretory vesicle transport to the apex.

Actins↗
PubMed1996

Dynamic expression of cell-surface antigens probed with Candida albicans-specific monoclonal antibodies.

IgG hybridomas were produced with preferentially reacted with cell-surface antigens of either yeast cells or hyphae of Candida albicans. Four mAbs were used in an immunostaining procedure to follow the expression dynamics of these antigens in media supplemented with glucose or galactose. Yeast cell growth was analysed during the lag phase, the early- and late-exponential phases and the stationary phase, and mycelium formation was analysed between 0.5 and 24 h induction at 37 degrees C. It appears that yeast cell-surface antigens 5C11 and 2E11 are expressed throughout all phases of yeast cell growth as well as on young hyphae after up to 1 h induction. Longer hyphae only faintly react with these two mAbs as they switch to hyphal cell-surface antigens 2G8 and 4E1 after 3 h induction. The reactivity to mAbs 2G8 and 4E1 was induced after a 3 h temperature shift and was confined to the terminal third of growing mycelia. Growth and hyphae induction in galactose prolonged the reactivity of young hyphae with the two anti-yeast-cell mAbs, whereas the expression of surface antigens 2G8 and 2E11 appeared delayed and desynchronized on hyphae. Whereas a similar reactivity was found with ten ATCC strains of C. albicans, four clinical isolates had a unique pattern of reactivity. Immunoblot analyses of DTT extracts of cell-surface constituents indicated that the antigens were proteinaceous in nature and showed that yeast-cell antigens 5C11 and 2E11 are detected in four bands between 68 and 104 kDa, whereas mycelial antigens 4E1 and 2G8 are detected in 117 kDa and 104 kDa bands found in mycelial but not in yeast-cell extracts. Present data support the concept of a dynamic balance in the expression of phase-specific antigens in C. albicans.

Antibodies, Fungal↗
PubMed1993

Interfacial Self-Assembly of a Fungal Hydrophobin into a Hydrophobic Rodlet Layer.

The Sc3p hydrophobin of the basidiomycete Schizophyllum commune is a small hydrophobic protein (100 to 101 amino acids) containing eight cysteine residues. Large amounts of the protein are excreted into the culture medium as monomers, but in the walls of aerial hyphae, the protein is present as an SDS-insoluble complex. In this study, we show that the Sc3p hydrophobin spontaneously assembles into an SDS-insoluble protein membrane on the surface of gas bubbles or when dried down on a hydrophilic surface. Electron microscopy of the assembled hydrophobin shows a surface consisting of rodlets spaced 10 nm apart, which is similar to those rodlets seen on the surface of aerial hyphae. When the purified Sc3p hydrophobin assembles on a hydrophilic surface, a surface is exposed with high hydrophobicity, similar to that of aerial hyphae. The rodlet layer, assembled in vivo and in vitro, can be disassembled by dissolution in trifluoroacetic acid and, after removal of the acid, reassembled into a rodlet layer. We propose, therefore, that the hydrophobic rodlet layer on aerial hyphae arises by interfacial self-assembly of Sc3p hydrophobin monomers, involving noncovalent interactions only. Submerged hyphae merely excrete monomers because these hyphae are not exposed to a water-air interface. The generally observed rodlet layers on fungal spores may arise in a similar way.

Journal Article↗
PubMed1992

Allergic contact dermatitis in shiitake (Lentinus edodes (Berk) Sing) growers.

A 42-year-old female shiitake grower was investigated to clarify the etiology of skin lesions which developed during the planting of shiitake hyphae into bed logs. She complained of repeated eczematous skin lesions during the planting season, from March to July, for 10 years. She handled 7,000 pieces of small conic blocks made of beech, with shiitake hyphae attached to their surface, per day, and 300,000 pieces altogether per season. She was positive on patch testing with extracts of shiitake hyphae. In contrast, female shiitake growers with skin lesions associated with work other than planting, and without skin lesions, were negative on patch testing to the hyphae. Moderate allergenicity was observed to extracts of shiitake hyphae in a guinea pig maximization test. These findings indicated the etiology of skin lesions in shiitake growers to be allergic contact dermatitis induced by shiitake hyphae.

Adult↗