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Different temporal and spatial expression of two hydrophobin-encoding genes of the edible mushroom Agaricus bisporus.

In a search for genes that are only expressed in fruit bodies of the basidiomycete Agaricus bisporus, two cDNAs, hypA and hypB that encode hydrophobins have been isolated previously. In this study, the structure of hypB is resolved and it is shown that the two genes are differentially expressed, indicating that the encoded hydrophobins serve different functions in A. bisporus mushrooms. hypB encodes a polypeptide (HYPB) of 119 aa that shows little sequence identity with HYPA apart from the characteristic arrangement of eight cysteines found exclusively in hydrophobins. The temporal and spatial expression of the two hydrophobin-encoding genes during fruit body development was compared using Northern analysis and in situ hybridization. Accumulation of hypA mRNA was found in tissue fractions consisting of undifferentiated white hyphae. In situ hybridization showed that the highest hypA mRNA levels are not found in the outermost cell layers of the pileipellis but in the cell layers adjacent to that. The highest level of expression of hypB occurs early in development when the primordium differentiates into densely packed, randomly oriented cap hyphae and loosely packed, vertically oriented stipe hyphae. In mature mushrooms, a strong accumulation of hypB transcripts was found only in the transitional zone between cap and stipe tissue, demonstrating that transcription regulation of hypB is clearly distinct from hypA.

Agaricus↗

In vitro survival and germination of Candida albicans in the presence of nitrogen compounds.

The in vitro effect of nitric oxide (NO) and nitrite on blastoconidia and hyphae of Candida albicans was studied. Sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) were used as NO donors. Both minimal and complex media at two pH values, 7.0 and 4.5, were used for the assays. Blastoconidia were more susceptible than hyphae to NO. The NO effect on blastoconidia was greater at acidic pH. Nitrite affected the viability of blastoconidia in complex medium. The percentage germination and the relative rate of elongation of hyphae were both enhanced when NO was present in acidic conditions.

Candida albicans↗

The Aspergillus fumigatus cell wall is organized in domains that are remodelled during polarity establishment.

Aspergillus fumigatus is a life-threatening and increasingly frequent pathogen of the immunocompromised. Like other filamentous fungi A. fumigatus grows in a highly polar manner, adding new cell wall to the apical region of hyphae. mAbs were raised against isolated A. fumigatus cell walls. Fifteen antibodies bound reproducibly to isolated cell walls in ELISAs and to the walls of intact cells in immunofluorescence experiments. Surprisingly, individual mAbs showed distinct patterns of localization. Six antibodies labelled exclusively conidial or basal regions, seven labelled apical regions and a single antibody labelled both basal and apical regions of hyphae. Ten antibodies did not label the walls adjacent to septa. In double labelling experiments with representative mAbs there was little or no overlap between epitopes recognized. These labelling patterns suggest that the wall is made up of basal and apical domains that differ in composition or organization and that the wall region flanking septa differs from other regions of the lateral wall. In time-course experiments of early A. fumigatus growth, mAb16C4 failed to label isotropically expanding cells and labelled emerging germ tubes and branches. The same mAb failed to label the Aspergillus nidulans swoC mutant, which is defective in polarity establishment. However, mAb16C4 did label the A. nidulans swoA mutant, which completes polarity establishment, but is defective in polarity maintenance. Thus, mAb16C4 appears to recognize a cell wall change that occurs during polarity establishment. In immunogold labelling and transmission electron microscopy (TEM) experiments, conidia, basal regions and apical regions of thin-sectioned cells labelled with mAb16C4. That only apical regions labelled in intact cells (immunofluorescence) while conidial, basal and apical regions labelled in thin-sectioned cells (TEM) suggests that the 16C4 epitope is present along the whole hypha, but is masked everywhere except the tip until polarity establishment. That is to say, some remodelling of the wall during polarity establishment exposes the 16C4 epitope. The 16C4 epitope was partially purified from A. fumigatus total protein by passage through hydrophobic interaction and anion-exchange columns. The resulting single ELISA-positive fraction showed relatively few bands by SDS-PAGE and silver staining and a strong band by Western blotting with the16C4 mAb. Sequencing of the fraction yielded a predicted peptide with a 6-amino acid exact match to a region of the Cat1 protein previously identified as an immunodominant A. fumigatus catalase that localizes to the cell wall and is secreted to the medium. Experiments are under way to determine if mAb16C4 recognizes Cat1 or another protein that co-purifies with Cat1.

Antigens, Fungal↗

Induction of Defense-Related Ultrastructural Modifications in Pea Root Tissues Inoculated with Endophytic Bacteria.

The stimulation exerted by the endophytic bacterium Bacillus pumilus strain SE34 in plant defense reactions was investigated at the ultrastructural level using an in vitro system in which root-inducing T-DNA pea (Pisum sativum L.) roots were infected with the pea root-rotting fungus Fusarium oxysporum f. sp. pisi. In nonbacterized roots, the pathogen multiplied abundantly through much of the tissue including the vascular stele, whereas in prebacterized roots, pathogen growth was restricted to the epidermis and the outer cortex In these prebacterized roots, typical host reactions included strengthening the epidermal and cortical cell walls and deposition of newly formed barriers beyond the infection sites. Wall appositions were found to contain large amounts of callose in addition to being infiltrated with phenolic compounds. The labeling pattern obtained with the gold-complexed laccase showed that phenolics were widely distributed in Fusarium-challenged, bacterized roots. Such compounds accumulated in the host cell walls and the intercellular spaces as well as at the surface or even inside of the invading hyphae of the pathogen. The wall-bound chitin component in Fusarium hyphae colonizing bacterized roots was preserved even when hyphae had undergone substantial degradation. These observations confirm that endophytic bacteria may function as potential inducers of plant disease resistance.

Journal Article↗

Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis.

We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic alpha-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant-fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.

Amino Acid Sequence↗

Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae. Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene (glkA). Co-transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis. The size of the deletion was found to be 7.4 kb. Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.

Bacterial Proteins↗

Ultrastructure of Trichophyton violaceum.

BACKGROUND: The ultrastructure of different fungi have been described in recent literature; however, few reports have concerned dermatophytes. METHODS: Fine structure of Trichophyton violaceum was observed using electron microscopy. RESULTS: Hyphae appeared as branched segmented filaments with a cell wall consisting of two layers. Thin electron dense plasma membrane was evident continuous with the inner surface of the cell wall. Septal walls with central pores and a uniform electron opacity plugs were seen. The hyphae were multinucleated and the cytoplasm contained endoplasmic reticulum, mitochondria, and vacuoles. CONCLUSIONS: Although these findings showed ultrastructural similarities of the cell wall with Microsporum canis, Microsporum gypseum and Microsporum audouinii, there were some points of difference in the ultrastructure of the hyphae in Epidermophyton floccosum, and in the mitochondria of Trichophyton mentagrophytes as described by other investigators.

Microscopy, Electron↗

Outbreak of disseminated zygomycosis and concomitant pulmonary aspergillosis in breeder layer cockerels.

Disseminated zygomycosis and concomitant pulmonary aspergillosis were diagnosed in breeder layer cockerels. Five- to 9-week-old breeder layer cockerels with a history of an increased mortality rate were presented to a diagnostic laboratory for examination. On necropsy, large, multifocal, firm, tan, nodules were observed in the lungs, air sacs, peritoneum, livers, spleens and kidneys. On histopathology, mixed populations of zygomycetes and Aspergillus hyphae were observed in the granulomas in the lungs, and zygomycete hyphae were observed in the granulomas in the air sacs, peritoneum, livers, spleens and kidneys. No bacteria were isolated from any of the lesions. Aspergillus fumigatus was isolated from the lung lesions only and hyphae that were consistent with those of a Rhizopus spp. were isolated from the lesions in several organs. Pullets, which were kept together with the cockerels from the day they were hatched, were not affected. The absence of infection in the pullets, which were kept together with the cockerels, suggests that the cockerels were either infected during incubation, with the fungi penetrating the egg shell, or that they were infected during hatching before they were mixed with the pullets.

Animals↗

[The significance of secretory and structure-associated proteases of Aspergillus fumigatus for the pathogenesis of invasive aspergillosis].

In the course of invasive aspergillosis, Aspergillus fumigatus is capable of penetrating any tissue of the host. Secretory proteinases of the fungus might facilitate the hyphae to grow through fibrillar proteins like elastin and collagen. However, using systemic infection models, no significantly reduced virulence could be shown with fungal mutants deficient for all known secretory proteinases. Thus, secretory proteinases might be of minor relevance for the pathogenesis of invasive aspergillosis. In addition, microscopic examination of aspergilli penetrating vessel walls did not reveal obvious lysis of wall proteins, thus emphasizing a mechanical disruption of fibrillar proteins by the growing hyphae. However, a strictly localized proteolysis at the tips of growing hyphae caused by wall associated proteinases might be involved. Candidates for such a mechanism are the activities of aspartic and serine proteinases which we have discovered in the cell wall fraction of A. fumigatus.

Aspergillosis↗

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells.

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.

Adaptation, Physiological↗

Stimulation of hyphal growth in anaerobic cultures of Mucor rouxii by extracellular trehalose. Relevance of cell wall-bound activity of acid trehalase for trehalose utilization.

In fungi, the hydrolysis of extracellular trehalose is carried out by acid trehalases. These secretory glycoproteins may be more abundant either in the vacuolar compartment, like in yeast, or at the cell surface, such as in many filamentous fungi. The relative efficiency of these two compartments for the utilization of extracellular trehalose was investigated using as a model the dimorphic fungus Mucor rouxii, which produces yeast-like cells under a CO(2) atmosphere, or hyphae in the presence of air. Under CO(2), cultures supplemented with glucose produced yeast-like cells devoid of acid trehalase activity. On the other hand, trehalose-supplemented cultures developed hyphae exhibiting cell wall-bound and intracellular acid trehalase activity. Glucose-grown yeast-like cells supplemented with trehalose after glucose exhaustion, induced intracellular activity of acid trehalase, but no activity was detected at the cell surface. Even endowed of significant intracellular activity of acid trehalase, these cells did not grow further. When exposed to air these yeast-like produced germ tubes exhibiting cell wall-bound acid trehalase activity. These results suggest that the utilization of extracellular trehalose as a source of carbon for growth requires the localization of acid trehalase activity at the cell surface. Our results also show that extracellular trehalose elicits a morphogenetic phenomenon, inducing the formation of hyphae which are the physiological support for acid trehalase activity.

Anaerobiosis↗

Onychomycosis (trichophyton mentagrophytes). A scanning electron microscopic observation.

Nails from four patients, infected with dermatophytes, were investigated with the scanning electron microscope (SEM) to gain insight into the spatial arrangements of the dermatophytes within the nail. Fungal hyphae could be detected in nails of all four patients. A toenail from one patient infected with Trichophyton mentagrophytes was more extensively studied and the results are presented in this paper. Light microscopic observations with bright field illumination and Nomarski interference contrast confirmed the dermatophytic infection. Fungal hyphae found with the SEM distally on the ventral part of the toenail showed typical T. mentagrophytes structures and the comparison with cultured material clearly demonstrated the correspondence in morphology and size. Besides the fine structural morphology of the invasion of fungal hyphae into the nail plate between the horny cells and/or directly into corneocytes, "tunnel"-like holes were observed in paraffin embedded sections after removal of the paraffin. Scanning electron microscopy, with its enormous focal depth, yielded far more information than light microscopic techniques about the three dimensional behavior of dermatophytes in the nail.

Foot Dermatoses↗

Langerhans cells in candidal leukoplakia.

Using histochemical and immunohistochemical methods, we examined specimens of candidal leukoplakia from the oral mucosa of 5 smokers to determine the morphological relationships between Candida and Langerhans cells (LC) in tissue sections. LC were fairly evenly distributed in control sections, but had a patchy distribution in lesions. Fewer LC were found in lesions than in control tissue, but the difference was not statistically significant. Candidal antigens were not detected on LC by the methods used, but we found ATPase-positive LC among, or at least near, intraepithelial candidal hyphae. However, sections double-reacted with anti-Candida and T6 antibodies to label candidal antigens and LC, respectively, showed a clear zone of epithelium between the T6-positive LC and the candidal hyphae. The difference in distribution of ATPase-positive and T6-positive LC may indicate locations of 2 subtypes of LC, or a change in T6 antigen expression by the LC closest to the candidal hyphae.

Adult↗

Denture stomatitis-yeast occurrence and the pH of saliva and denture plaque.

The study comprised 30 denture wearers with generalized simple or granular inflammation in the palate and 30 without (controls). )easts, mostly Candida species, were cultivated from the maxillary dentures of all subjects with inflammation and of 23 controls. Hyphae were found in the maxillary denture smears from 28 subjects with inflammation and from 18 controls. Thus it seems unjustified to consider the occurrence of hyphae pathognomonic of denture stomatitis. The pH of whole saliva did not differ in the two groups (inflammation:mean pH 6.5, control:6.6). There was no clear relation between the pH of resting saliva and the amount of fungal cultures. Thirty minutes after a mouthrinse with 10 ml of a 25% sucrose solution, the mean saliva pH had dropped equally in both groups. With regard to the denture plaque, samples taken 40 min after the rinse indicated a more pronounced acid production in the plaque associated with inflammation. The pH of "resting" plaque was also lower in the inflammation than in the control group (mean maxillary pH 5.7 and 6.3 respectively, a=0.002). No association was found between the pH and the occurrence of hyphae in "resting" denture plaque. This supports the view that the pH is of no major importance for filamentation in vivo.

Adult↗

Diagnosis of canine nasal aspergillosis by cytological examination: a comparison of four different collection techniques.

OBJECTIVES: To compare the efficacy and diagnostic value of four different sample collection techniques for cytological identification of nasal aspergillosis-penicilliosis in dogs. METHODS: Fifteen dogs with a history of persistent nasal discharge and clinical and radiographic findings suggestive of aspergillosis were evaluated using four different cytological sampling techniques. These were a direct smear from the nasal discharge, blind swab collection under general anaesthesia, brushing from suspect lesions under direct endoscopic visualisation and a squash technique of mucosal biopsies from suspect lesions obtained under direct endoscopic visualisation. RESULTS: Direct smear collection and blind swab collection detected fungal hyphae in 13.3 and 20 per cent of examined cases, respectively; brush samples detected fungal hyphae in 93.3 per cent and fungal spores in the 45 per cent of examined cases and squash samples detected fungal hyphae in 100 per cent and fungal spores in 36 per cent of examined cases. CLINICAL SIGNIFICANCE: This study confirmed the high accuracy of cytology samples in the diagnosis of nasal aspergillosis-penicilliosis when collected under direct endoscopic visualisation and showed the poor value of samples that were collected by blind swabs or prepared from samples of nasal discharge.

Animals↗

Systemic mycosis due to Aspergillus deflectus in a dog.

A 4-year-old, entire female, German Shepherd Dog was referred with a 3-month history of right foreleg lameness that partially responded to nonsteroidal anti-inflammatory and antimicrobial therapy. The bitch lost weight, was polydipsic and had reduced exercise tolerance. On referral, the animal was in poor condition, pyrexic and exhibited moderate pain on full extension of the right shoulder. Blood, urine and joint fluid were obtained and radiographs were taken of the right shoulder and chest. The bitch was lymphopaenic, hyperfibrinogenaemic, hyperglobulinaemic, mildly azotaemic, mildly proteinuric and isosthenuric. Branching fungal hyphae were present in the urine. On radiography, the thorax contained a large ventral mediastinal mass and the humeral head had extensive areas of radiolucency. An aspirate from the right humeroscapular joint exhibited branched fungal hyphae and numerous neutrophils and macrophages. A diagnosis of disseminated mycosis was made and euthanasia was performed. At necropsy, numerous caseating granulomas were present, especially in the kidneys, adrenal glands, heart and lymph nodes. Extensive osteomyelitis involved the head of the right humerus, the sternebrae and the fifth intervertebral disc. Fungal hyphae were detected in sections of granulomas in all affected organs and a diagnosis of disseminated fungal granulomatosis was made. Aspergillus deflectus was readily isolated from affected lymph nodes, but confirming its identity as A deflectus using standard procedures proved difficult. The identity of the fungus was finally confirmed by sequencing part of the 185 rRNA of the isolate. This is the first report in Australia of a disseminated mycosis caused by A deflectus. Previously, the involvement of A deflectus as a cause of disseminated mycosis was limited to 5 cases from the West Coast of the USA, four of which occurred in German Shepherd Dogs.

Animals↗

Dermal mass aspirate from a Persian cat.

A 1-year-old spayed female Persian cat with alopecia and weight loss had numerous variably ulcerated dermal nodules. Cytologic examination of an aspirate of one of the nodules revealed pyogranulomatous inflammation along with septate hyphae and basophilic round bodies, 0.5-1.0 microm in diameter, surrounded by a thin clear halo (arthrospores). The cytologic diagnosis was dermatophytic pseudomycetoma. Histologically, there were dermal granulomas containing poorly staining, septate hyphae with bulbous spores embedded within abundant amorphous eosinophilic material (Splendore-Hoeppli reaction), and the histologic diagnosis was pseudomycetoma-associated chronic multifocal severe granulomatous dermatitis with lymphocytic perifolliculitis and furunculosis. Microsporum canis was cultured from the lesion. Pseudomycetomas are distinguished from fungal mycetomas, or eumycotic mycetomas, by the findings of multiple lesions, lack of a history of skin trauma, an association with dermatophytes, most commonly Microsporum canis, and, histologically, lack of true cement material and a more abundant Splendore-Hoeppli reaction in pseudomycetomas. Additionally, pseudomycetomas differ from dermatophytosis, in which lesions are restricted to epidermal structures. Persian cats have a high incidence of pseudomycetoma formation, suggesting a heritable predisposition. The prognosis is fair with systemic antifungal therapy. When examining cytologic specimens from Persian cats with single or multiple dermal nodules, especially if pyogranulomatous inflammation is present, a diagnosis of pseudomycetoma should be suspected and is warranted if arthrospores and refractile septate hyphae are present.

Alopecia↗

"Carry-on" dermal baggage: a nodule from a dog. Pyogranulomatous inflammation with intralesional fungal agents.

A 2-year-old intact female Australian Cattle Dog presented with a 1-cm diameter nonexudative dermal nodule on the medial aspect of the right thigh. Fine-needle aspiration revealed pyogranulomatous inflammation and many ovoid, 2-4 microm diameter, thin-capsulated, basophilic bodies that appeared to be fungal spores or yeast. Results of CBC, serum chemistry panel, lymph node palpation, and radiographs were unremarkable. Excisional biopsy and histopathology revealed pyogranulomatous folliculitis, furunculosis, and perifolliculitis. Rare fungal hyphae and spore forms were intimately associated with, and occasionally within, hair shafts. A morphologic diagnosis of dermatophytosis was made and Microsporum canis infection was confirmed by fungal culture. M canis is a common infectious agent found within the follicles and superficial keratin layers of canine skin. The kerion-type clinical presentation observed in the dog of this case is uncommonly observed with M canis. Additionally, the cytologic finding of multiple arthroconidia without hyphae is unusual. In the absence of hyphae, M canis arthroconidia may be confused with other fungal yeast bodies; therefore close scrutiny of a cytologic sample for arthroconidia associated with keratin, hair fragments, or hyphal structures is recommended.

Animals↗