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PubMed1980

Candida albicans ultrastructure: colonization and invasion of oral epithelium.

The colonization and invasion of various animal oral mucosae by Candida albicans were examined in an organ culture model. Scanning and transmission electron microscopy of the oral epithelium between 12 and 30 h after inoculation with the fungus revealed the morphological relationships between host and parasite. Examination of the fungi in thin sections showed five distinct layers in the cell wall of C. albicans within the epithelium, but changes were evident in the organization and definition of the outer cell wall layers in budding hyphae and in hyphae participating in colonization and invasion of the epithelial cells. Adherence of the fungus to the superficial cells of the oral mucosa appeared to involve intimate contact between the epithelial cell surface and the deeper layers of the fungal cell wall. During invasion a close seal was maintained between the invading hyphae and the surrounding epithelial cell envelope, there being no other evidence of damage to the host cell surface except at the site of entry. Within the epithelial cells there was only occasional loss of cytoplasmic components in the vicinity of the invading hyphae. These findings would suggest that enzymatic lysis associated with the invasive process is localized and that the mechanical support provided by surface adherence and the intimate association between the fungus and the epithelial cell envelope may permit growth of Candida on through the epithelium.

Animals↗
PubMed1992

Lack of vessel wall elastolysis in human invasive pulmonary aspergillosis.

In experimental studies, the apparent ability of Aspergillus fumigatus isolates to produce elastase in agar plates correlates with their ability to cause invasive pulmonary aspergillosis in mice pretreated with cortisone. Thus, elastase production may govern the pathogenicity of particular isolates. If this is so, then disruption of the elastic layers within blood vessel walls in invasive aspergillosis would be expected. To test this hypothesis, tissue blocks were prepared from nine patients with invasive pulmonary aspergillosis. Separate but immediately adjacent histological sections were stained by the Grocott and periodic acid-Schiff methods for fungal hyphae and by the elastic van Gieson technique for elastic tissue. Comparison of those segments of vessel walls infiltrated by hyphae with those not infiltrated by hyphae showed no overall loss of elastic tissue. Material from five of the cases was also stained with an unconventional combination of histochemical stains, allowing accurate identification of both fungal hyphae and elastic laminae in the same histological sections. The results showed no more disruption of elastic laminae than would be expected from simple physical displacement of elastic laminae. We conclude that if elastolysis contributes at all to invasion of vessel walls by aspergilli, then it seems to be very localized and/or transient.

Adult↗
PubMed2006

DevA, a GntR-like transcriptional regulator required for development in Streptomyces coelicolor.

The gram-positive filamentous bacterium Streptomyces coelicolor has a complex developmental cycle with three distinct phases: growth of the substrate mycelium, development of reproductive structures called aerial hyphae, and differentiation of these aerial filaments into long chains of exospores. During a transposon mutagenesis screen, we identified a novel gene (devA) required for proper development. The devA mutant produced only rare aerial hyphae, and those that were produced developed aberrant spore chains that were much shorter than wild-type chains and had misplaced septa. devA encodes a member of the GntR superfamily, a class of transcriptional regulators that typically respond to metabolite effector molecules. devA forms an operon with the downstream gene devB, which encodes a putative hydrolase that is also required for aerial mycelium formation on R5 medium. S1 nuclease protection analysis showed that transcription from the single devA promoter was temporally associated with vegetative growth, and enhanced green fluorescent protein transcriptional fusions showed that transcription was spatially confined to the substrate hyphae in the wild type. In contrast, devAB transcript levels were dramatically upregulated in a devA mutant and the devA promoter was also active in aerial hyphae and spores in this background, suggesting that DevA might negatively regulate its own production. This suggestion was confirmed by gel mobility shift assays that showed that DevA binds its own promoter region in vitro.

Amino Acid Sequence↗
PubMed1969

Ultrastructure of dimorphic transformation in paracoccidioides brasiliensis.

The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M right harpoon over left harpoon Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M --> Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y --> M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M --> Y, and intrayeast hyphae during the Y --> M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon.

Fungi↗
PubMed1972

Model for branch initiation in Aspergillus nidulans based on measurements of growth parameters.

The rate of hyphal elongation and the number of branches per hypha were measured on short sporelings of Aspergillus nidulans growing at different rates. The rate of elongation was proportional to total length in unbranched and branched hyphae. At each growth rate, the number of branches per hypha increased with increasing length and gave approximately straight-line graphs when plotted against length. The average number of branches per unit of hyphal length was quite different for the various growth rates and increased in direct proportion to the growth rate. The results are interpreted to mean that (i) growing tips have a maximum rate at which they can elongate and which is reached at hyphal lengths characteristic of the particular growth rate and (ii) a new branch is formed when the capacity of the hypha to elongate exceeds that of the existing tips.

Acetates↗
PubMed1974

Characterization of dimorphism in Cladosporium werneckii.

Yeast forms of the dimorphic fungus Cladosporium werneckii grow by polar budding and yield a homogeneous yeast phase when cultured at 21 C in an agitated sucrose-salts medium (Czapek-Dox broth). Yeast extract enrichment of such a yeast phase consisting of 10(4) yeasts per ml induces a quantitative conversion of the yeasts to true hyphae. This conversion is not mediated by a transition cell and is often attended by capsule formation. When 10(5) or 10(6) yeasts per ml receive enrichment, a nonquantitative conversion to moniliform hyphae is effected and no capsule formation is observed. Rapid agitation compared to slow agitation or stationary incubation of the nutritionally mediated conversion cultures greatly accelerates the production of lateral hyphal buds or their yeast progenies. These cells appear incapable of undergoing nutritional conversion to hyphae, but instead must grow for several generations in the unenriched sucrose-salts medium to restore conversion competence. Temperature shifts affect directly the morphology and morphogenesis of the yeast in unenriched medium; at 17 C yeasts are smaller and more ovoid than at 21 C, and at 30 C marked conversion of yeasts to moniliform hyphae occurs. A methodology employing the Coulter counter and Coulter channelizer provides evidence that direct correlations do not always exist between the optimum conditions for the growth of C. werneckii and the optimum conditions for its yeast-to-mold conversion.

Cell Count↗
PubMed1978

Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms.

Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.

Candida albicans↗
PubMed1986

Effect of L-amino acids on Mucor rouxii dimorphism.

Mucor rouxii organisms growing aerobically and exponentially on a well-defined minimal medium are able to differentiate as yeasts or as mycelia, depending on the amino acid as the nitrogen source. When certain amino acids were used as the nitrogen source, spores differentiated only as hyphae, whereas other amino acids gave rise to other morphological forms having different ratios of yeasts to hyphae. In both hyphal and yeast cultures, an aerobic metabolism was predominant, as shown by determining several metabolic parameters such as oxygen tension, glucose consumption, ethanol production, and CO2 release. A complete conversion of yeasts to hyphae was obtained by the appropriate change in the amino acid used as nitrogen source. By preparing spheroplasts from mycelial cultures and transferring them to media with amino acids that induce yeast formation, a 50% yield in the reverse transformation was achieved. A correlation between the change in pH of the medium and cell morphology was observed in different growth conditions. Decrease in the pH of the medium preceded the appearance of hyphae. Also, when the initial pH of the medium was increased, aspartate-containing cultures developed mainly as mycelia, instead of yeasts, with a corresponding decrease in the final pH.

Amino Acids↗
PubMed1967

Ultrastructural studies of microconidium formation in neurospora crassa.

Microconidiating cultures of "peach-fluffy" (pe, fl; Y8743m, L; FGSC #569) were fixed at various times after the initiation of growth and examined with an electron microscope. Hyphae from which microconidia form are markedly vacuolated and show a much more extensive system of rough endoplasmic reticulum than young vegetative hyphae. A bulge in the hypha presages the start of microconidium formation, followed by the rupture of the outermost wall layers. A thick collar forms around the protruding microconidium due to extensive thickening of the inner wall layer of the parent hypha. At this stage, the cytoplasm of the developing microconidium is still continuous with that of the microsporophore cell from which it arises and is contained by a wall which is derived from the thickened collar. The microconidium is finally isolated from the cytoplasm of the microsporophore by a centripetal extension of the collar. Microconidia differ from macroconidia in having a more extensive endoplasmic reticulum and fewer mitochondria, in addition to being smaller and having a single nucleus.

Cell Nucleus↗
PubMed1999

A G1 cyclin is necessary for maintenance of filamentous growth in Candida albicans.

Candida albicans undergoes a dramatic morphological transition in response to various growth conditions. This ability to switch from a yeast form to a hyphal form is required for its pathogenicity. The intractability of Candida to traditional genetic approaches has hampered the study of the molecular mechanism governing this developmental switch. Our approach is to use the more genetically tractable yeast Saccharomyces cerevisiae to yield clues about the molecular control of filamentation for further studies in Candida. G1 cyclins Cln1 and Cln2 have been implicated in the control of morphogenesis in S. cerevisiae. We show that C. albicans CLN1 (CaCLN1) has the same cell cycle-specific expression pattern as CLN1 and CLN2 of S. cerevisiae. To investigate whether G1 cyclins are similarly involved in the regulation of cell morphogenesis during the yeast-to-hypha transition of C. albicans, we mutated CaCLN1. Cacln1/Cacln1 cells were found to be slower than wild-type cells in cell cycle progression. The Cacln1/Cacln1 mutants were also defective in hyphal colony formation on several solid media. Furthermore, while mutant strains developed germ tubes under several hypha-inducing conditions, they were unable to maintain the hyphal growth mode in a synthetic hypha-inducing liquid medium and were deficient in the expression of hypha-specific genes in this medium. Our results suggest that CaCln1 may coordinately regulate hyphal development with signal transduction pathways in response to various environmental cues.

Actins↗
PubMed1987

Position-dependent and -independent mechanisms regulate cell-specific expression of the SpoC1 gene cluster of Aspergillus nidulans.

Many genes that are expressed specifically in the differentiating asexual spores (conidia) of Aspergillus nidulans are organized into clusters. We investigated the effects of altered chromosomal position on expression of a gene from the conidiation-specific SpoC1 gene cluster. The gene became deregulated when integrated at nonhomologous chromosomal sites, in that transcript levels were elevated in vegetative cells (hyphae) and variably altered in conidia. We also investigated the effects on expression of insertion of the nonregulated argB gene into the SpoC1 region. Levels of argB transcripts were markedly reduced in hyphae. The results suggest that a cis-acting regional regulatory mechanism represses transcription of SpoC1 genes in hyphae. They also indicate that expression of individual SpoC1 genes is modulated during conidiation by trans-acting factors. We propose that the two types of regulation act together to produce the major differences in transcript levels observed in hyphae versus conidia.

Aspergillus nidulans↗
PubMed1989

Aspergillosis of the nose and paranasal sinuses.

Fulminant aspergillosis was diagnosed on nasal biopsy in a 49 year old man who had features of an aspergilloma. Further postmortem examination of this area was performed and the results were contrasted with the histological features of other Aspergillus infections. The nasal biopsy specimen and postmortem examination showed infiltrating Aspergillus hyphae with tissue necrosis and little inflammatory response. The hyphae were easily seen with routine stains. This contrasts with the findings in invasive aspergillosis where there is fibrosis and a granulomatous response to the Aspergillus hyphae. The hyphae are seen in giant cells using fungal stains. In the saprophytic infections aspergilloma and allergic Aspergillus sinusitis there is no tissue invasion or destruction. Aspergillus infections of the nose and paranasal sinuses often require biopsy for accurate diagnosis. As treatment varies pathologists need to be able to distinguish the different patterns of infection.

Aspergillosis↗
PubMed1999

Microscopic features of vaginal candidiasis and their relation to symptomatology.

OBJECTIVES: To document the microscopic features of vaginal candidiasis and to examine the relation between yeast morphology and patient symptomatology. METHOD: The study population comprised women undergoing screening for genital infection at a department of genitourinary medicine. RESULTS/CONCLUSION: Data were collected on 267 women of whom 234 were found to have vaginal candidiasis by vaginal culture. The remaining 33 patients had microscopic features of candidiasis (spores and/or hyphae) but were culture negative. Of the culture positive women, microscopy was positive in 182 (78%). "Spores only" were identified in 65 (28%), "hyphae only" in 16 (7%), and both "spores and hyphae" in 101 (43%). 68% of culture positive women were symptomatic, the commonest symptoms being irritation alone (27%) or irritation plus vaginal discharge (25%). No association was found between yeast morphology (spores, budding/non-budding; hyphae, branching/non-branching) as identified on microscopy of vaginal secretions and symptomatology.

Candida↗
PubMed1978

Schizophyllum commune Fr. as a destructive mycoparasite.

Schizophyllum commune Fr. was shown, by light, scanning, and transmission electron microscopy, to be a destructive mycoparasite on several phytopathogenic and nematode-trapping fungi. The hyphae of S. commune coiled around host hyphae and fruiting structures and penetrated them by means of either unspecialized hyphae or by penetration pegs that developed from terminal appressoria. The host cell walls were usually chemically degraded after which the parasite grew through an electron-dense, papillate, reaction region and its underlying membrane(s) produce trophic hyphae inside the host cells.

Agaricales↗
PubMed1984

The comparative pathology of equine cutaneous phycomycosis.

Pythiosis (204 cases, 77%), basidiobolomycosis (48 cases, 18%), and conidiobolomycosis (14 cases, 5%) were diagnosed morphologically from 266 horses with phycomycosis. All lesions were cutaneous ulcerative granulomas and three horses with pythiosis had metastatic lesions in regional lymph nodes. Lesions of pythiosis contained characteristic yellow, coral-like coagula and had a fibrotic surface containing sinus tract openings. Basidiobolomycosis was characterized by infrequent small yellow coagula and a yellow line of fungal invasion beneath an edematous surface. Lesions of conidiobolomycosis had numerous small coagula and a nasal location. There were minor differences in inflammatory cell populations within the granulomatous lesions. Most differences were associated with coagula size and fungal morphology. Coagula were composed of collagen, fibrin, cellular debris, degranulated and whole eosinophils and hyphae. Histochemistry revealed no major differences among the three diseases. Pythium sp hyphae were 2.6 to 6.4 micrometers in diameter, had thick walls, and occasionally were septate. Basidiobolus haptosporus hyphae were 5.1 to 20.5 micrometers in diameter, had thin walls, and commonly were septate. Conidiobolus coronatus hyphae were 5.1 to 12.8 micrometers in diameter, had thin walls, and commonly were septate. A perihyphal eosinophilic cuff ( Splendore - Hoeppli phenomenon) with a radius of up to 20 micrometers was associated with the latter two fungi. Ultrastructurally, Pythium sp was composed of a thick, single density cell wall while B. haptosporus and C. coronatus had thin, double-layered cell walls.

Animals↗
PubMed2006

The RNA-binding protein Rrm4 is essential for polarity in Ustilago maydis and shuttles along microtubules.

Formation of polar-growing hyphae is essential for infection by the plant pathogen Ustilago maydis. Here we observe that loss of RNA-recognition motif protein Rrm4 caused formation of abnormal hyphae. The insertion of septa at the distal pole was abolished and a significantly increased number of hyphae grew bipolarly. UV-crosslinking experiments revealed that Rrm4 bound RNA via its N-terminal RRMs and that its RNA-binding activity was substantially increased during filamentation. Rrm4 assembled into particles that shuttled bidirectionally along microtubules to both poles. Recruitment of Rrm4 into particles increased during filamentation, and mutations in the peptide-binding pocket of its PABC domain caused abnormal particle formation as well as polarity defects. Shuttling was mediated by active transport because loss of conventional kinesin, which interferes with the balance of microtubule-dependent motors, caused accumulation of particles at the poles resulting in disturbed polarity. Thus, constant transport of the RNA-binding protein towards the poles is needed to orchestrate hyphal growth. Since a mutation of the N-terminal RRM that leads to reduced RNA binding in vivo also affected polarity, Rrm4 might regulate polarity of the infectious hyphae by transporting RNA from the nucleus to cell poles.

Amino Acid Sequence↗
PubMed1995

The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa.

Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytoplasmic Ca2+, which was absent in non-growing hyphae and was insensitive to Gd3+ in the medium. Patch clamp recordings in the cell-attached mode, from the plasma membrane of these hyphae, showed two types of channel activities; spontaneous and stretch activated. The spontaneous channels were identified as inward K+ channels based on inhibition by tetraethylammonium. The stretch activated channels had increased amplitudes in response to elevated Ca2+ in the pipette solution, and thus are permeable to Ca2+ and mediate inward Ca2+ movement. Gd3+, which is an inhibitor of some stretch activated channels, incompletely inhibited stretch activated channel activity. Both tetraethylammonium and Gd3+ only transiently reduced the rates of tip growth without changing tip morphology, thus indicating that the channels are not absolutely essential for tip growth. Furthermore, in contrast to the hyphae of another tip growing organism, Saprolegnia ferax, tip-high gradients of neither spontaneous nor stretch activated channels were found. Voltage clamping of the apical plasma membrane potential in the range from -300 to +150 mV did not affect the rates of hyphal elongation. Collectively, these data suggest that ion transport across the plasma membrane at the growing tip in Neurospora is not obligatory for the maintenance of tip growth, but that a gradient of Ca2+, possibly generated from internal stores in an unknown way, is required.

Calcium↗
PubMed1997

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells.

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/-0.03 and +/-0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

Benzopyrans↗