Mycelial Phase Research Articles

Immunochemical studies of heat-shock protein 80 ofHistoplasma capsulatum

A monoclonal antibody (MAb) of the IgG1 subclass, with greater activity to the yeast than the mycelial phase of Histoplasma capsulatum was raised and was found to predominantly recognize a molecule of 80 kDa by immunoblot. Enzymatic deglycosylation and chemical degradation, followed by reaction with MAb 69F on Western blots showed the molecule to be O-glycosylated, and immunofluorescence studies showed it to be heat-inducible and its distribution to be cytoplasmic and possibly cell membraneous. There was no apparent staining of the cell wall. Culture filtrate was positive by ELISA and Western blot when reacted with MAb 69F. In addition, ELISA and Western blot demonstrated that a similar epitope was present in other fungal species. The glycoprotein had a pI of approximately 4.7. N-terminal amino acid sequencing revealed this molecule to be homologous to members of the heat-shock protein 70 family and to a recently described antigen from H. capsulatum.

Diagnosis of systemic candidiasis by enzyme immunoassay detection of specific antibodies to mycelial phase cell wall and cytoplasmic candidal antigens.

Diagnosis of systemic Candida infections was attempted by the use of an enzyme-linked immunosorbent assay (EIA) to detect IgG antibodies towards cell wall-bound and cytoplasmic candidal antigens. Cell wall antigens were sequentially solubilized by treatment of germinated blastoconidia of Candida albicans (ATCC 26555 strain) with beta-mercaptoethanol (beta ME extract) and digestion with Zymolyase 20T, a beta-glucanase preparation (Zymolyase extract). Protoplasts obtained after treatment with Zymolyase were osmotically lysed (cytoplasmic antigens). Sera were obtained from patients with systemic (n = 28) and superficial (n = 46) candidiasis. Control sera were obtained from normal healthy individuals (n = 31) and from hospitalized patients at low (n = 36) and at high (n = 13) risk of developing systemic candidiasis yet showing no symptoms of candidal infection. Detection of antibodies in crude sera samples by EIA using all of these antigenic extracts was highly specific (98-100%), but sensitivity of the method was low (3.5-17.8%). However, adsorption of sera with latex microspheres coated with purified Candida mannan in order to selectively remove anti-mannan antibodies prior to EIA improved the diagnostic efficiency of this test. Improvement was particularly noticeable when the beta ME extract was used as antigenic preparation, yielding a sensitivity of 89.2% and a specificity of 98.6%.

Aberrant Histoplasma capsulatum confirmation of identity by a chemiluminescence-labeled DNA probe

A cottony, light tan, filamentous fungus with pear-shaped microconidia and lacking tuberculated macroconidia was isolated from a bronchial lavage specimen. Subculture on several media at 37°C failed to convert the fungus to a yeast form after several weeks; attempts at in vivo conversion in mice were also unsuccessful. Sera obtained several months apart showed M bands with Histoplasma capsulatum (HC) antigen by immunodiffusion and an increase in complement fixation titers with mycelial and yeast phase antigens of HC. Parallel identity was obtained on two occasions with exoantigen culture confirmation reagents for HC from Immuno-Mycologics as well as one of identity with Nolan reagents. Extracts from four Chrysosporium spp. strains had no identity reactions with HC with either kit. The fungus was identified as HC by the Accuprobe Histoplasma chemiluminescence-labeled DNA probe directed at ribosomal RNA, whereas all four Chrysosporium spp. isolates tested negative. DNA probes are a fast and accurate method to confirm the identity of aberrant fungal isolates.

Identification and immunochemical characterization of a germ tube specific antigen of Candida albicans.

Germ tube specific fractions of the dimorphic pathogenic fungus Candida albicans were fractionated according to their ability to link to fibrinogen. These fibrinogen binding factors were used as immunogens to prepare monoclonal antibodies (mAbs) with BALB/c mice. Among the resulting mAbs, one (mAb 3D9.3) was shown by indirect immunofluorescence to be specific to the surface of the mycelial phase of the C. albicans species. No labelling of the cell wall of any other Candida species was observed. This morphological shape specificity was confirmed by immunoblotting where a polydispersed high molecular mass component was identified. The molecular mass varied with the extraction procedure used; over 210 kDa with EDTA-2ME treatment, and ranging from 110 to 220 kDa after Zymolyase digestion. This phase-specific epitope was sensitive to proteolysis with pronase E, proteinase K and trypsin, but not to periodate treatment. Further purification of this material would allow further development of new serodiagnostic assays that might be more specific for invasive disease than currently available tests.

Protoplast formation and regeneration in Streptomyces flavopersicus.

Protoplasts of Streptomyces flavopersicus with the highest regeneration frequency were isolated from late log phase mycelium grown in a two-stage culture system with 2% glycine in the medium. Of the 11 regeneration media tested, R9 was selected as the most efficient with 29 degrees C as the best temperature.

Influence of culture conditions on mycelial growth and phialospore production and germination in Chalara elegans

Mycelial growth, both linear extension and biomass accumulation, and phialospore production in three Chalara elegans strains from diverse geographical areas were greatest at 20–25 °C. Growth and sporulation were minimal at 10 °C and nil at 35 °C. The optimal pH range for growth was 4.0–6.5, and no growth was observed at pH 3.0 or 7.75. Phialospore production was most abundant at pH 5.5–6.5. A reduction in the osmotic potential of the medium from −0.2 to −0.6 MPa using PEG 8000- or CaCl2-enhanced phialospore production by twofold to threefold and increased mycelial biomass by approximately 35%. With further reductions in osmotic potential, there were corresponding decreases in growth and sporulation, both of which were nil at −1.8 MPa. Cultures grown in continuous darkness had slightly greater growth and sporulation than cultures exposed to light. Phialospores germinated on 1 % Noble water agar in the absence of exogenous nutrients, with 80–96% germination at pH 4.0–6.0, temperatures of 15–25 °C, and an osmotic potential of −0.2 to −0.4 MPa. The germ tubes either developed into extensive hyphae or terminated in short phialides within which secondary phialospores were produced without an intervening mycelial phase (microcycle conidiation). Germination to produce microcycle conidiation was strain dependent, was greater in older (8 week) than in younger (3 week) cultures, and was not affected by the density of spores plated onto the agar surface. The addition of 50 mM glucose enhanced the hyphal form of germination in all strains, whereas heat stress (40 °C for 22 h) enhanced microcycle conidiation in one strain. An osmotic stress provided by 50 mM NaCl or CaCl2 had no effect on the two forms of germination. These findings are discussed in relation to the development of root diseases caused by C. elegans. Key words: black root rot, dematiaceous hyphomycete, endoconidia, microcycle conidiation, Thielaviopsis basicola.

Detection of proteolytic enzymes released by the dimorphic fungusParacoccidioides brasiliensis

The release of proteolytic enzymes with elastinolytic and collagenolytic activity was investigated in 10 different isolates of Paracoccidioides brasiliensis. The study encompassed both the mycelial and the yeast phase of the fungus. In the mycelial phase, secretion of elastinolytic and collagenolytic enzymes was demonstrated in 70% and 80% of the isolates, respectively. None of the isolates in the yeast phase exhibited elastinolytic activity, but 90% showed collagenolytic activity. These results demonstrate that the dimorphic fungus P. brasiliensis has the ability to release proteolytic enzymes in both growth phases, although with apparently different specificities.

A preliminary study ofin vitroantibiotic activity of saperconazole and other azoles onParacoccidioides brasiliensis

Saperconazole, a new triazole related to itraconazole, was tested against Paracoccidioides brasiliensis and results compared with ketoconazole and itraconazole. The fungus was highly sensitive to the action of these compounds, particularly saperconazole, with minimum inhibitory concentrations and minimum fungicidal concentrations ranging from 10(-7) to 10(-10) M (equivalent to 6.7 x 10(-2)-6.7 x 10(-5) micrograms ml-1 for saperconazole), according to the morphological phase and the antifungal tested. The yeast phase was more sensitive than the mycelial phase to any of the azoles. Morphological changes were observed in the cell membranes, particularly when saperconazole was used as the antifungal agent.

Dimorphism in Benjaminiella poitrasii: cell wall chemistry of parent and two stable yeast mutants.

In the dimorphic zygomycetous fungus Benjaminiella poitrasii, the cell wall compositions of mycelial phase (M), yeast phase (Y) and its yeast form mutants (Y-2 and Y-5) were studied. Chitosan was abundant in M-phase (26.6%) whereas lesser amounts were present in Y-phase (17.3%) and in mutants Y-2 (19.6%) or Y-5 (17.3%). Although chitin was present as a smaller fraction of the total glucosaminoglycan in each of different cell wall preparations, it was almost 3 times more prevalent in M-phase than the Y-phase cells. Cross-linking studies among the various cell wall components in B. poitrasii, suggest linkages among mannans and proteins and glucans and glucosaminoglycans.

O-glycosidically linked oligosaccharides from peptidorhamnomannans of Sporothrix schenckii.

beta-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases of Sporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(alpha 1-2)Man-ol, Rha(alpha 1-3)Man(alpha 1-2)Man-ol, Rha(alpha 1-4)GlcA(alpha 1-2)Man(alpha 1-2)Man-ol, and Rha(alpha 1-4)[Rha(alpha 1-2)] GlcA(alpha 1-2)Man(alpha 1-2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.

Small subunit ribosomal RNA of Blastomyces dermatitidis: sequence and phylogenetic analysis.

We determined the small subunit (18S) ribosomal RNA sequence of the dimorphic fungus Blastomyces dermatitidis. The sequence was compared to that of fourteen other eukaryotic organisms, ten of which were higher fungi, and an evolutionary tree was constructed based on these sequences. B. dermatitidis aligned most closely with the Ascomycetes Neurospora crassa and Podospora anserina, in agreement with previous phylogenetic analysis based on morphological criteria. Phase-specific cDNA clones derived by reverse transcription of RNA isolated from the yeast and mycelial phases of B. dermatitidis were also sequenced. The 18S ribosome sequence was found to be the same in both phases. Heterogeneity was found at both the genomic and RNA level at position 1352.

Interaction of azoles with rifampin, phenytoin, and carbamazepine: in vitro and clinical observations.

Twelve patients receiving therapy with an azole agent (ketoconazole, itraconazole, and/or fluconazole) for systemic mycoses experienced drug interactions with rifampin, phenytoin, and/or carbamazepine resulting in substantial decreases in azole concentrations in serum. All four patients receiving azoles and concurrent phenytoin and/or carbamazepine failed to respond to treatment or suffered a relapse of their fungal infection. Four of five patients with cryptococcosis who received itraconazole and rifampin responded despite decreases in their serum itraconazole concentrations; synergy between itraconazole and rifampin was documented by in vitro analysis of inhibition and of killing of Cryptococcus neoformans isolates from all patients receiving this combination. In contrast, two patients with coccidioidomycosis failed to respond to itraconazole/rifampin. Moreover, two patients with cryptococcosis suffered a relapse or persistence of seborrheic dermatitis while receiving itraconazole/rifampin. The latter combination showed synergy in vitro in the inhibition of the mycelial phase of Coccidioides immitis and, to a lesser extent, of the pathogenic spherule phase of this fungus; synergy in the killing of C. immitis was not noted, nor was synergy seen against Malassezia furfur, the purported etiologic agent of seborrheic dermatitis. These findings illustrate several drug interactions that may affect clinical outcome and that must be considered in the management of antifungal therapy.

Mutants ofParacoccidioides brasiliensisstrain IVIC Pb9 affected in dimorphism

Morphological mutants were isolated after nitrosoguanidine treatment of Paracoccidioides brasiliensis strain IVIC Pb9. Two of these mutants, Pb257 and Pb258, developed a typical mycelia at 23 degrees C, however, the yeast cells which developed at 37 degrees C were indistinguishable from those of the parental strain. A third mutant, strain Pb267, was thermosensitive, grew as yeast-like cells at 23 degrees C, but was unable to survive at 37 degrees C. Morphological observations as well as serological and segregation tests confirmed that the mutant strains originated from P. brasiliensis. Cell wall chemical analyses of the mutant strains grown at 23 degrees C indicated the presence of alkali-soluble, acid-insoluble polysaccharides absent in the parental wild-type strain Pb9 grown under the same conditions. The phenotypes shown by the mutant strains may be related to deficiencies in the proper synthesis of cell wall components of the mycelial phase of this fungus.

A new genotype of Neurospora crassa that selectively produces abundant microconidia in submerged shake culture

A new microcycle microconidiating strain ( mcm) of Neurospora crassa was derived by successive backcrosses of a soil isolate to a laboratory wild-type strain. Surface grown cultures show normal wild-type vegetative morphology. However, under continuous agitation in liquid cultures at 22°C, macroconidial germlings bypass the usual mycelial phase and produce abundant numbers of uninucleate microconidia within 24 h. Temperature and culture media affect the production of microconidia.

Ontogenetic stages from coenocyte to basidiome and their relation to phenoloxidase activity and colonization processes in Phanerochaete magnoliae

Three separate collections were made in southern England of Phanerochaete magnoliae forming hydnoid hymenial surfaces through the tubes of Datronia mollis basidiomes. Isolates of P. magnoliae exhibited unusual patterns of development and interaction with other fungi. Germinating basidiospores produced an extensive, sparsely branched, rapidly extending and fully coenocytic mycelial phase with a low fractal dimension. This phase was followed by events of septation, lateral branching, anastomosis, hymenium formation and autolysis in a strongly ordered ontogenetic sequence. These events were correlated with changes in constitutive and inducible laccase and tyrosinase activity. P. magnoliae also had a non-outcrossing breeding strategy and was able specifically to replace colonies of D. mollis without producing dense invasive mycelium.

A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function

Enzymological evidence has been sought for the purported involvement of chitinolysis in vegetative growth of filamentous fungi. A procedure has been developed for the production of fast growing and morphologically homogeneous exponential phase mycelium of the non-septate dimorphic zygomycete Mucor rouxii. A partially purified extract of this material has been subjected to gel-permeation chromatography and the chitinolytic activity of eluate fractions has been assessed using colloidal and nascent chitin and 3,4-dinitrophenyl tetra-N-acetylchitotetraoside [3,4-DNP-(GlcNAc)4] as substrates. Exponentially growing (td = 1.1 h) mycelium consisting of single short-branched hyphae contains at least seven chitinases. The two particulate ones have not been studied in detail. The soluble chitinases hydrolyse (pseudo)chito-oligomers by random cleavage of internal beta-1,4-bonds (and not by processing) and have a minimum chain-length requirement of n = 4. They are clearly distinct from beta-N-acetylglucosaminidase (beta-GlcNAc'ase) with respect to their chromatographic behaviour, substrate chain-length specificity, inhibition by chitobionolactone oxime (Ki = 175 microM), and non-inhibition by the specific beta-GlcNAc'ase inhibitor N-acetylglucosaminono-1,5-lactone oxime. Their pH optima are similar (6.5-7.0), and all can hydrolyse 3,4-DNP-(GlcNAc)4 as well as nascent chitin. With respect to their charge, response to protease treatment, behaviour upon gel-permeation chromatography and ability to use colloidal chitin as a substrate, the soluble chitinases do, however, represent two distinct groups. Type A chitinases are acidic, display partial latency, show an unusual affinity to dextran gel and act weakly on colloidal chitin. Type B chitinases are basic (or neutral) and non-zymogenic, do not behave anomalously upon gel filtration and can degrade performed chitin. An hypothesis is presented for the function of the complex chitinolytic system of the fungal hypha in branching and, possibly, also in apical growth.

Microcyclic Conidiation Cycles in Epichloe typhina

Germination of ascospores in Epichloe typhina was described earlier as iterative germination in which each part spore of an ascospore germinated and produced conidia without an intervening mycelial phase. In the present study it was determined that the conidia produced by iterative germination also undergo an additional series of microcyclic conidiations. Thus, conidia produced from the ascosporic microcyclic structure germinated and produced other conidia, which also germinated producing yet another microcyclic structure. It was only after the third stage of microcyclic conidiations that mycelial development occurred. Microcycle conidiation in E. typhina is viewed as an obligate developmental sequence since no alternative process was observed. This distinguishes it from microcycle conidiation reported in other fungi where microcycle conidiation is facultative, being inducible only under certain laboratory conditions. Although unexplored, microcyclic conidiation may be a fundamentally important aspect of the life history of E. typhina and a variety of other fungi.

Coccidioides immitis fractions which are antigenic for immune T lymphocytes

The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity. We have generated a Coccidioides immitis antigen-specific murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays. The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components. The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay. Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive. In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C. immitis, which was constructed in lambda gt11 to identify clones that encoded T-cell-reactive fusion proteins (FPs). The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components. The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-cell response was determined. The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C. immitis. Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption. The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF. The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C. immitis. Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way. The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C. immitis.

HSP 90, yeasts andCorynebacterium jeikeium

Recovery from disseminated candidosis is associated with seroconversion to a 47 kDa breakdown product of the Heat Shock Protein (HSP) 90 of Candida albicans. Cloning, sequencing and epitope mapping has allowed the delineation of the immunodominant epitopes LKVIRKNIVKKMIE and STDEPAGESA. Monoclonal and polyclonal antibodies specific to these epitopes are used to show that all strains of C. albicans tested produce HSP 90 in both the yeast and mycelial phases. Homologous proteins are demonstrated in Saccharomyces cerevisiae, Candida parapsilosis and Corynebacterium jeikeium but not in Torulopsis glabrata. Evidence is presented for the existence of two distinct HSP 90s in C. albicans. The first of these is expressed constitutively whilst the second is produced on heat shocking the yeast from 23 to 37 degrees C.

Microcycle conidiation and its genetic basis in Neurospora crassa

Some wild isolates of Neurospora show microcycle conidiation in liquid culture under continuous agitation. Macroconidia from agar-grown mycelial cultures germinated in liquid and the germlings spontaneously produced conidia with no intervening mycelial phase. Three types of microcycle conidiation were seen among progeny of N. crassa Vickramam A x N. crassa a wild-type: (1) multinucleate blastoconidia produced by apical budding and septation, (2) multinucleate arthroconidia produced by holothallic septation and disarticulation of cells, and (3) uninucleate microconidia produced directly from conidiogenous cells of the germlings. Two genes were identified which control specific patterns of microcycle conidiogenesis. A single gene mcb in linkage group VR near al-3 (3.2% recombination) controls blastoconidiation. This gene is epistatic to gene mcm located in linkage group IIL, very near ro-7 (1.4%). mcm controls both microconidiation and arthroconidiation depending on temperature. Strains of genotype mcm produce microconidia almost exclusively at 18-22 degrees C, but arthroconidia with few or no microconidia at 30 degrees C. Because they result in rapid and synchronized conidiation in liquid culture, the two genes should be useful for studies of developmental gene regulation. mcm makes it possible to obtain large quantities of pure microconidia rapidly for experimentation.