Mycelial Phase Research Articles

Histoplasmosis in Dogs and Cats

Histoplasma capsulatum is endemic throughout most of the United States with a high prevalence of infections in the Midwest and South. Histoplasmosis is the second most common systemic fungal disease in cats that may be more susceptible than dogs. Infection occurs by inhalation of conidia from the mycelial phase, which subsequently convert to the yeast form. Histoplasma capsulatum is phagocytized and harbored by cells of the mononuclear phagocyte system. Infection may be subclinical or cause clinical pulmonary granulomatous disease or dissemination. Disseminated disease predominantly affects the liver, spleen, gastrointestinal tract, bone and bone marrow, integument, and eyes. Primary gastrointestinal histoplasmosis also occurs. Clinical signs of histoplasmosis often are nonspecific, including chronic wasting, fever, anorexia, respiratory signs, and lameness. Gastrointestinal signs (eg, diarrhea with hematochezia or melena) are common in dogs. The definitive diagnosis is made by identification of the yeast in tissue samples. Itraconazole is the treatment of choice.

Mycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves

BackgroundThe current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface.ResultsThe work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases.ConclusionAn early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.

Functional genome of the human pathogenic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.

Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Characterisation and biosynthesis of d-erythroascorbic acid in Phycomyces blakesleeanus

d-Erythroascorbate and d-erythroascorbate glucoside have been identified in the Zygomycete fungus Phycomyces blakesleeanus. Ascomycete and Basidiomycete fungi also synthesise d-erythroascorbate instead of l-ascorbate, suggesting that d-erythroascorbate synthesis evolved in the common ancestor of the fungi. Both compounds accumulate in P. blakesleeanus at higher levels than observed in other fungal species. d-Erythroascorbate glucoside reduced dichlorophenolindophenol as effectively as l-ascorbate, but was more stable to autoxidation. d-Erythroascorbate glucoside predominated in spores and stationary phase mycelium. Free d-erythroascorbate accumulated during the exponential phase of mycelial growth and decreased to very low levels in the stationary phase. This suggests an association between growth and free d-erythroascorbate. P. blakesleeanus converted exogenous d-arabinose to d-erythroascorbate and its glucoside. A monomeric NAD-dependent d-arabinose dehydrogenase of 41 kDa was purified to near homogeneity. The enzyme oxidised d-arabinose, l-galactose, and l-fucose. Correspondingly, mycelium converted exogenous l-galactose and l-fucose to l-ascorbate and 6-deoxyascorbate, respectively. The antioxidant role of d-erythroascorbate and its glucoside is discussed.

α-galf1→6-α-mannopyranoside side chains inParacoccidioides brasiliensiscell wall are shared by members of the Onygenales, but not by galactomannans of other fungal genera

The water-soluble polysaccharide fraction of the cell wall alkali extract (F1SS) from the mycelial phase of the dimorphic fungus Paracoccidioides brasiliensis is compared with F1SS polysaccharides obtained from the Onygenalean mycelial fungi Ascocalvatia alveolata, Onygena equina and Aphanoascus terreus. These polymers were exclusively composed of mannose and galactose. Data from methylation and NMR analyses reveal that F1SS polysaccharides from the four fungi contain the same residues although in different proportions: [-->2,6)-alpha-D-Manp-(1 -->]; [2)-alpha-D-Manp-(1 -->]; [ -->6)-alpha-D-Manp-(1 -->]; and [alpha-D-Galf-(1 -->]. In P. brasiliensis, the repeating unit of the polysaccharide consists of a backbone of [(1 -->6)-alpha-D-Manp] substituted at the 0-2 position by the disaccharide [alpha-D-Galf-(1 -->6)-alpha-D-Manp-(1 -->], while the remaining 0-2 positions are substituted by single residues of mannose or short chains of (1 -->2)-mannose. The other species had a lower proportion of galactofuranose-containing side chains and higher proportion of mannose-containing side chains. The similarities found among the F1SS polysaccharides from P. brasiliensis and the Onygenalean A. alveolata, A. terreus and O. equina, reveal the close relatedness of all these fungi, show differences with polysaccharides from other fungal genera and agree with the molecular evidence provided in the scientific literature for the placement of P. brasiliensis within the Onygenales.

Direct detection of Taphrina deformans on peach trees using molecular methods

The ascomycetous fungus Taphrina deformans is the agent of peach leaf curl, a worldwide disease of peach potentially devastating to both crop yields and tree longevity. Conspicuous leaf curl symptoms result from the invasion of host tissue by the strictly parasitic mycelial phase of the T. deformans dimorphic life-cycle. Successful isolation of the fungus in pure culture is cumbersome and limited to late spring/early summer (time of ascospore discharge from infected leaves) and only rarely has the asymptomatic yeast phase been isolated from buds. Molecular methods, namely those based on the hybridisation of nucleic acids, are advantageous for diagnostic purposes since they do not require isolation of the fungus on culture media. Direct amplification using the polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) were tested for diagnosis of peach leaf curl disease in order to provide a fast and reliable method for disease risk assessment. Specific primers and probes were designed based on available ribosomal DNA sequence data. Positive and specific diagnoses of peach leaf curl were achieved with primer TDITS1, using PCR-detection, and probe TDE634, using FISH, both on infected leaves and in washings of asymptomatic peach buds.

Dibutyryl c-AMP as an inducer of sporidia formation: biochemical and antigenic changes during morphological differentiation of Karnal bunt (Tilletia indica) pathogen in axenic culture.

Effect of dibutyryl adenosine 3',5'-cyclic monophosphate (dbc-AMP), an analogue of c-AMP, was investigated on growth and morphological differentiation of Tilletia indica. Exponential growth was observed up to 21 days in both presence and absence of dbc-AMP; however, increasing concentration of dbc-AMP was deleterious to mycelial growth in liquid culture. A slow increase of mycelial biomass up to 21 days and decline at 30 days in the presence of 2.5 mM dbc-AMP was observed, therefore, this concentration was chosen in subsequent investigations. The inhibitory influence of dbc-AMP was further substantiated by decrease in soluble protein. The fungus on exposure to dbc-AMP experienced morphological differentiation from vegetative mycelial phase to sporogenous mycelial phase, and was induced to produce filiform sporidia. Use of quantitative ELISA further suggested that sporidia formation took more than 21 days in the presence of dbc-AMP. Variations of proteins during different stages of T. indica grown in the presence and absence of dbc-AMP suggested the expression of stage-specific proteins or differential expression of proteins induced by dbc-AMP. The changes in expression of cell surface antigens as evidenced from decrease and increase binding of anti-mycelial and anti-sporidial antibodies in dbc-AMP treated culture by ELISA was further interpreted on the basis of morphological differentiation from mycelial to sporidial phase

Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis.

Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.

Structural differences between the alkali-extracted water-soluble cell wall polysaccharides from mycelial and yeast phases of the pathogenic dimorphic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques. The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of -->6)-[alpha-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->. The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of -->6)-[-alpha-Manp-(1-->2)]-alpha-Manp-(1-->, alternating with a trisaccharide repeating block of -->6)-[beta-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->.

Identifying phase-specific genes in the fungal pathogen Histoplasma capsulatum using a genomic shotgun microarray.

A fundamental feature of the fungal pathogen Histoplasma capsulatum is its ability to shift from a mycelial phase in the soil to a yeast phase in its human host. Each form plays a critical role in infection and disease, but little is understood about how these two morphologic phases are established and maintained. To identify phase-regulated genes of H. capsulatum, we carried out expression analyses by using a genomic shotgun microarray representing approximately one-third of the genome, and identified 500 clones that were differentially expressed. Genes induced in the mycelial phase included several involved in conidiation, cell polarity, and melanin production in other organisms. Genes induced in the yeast phase included several involved in sulfur metabolism, extending previous observations that sulfur metabolism influences morphology in H. capsulatum. Other genes with increased expression in the yeast phase were implicated in nutrient acquisition and cell cycle regulation. Unexpectedly, differential regulation of the site of transcript initiation was also observed in the two phases. These findings identify genes that may determine some of the major characteristics of the mycelial and yeast phases.

Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes.

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5' and 3' ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.

Ammonium fumarate production by free or immobilised Rhizopus arrhizus in bench‐ and laboratory‐scale bioreactors

AbstractAmmonium fumarate production from glucose‐based media by Rhizopus arrhizus NRRL 1526 with mycelial growth controlled by phosphorus limitation exhibited mixed‐growth‐associated product formation kinetics, with growth‐associated production related to secondary mycelial growth only. The contribution of the primary mycelial growth phase was minimised by resorting to prolonged batch production using free mycelia under intermittent glucose feeding or repeated batch production using immobilised mycelia. The metabolic activity of free or immobilised mycelia was limited by fumarate accumulation or by oxygen diffusion phenomena, respectively. For batch cultures in a 15 dm3 stirred bioreactor the peripheral impeller speed (vI) was increased from 1.88 to 3.3 m s−1, and the fumarate yield coefficient on glucose increased from 0.25 ± 0.01 to 0.42 ± 0.02 g g−1, while the malate yield coefficient on fumarate (YM/F) reduced from 0.46 ± 0.01 to 0.14 ± 0.01 g g−1. With a net increase in the fumarate‐to‐malate ratio from 2 to 6.5, a vI value of 3.3 m s−1 gave the best fermentation performance and provided a basis for further scale‐up studies.© 2002 Society of Chemical Industry

Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases.

Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species. Evidence is presented here for two additional catalase isozymes in H. capsulatum. Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts. Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated. The transcriptional expression of the H. capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA. Results at the transcript levels for these genes are shown for three conditions: cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H(2)O(2)) and carbon source (glucose vs glycerol). Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress. A phylogenetic analysis of presently available catalase sequences and intron residences was done. The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes.

Production of the mycelial phase of Malassezia in vitro

Production of the mycelial phase of Malassezia in vitro

A nutrient-regulated, dual localization phospholipase A(2) in the symbiotic fungus Tuber borchii.

Important morphogenetic transitions in fungi are triggered by starvation-induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up-regulation of TbSP1, a surface-associated, Ca(2+)-dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the first phospholipase A(2) to be described in fungi and identifies a novel class of phospholipid-hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a pre-protein, is processed efficiently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free-living and symbiosis-engaged hyphae. It thus appears that a dual localization phospholipase A(2) is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1-related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non-filamentous microorganisms, points to a general role for TbSP1 phospholipases A(2) in the organization of multicellular filamentous structures in bacteria and fungi.

Antibiotic production, accumulation of intracellular carbon reserves, and sporulation inMicromonospora echinospora(ATCC 15837)

The physiology of the actinomycete Micromonospora echinospora was examined during growth. Biphasic accumulation of glycogen occurred, initially during the early exponential growth phase, and again following the onset of sporulation at 120 h. Lipid levels increased during growth eventually representing 25% of the cell mass. A significant proportion of the lipid was found to be in the form of triacylglycerols, which were found to accumulate markedly during the sporulation phase. The disaccharide trehalose was also found to accumulate during growth with levels rising to 5% of the dry weight during the mycelial production phase, then remaining constant during sporulation. Antibiotic was produced transiently by the cultures over the period preceding sporulation.

Features and Phylogenetic Status of an Enigmatic Clavicipitalean Fungus Neoclaviceps monostipa gen. et sp. nov.

A new species and genus of the gramini- colous Clavicipitaceae are described. Neoclaviceps monostipa infects individual florets in the same way as species of genus Claviceps but does not produce sclerotia. In culture it shows dimorphism in produc- tion of a yeast-like microcyclic conidiation (ephelidi- al) phase and a mycelial phase. Phylogenetic analysis of the 26S large subunit rDNA suggests that Neoclav- iceps along with genus Myriogenospora are evolution- arily intermediate between Claviceps and Balansia.

Features and phylogenetic status of an enigmatic clavicipitalean fungus Neoclaviceps monostipa gen. et sp. nov.

A new species and genus of the gramini-colous Clavicipitaceae are described. Neoclaviceps monostipa infects individual florets in the same way as species of genus Claviceps but does not produce sclerotia. In culture it shows dimorphism in production of a yeast-like microcyclic conidiation (ephelidial) phase and a mycelial phase. Phylogenetic analysis of the 26S large subunit rDNA suggests that Neoclaviceps along with genus Myriogenospora are evolutionary intermediate between Claviceps and Balansia.

Cell-mediated immunity to the mycelial phase of Malassezia spp. in patients with pityriasis versicolor and controls.

Malassezia is the aetiological agent of pityriasis versicolor. The mycelial phase of the organism predominates in lesions of pityriasis versicolor. To evaluate the cell-mediated immune (CMI) response to the mycelial phase of Malassezia in patients with this disease, which has not previously been studied. The CMI status of 12 patients with pityriasis versicolor and 12 age- and sex-matched controls to mycelial antigen(s) of the organism was examined. The responses to the mycelial form of three strains of the organism were assessed using lymphocyte transformation and leucocyte migration inhibition assays. The transformation responses of the lymphocytes from both patients and controls gave transformation indices < or = 3, although the responses of lymphocytes from patients with pityriasis versicolor to the mycelial form of Malassezia strains were generally higher than those of the controls. In the leucocyte migration inhibition assay, leucocytes from patients with pityriasis versicolor and controls responded to the mycelial antigens of three different Malassezia strains; however, there was no significant difference in leucocyte response between patients with pityriasis versicolor and controls. Patients with pityriasis versicolor do not therefore have a CMI deficiency to Malassezia mycelial antigens but fail to generate a protective CMI response to mycelial antigens over and above that of control individuals during active disease.