Yeast-like Phase Research Articles

Paracoccidioidomycosis: a sequential histopathologic study of lesions in experimentally-infected rats.

Female albino rats were used for the sequential histopathological study of experimental paracoccidioidomycosis. The animals were inoculated intraperitoneally with a strain of Paracoccidioides brasiliensis in the yeast-like phase, and sacrificed at given intervals from 1 to 168 days after inoculation; each animal received an inoculum of 4 x 10(6) cells in 0.8 ml of saline. The control group received saline containing scrapings of the culture medium. Tissue from the inoculation site was examined. The cellular population, the extracellular matrix, and the presence and characteristics of fungi were analysed in the inflammatory granulomatous process by light microscopy. The results allowed to separate the kinetic of the inflammatory response into three stages: 1) neutrophilic or macrophagic-neutrophilic; 2) pre-granulomatous; 3) granulomatous. Synthesis of the extracellular matrix began with the depositing of fibrin-like material, and increased gradually with deposits of collagen, proteoglycans, and glycoproteins. Parasites were present in all of the examined periods. Recurrences of the disease were clearly shown through the concurrence of recently-formed granulomas with older granulomas, implying that this type of granulomatous process does not eliminate the disease, nor is it able to limit fungal dissemination over a prolonged period of time.

A novel method for estimating killing ability and digestion ofParacoccidioides brasiliensisby phagocytic cellsin vitro

We describe a novel method by which phagocytosis, digestion and killing of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated simultaneously. Suspensions of P. brasiliensis (yeast-like phase) were sonicated, counted and incubated at 37 degrees C with known numbers of phagocytes. Control preparations contained no phagocytic cells. At given intervals samples were incorporated into gelatin nutrient medium and droplets of the mixtures were incubated at room temperature. Live yeast-like P. brasiliensis germinate in vitro and produce filaments. After incubation, droplets may be melted and examined under phase contrast optics, or the cells may be washed and stained by a variation of Papanicolaou's method. Digested P. brasiliensis, intact but non-germinating yeasts and filamented (viable) yeasts may be identified and counted. Killing and digestive abilities of phagocytes may be estimated by the difference between values obtained from phagocyte-containing and control preparations.

Nutritional studies on Paracoccidioides brasiliensis: the role of organic sulfur in dimorphism.

The nutritional requirements of the mycelial and yeast-like phases of the dimorphic fungus Paracoccidioides brasiliensis, a human pathogen, were investigated. For all nine isolates tested, mycelial cells were prototrophic, whereas yeast-like cells required a sulfur-containing amino acid for growth. Moreover, changing the source of nitrogen greatly affected the morphology of the yeast-like cells.

Ultrastructural changes produced by ketoconazole in the yeast-like phase of Paracoccidioides brasiliensis and Histoplasma capsulatum.

The ultrastructural changes produced by ketoconazole in the yeast-phase of H. capsulatum and P. brasiliensis were studied by means of scanning and transmission electron microscopy. The observed alterations on both fungi were very similar to those induced by the same drug on the ultrastructure of C. albicans. These alterations include surface changes, abnormal membrane proliferation, fatty degeneration of the cytoplasm and lysis of the subcellular organelles. P. brasiliensis seems to be more sensitive to ketoconazole than H. capsulatum, since the necrosis of most of the cells was obtained in the former at a concentration of 0.1 microgram/ml and in the latter at 1 microgram/ml.

Acid phosphatase observed in the intramembranous system of the yeast-like phase of Paracoccidioides brasiliensis

Acid phosphatase observed in the intramembranous system of the yeast-like phase of Paracoccidioides brasiliensis

New method for estimating digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro.

We describe a method by which phagocytosis and digestion of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated. Suspensions of P. brasiliensis in its yeastlike phase were sonicated, counted, and incubated with known numbers of peripheral blood polymorphonuclear leukocytes. At given intervals, cytocentrifuge droplets were stained by a variation of Papanicolaou's method. Stained preparations were examined with phase-contrast optics. Digested organisms showed total or partial disappearance of protoplasm. Green-stained cell walls resisted digestion. The proportion of digested cells as a function of time was estimated.

Filobasidiella arachnophila sp. nov.

A new species of Filobasidiella Kwon-Chung is described and illustrated. This species, F. arachnophila, was isolated from a dead spider and appears to be nonpathogenic. It differs from previously described species in being homothallic and in lacking a yeast-like phase of growth.

Electron microscopy of yeastlike cell development from the microconidium of Histoplasma capsulatum.

Fine details of the sequential morphological events occurring during transition of microconidia (spores less than 5 micrometer in diameter) to the yeastlike phase of Histoplasma capsulatum as seen in ultrathin section are described and illustrated by electron micrographs. Masses of microconidia were obtained when the fungas was grown on a garden soil extract medium. Spores were incubated under in vitro environmental conditions conducive for phase transition (an enriched medium at 37 degrees C). Within 48 h of incubation, the microconidia either germinated to give rise to a short mycelium or the germ tube process became a yeast mother cell without further extension. The wall of the yeast mother cell was thin and smooth, and its cytoplasmic content was ultrastructurally complex, consisting of numerous lipid bodies, vacuoles, glycogen-like deposits, and membrane systems. Within 96 h, the mother cell underwent multipolar budding to form simultaneously linear hyphal and/or ovate yeastlike daughter cells. During the transition, new cell wall materials of the germ tube, the mother cell, and yeastlike daughter cells arose by blastic action from the innermost layer(s) of the wall of the precursor form. Lomasome-like vesicles were often seen in association with areas of new cell wall formation. After organellar migration into and septation of the daughter cells, the yeast mother cell's cytoplasmic content underwent marked degenerative changes.

Enzyme and Respiration Activities on the Morphological Change of <i>Geotrichum candidum</i> belonging to Sludge Fungi

Respiration and enzyme activities of Geotrichum candidum showing filamentous phase in bulking sludge was examined in comparison with yeast-like phase.1) RQ value of endogenous respiration in F phase cells for the early period was slightly greater than that of Y-like phase cells, but considerable differences were not observed between both phases. NaN3, CH2ICOOH strongly inhibited the endogenous respiration of F phase cells.2) The dissolved oxygen (DO) consumption of G. candidum on various substrates was generally greater in Y-like phase cells than in F phase cells, and there was, in particular, a remarkable difference of DO consumption when the organism was grown in the medium added with amino acid such as glycine, alanine and arginine. As3+ and Hg2+ showed almost equal inhibition on pyruvate oxidation in both phase cells, but Cu2+, Pb2+, CN- and HCHO inhibited strongly the respiration of Y-like phase cells.3) On enzymes of Leucine aminopeptidase, Alcohol dehydrogenase, G-6-P dehydrogenase and Glutathione reductase, except 5'-Nucleotidase, F phase cells showed high activities. Acid phosphatase (PMase) in cells of Y-like phase showed a marked lower activity than in F phase cells. However, Acid PMase in cell free extracts from Y-like phase showed opposite results.4) Acid PMase was activated by the addition of Ca2+, Mg2+ and Ba2+, and it was noted that the activity was markedly accelerated by Pb2+ and Cu2+ Mo6+ and W6+ showed complete inhibition by the activity.

14C‐labeled aflatoxin B1prepared with yeastlike cultures ofAspergillus parasiticus

A simple method was developed to produce 14C-labeled aflatoxin B1 by using the yeastlike phase of Aspergillus parasiticus NRRL 2999. Yeastlike cultures resulted from absence of manganese in a synthetic medium. Sodium acetate-1-14C had a 0.22% average incorporation; sodium acetate-1,2-14C, 0.70%. The average yield of labeled B1 was 10 mg/500 ml medium with an average specific activity of either 63.3 mCi/mole (C-1 label) or 194.3 mCi/mole (C-1, 2 label).

Ultrastructural studies of the mycelium-to yeast transformation of Sporothrix schenckii.

Fine details of the internal and external morphology of the in vitro mycelial phase (MP) to yeastlike phase (YP) transition of the dimorphic fungal pathogen Sporothrix schenckii are shown in electron micrographs of ultrathin sections. Morphological transformation at the ultrastructural level was observed to occur by direct formation of budlike structures at the tips and along the hyphae and by oidial cell formation. Direct budding of yeast from conidiospores was not observed. Early transitional forms arising by direct blastic action from the MP possessed conspicuous electron-dense microfibrillar material at the outer limits of the cell wall. The electron density of this microfibrillar material was enhanced by staining with acidified dialyzed iron. It is believed that this extracellular material may be composed in part of an acid mucosubstance. No acid phosphatase activity was associated with this microfibrillar material. This substance was found to be a characteristic of the outer limits of the cell wall of the YP of S. schenckii. Oidial YP cell formation occurred later during the transition. The cell wall of the developing oidial YP transitional form arose from an inner layer of the converting hyphae. No consupicuous alterations of the cytoplasmic content of the parent MP cell was observed during MP-to-YP transition. It is suggested that the MP-to-YP transition of S. schenckii may be regulated by at least two mechanisms involving alterations of the biochemical and/or biophysical nature of the cell wall of the MP cell in response to the conversional stimuli.

Yeast-like growth of Mucor alternans (van Tieghem) in tissue-culture medium 199

Mucor alternans(van Tieghem) was found to be a diphasic species in that it grew exclusively in the yeast-like budding phase under anaerobic conditions in the complex medium yeast extract-peptone-glucose broth and in tissue-culture medium 199. In the latter medium this growth form occurred also at 37C, at an initial pH of 7.2, and at glucose concentrations of 0.1 and 1.0%. The authors suggest that because of its synthetic nature the tissue-culture medium could be used with advantage in the study of nutritional requirements of dimorphic mucors.

Aspects of the dimorphism of Histoplasma farciminosum: a light and electron microscopic study.

Within 48h following the induction of mycelial to yeast-like phase conversion of Histoplasma farcininosum, randomly occurring hyphal cells were observed to contain multiple nuclei and markedly increased numbers of mitochondria. Yeast-like cells arose as buds from swollen tips of terminal hyphae, as sessile buds along the hyphae, and as buds from chlamydospores. Yeast-like cells were characterized by the presence of numerous buds over the surface of the mother cell. Bud scars were evident in the cell wall of the mother cell following abscission of the bud cell. Little similarity was noted between the fine structure of yeast-like H. farciminosum and that reported for H. capsulatum. The yeast-like cells of H. frciminosum underwent rapid transformation to the mycelial phase at 25 degrees C. The hyphal cell wall originated from the inner layer of cell wall of the yeast-like form. The cytoplasm of the hyphal cell usually contained a single nucleus, scattered mitochondria and occasional lipid storage bodies. Occasionally, Woronin bodies were observed at the septal pore.

The uptake and incorporation of leucine and cystine by the mycelial and yeastlike phases of Blastomyces dermatitidis.

Uptake and incorporation of L-leucine-C14 and L-cystine-S35 was studied in the mycelial [MP] and yeastlike [YP] phases of the dimorphic fungal pathogen,Blastomyces dermatitidis. Both amino acids entered the cells of the two morphological forms ofB. dermatitidis by a permease-like system at low external concentrations of substrate. At high substrate levels, the amino acids entered the cells by a simple diffusion-like process in addition to the permease-like system. Michaelis-Menten constants [Km] for L-leucine was found to be 1.1×10−5 M and 4.4×10−5 M for the MP and YP phases, respectively. The Km for L-cystine was found to be 1.0×10−5 M for the MP and 0.5×10−5 M for the YP. A requirement for energy supplied by metabolic activity was demonstrated by the inhibition of uptake and incorporation of the amino acids by cells incubated with either 2,4-dinitrophenol or sodium azide. Amino acid uptake was broadly tolerant of hydrogen ion concentration, but definite optima were demonstrated at pH 7.0 to 7.5.

Factors affecting mycelial to yeast phase conversion and growth of the yeast phase of Histoplasma capsulatum.

A procedure for rapid induction of mycelial to yeast phase (M→Y) conversion ofHistoplasma capsulatum has been devised. Exposure of mycelial fragments to low oxidation-reduction (O/R) potentials (+5 to+65 mv) either aerobically (ascorbic acid treatment) or anaerobically (nitrogen atmosphere) for 18 to 24 hours at 37° C resulted in induction of the M→Y conversion process whether or not an organic sulfur source was available. However, aerobic conditions and a suitable organic sulfur source, such as cysteine, cystine or lanthionine were found essential for outgrowth and maintenance in the yeastlike phase.

Electron microscopy of the yeast to mycelial phase conversion of Sporotrichum schenckii.

Ultrastructural events occurring during the yeast-like to mycelial phase conversion of the dimorphic fungal pathogen Sporotrichum schenckii were studied by electron microscopy of thin sections. Discrete intracytoplasmic membrane systems were formed as early as 6 h after induction of the conversion stimuli. Within 12 to 18 h, a transitional cell was formed from the parent yeast-like cell. Definite streaming of yeast-like cell cytoplasm and mitochondria was noted before septal formation in the intermediate cell. Although the cell wall of the parent yeast-like phase cell is present in two distinct layers, only the inner layer persists upon transitional cell formation. Yeast-like to mycelial phase reorganization of S. schenckii resembles closely that described for Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis, and it is suggested that yeast to mold transformation of these dimorphic fungi may proceed via a common conversional mechanism.

The uptake of low molecular weight sulfur-containing compounds by Histoplasma capsulatum and related dimorphic fungi.

A number of low molecular weight organic sulfur-containing compounds were tested for their effect on the respiratory activity of yeastlike and mycelialH. capsulatum. Of the compounds tested, L-cyst(e)ine was found to give maximum stimulatory effect on yeastlike phase respiration. The D- and meso isomers of cyst(e)ine as well as substituted derivatives were much less effective in the stimulation of respiratory activity of yeastlikeH. capsulatum. Respiration of homologous mycelial phase cell suspensions was depressed in the presence of L-cystine as substrate, while respiratory activity of yeastlikeB. dermatitidis andS. schenckii was unaffected.

Dimorphism of Sporotrichum schenckii; Experiment of its conversion into the yeast-like phase with studies on morphological comparison of the yeast-like cells

Dimorphism of Sporotrichum schenckii; Experiment of its conversion into the yeast-like phase with studies on morphological comparison of the yeast-like cells

Ultrastructural studies on the yeastlike and mycelial phases of Sporotrichum schenckii.

Fine details of the internal and external morphology of the yeastlike and mycelial phases of the dimorphic fungal pathogen Sporotrichum schenckii as seen in ultrathin sections are described and illustrated by electronphotomicrography. Comparisons of yeastlike phase ultrastructure were made using two different methods of fixation and embedding. The internal morphology of the two forms of yeastlike S. schenckii was in many ways similar to that of similarly dimorphic fungi and yeasts studied by other authors. However, the use of the glutaraldehyde-osmium in agar fixation technique suggests the presence of an electron transparent capsular or slime layer with associated electron dense microfibrils to be present external to the cell wall of the yeastlike phase but not the mycelial phase. Mycelial phase S. schenckii was found to contain many of the internal microstructures reported for other filamentous dimorphic fungi. Conidia production in agitated liquid culture was found to be restricted since only rare sessile conidia were observed.

Stimulation by phenethyl alcohol of aerobic fermentation in [formula omitted

In the absence of oxygen, some Mucor species exhibit a yeast-like morphology and reproduce by budding. Bartnicki-Garcia and Nickerson (1962 a,b) showed that the yeast-like phase of Mucor rouxii took place only when hexoses were fermented and that it could be converted to the hyphal phase after a few hours of exposure to air. Haidle and Storck (1966 a,b) found that this conversion which required the synthesis of RNA and proteins was accompanied by the appearance of cytochrome oxidase activity. Recently Terenzi and Storck (1967) reported that a yeast-like phase was obtained in aerobiosis provided 0.2% ( v v ) of phenethyl alcohol (PEA) and a hexose (2–5%) were included in the growth medium. In the present paper it is shown that aerobic yeast-like growth involves an increase of alcoholic fermentation and a concomitant decrease of respiration. The data further suggest that PEA acts like an uncoupling agent and stimulates a Crabtree effect.