Role of the conidium in dimorphism of Blastomyces dermatitidis

Fine details of yeastlike cell development of Blastomyces dermatitidis from its conidium are described and illustrated by electron micrographs. When cultured in an enriched medium at 37 °C, conidia of two strains of B. dermatitidis readily underwent ultrastructural changes consistent with mycelial to yeast dimorphism. Although hyphal cells contained in the conversion cultures were observed consistently to undergo profound degenerative changes, the conidia rapidly germinated to give rise to short germ tubes which subsequently enlarged to form intermediate yeast mother cells (YMC). The wall of the germ tube arose from the innermost layer of the wall of the germinant. During the transition globoid osmiophilic inclusions of unknown origin and function were observed in vacuolated areas of the germ tube and YMC cytoplasm. Yeastlike daughter cells then budded from the intermediate YMC. Since transformation was readily accomplished under in vitro conditions favoring mycelial to yeast dimorphism, it is suggested that the conidium of B. dermatitidis represents the primary infective unit of this pathogenic fungus.     

Speculative cell cycle in yeast-phase growth of Sporothrix schenckii: plasma membrane ultrastructure as revealed by freeze-fracture electron microscopy

The cell cycle in yeast-phase growth of Sporothrix schenckii was investigated by light microscopy and freeze-fracture electron microscopy after a 3- to 7-day cultivation on brain heart infusion agar medium at 37 degrees C. Mother yeastlike cells were able to bear daughter yeastlike cells. They were also able to produce germ tubes that had the potential to develop into pseudohyphae and hyphae. On the other hand, hyphae or pseudohyphae born from yeastlike cells were able to bear yeastlike cells directly. These results lead us to propose a hypothetical cell cycle for yeast-phase growth involving yeastlike vegetative cells, pseudohyphae, and hyphae.     

Molecular and cellular events during the yeast to mycelium transition in Sporothrix schenckii

Unbudded singlets from exponentially growing yeast cells of Sporothrix schenckii were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 degrees C. These conditions supported only the development of the mycelial form of S. schenckii in a reproducible manner which allowed further analysis of the early cellular events occurring during the yeast-to-mycelium transition. The relationship between macromolecular synthesis (DNA and RNA synthesis) and nuclear division, hyphal growth and septum formation were investigated during germ tube formation. RNA synthesis started 0 to 3 h after the induction of germ tube formation, followed by DNA synthesis and the first nuclear division, which took place between 3 and 6 h. Germ tube formation followed nuclear division and was first evidenced 6 h after the induction of germ tube formation, but was not completed until 12 h after inoculation. Septation was first observed in these germ tubes at the mother cell-germ tube junction 6 h after induction. Addition of hydroxyurea, an inhibitor of DNA synthesis, to the medium, also inhibited nuclear division and germ tube growth, suggesting that these processes in S. schenckii are dependent upon DNA synthesis.     

Induction of mycelial type of development in Candida albicans by low glucose concentration

A new simple method for synchronous germ tube production in Candida albicans has been described, based on the further incubation of cells released from stationary grown cultures in aerated mineral medium enriched with vitamins and low glucose concentration (5 mmol/l). At higher initial glucose (e.g. 250 mmol/l) the growth proceeded in yeastlike form. At low glucose concentration germ tubes developed at 28°C which is in contradiction with the results of many authors, considering 37°C besides other factors to be an inevitable requirement. On the other hand the cell population from stationary growth phase was the absolute prerequisite for massive germ tube production. Its importance for other inductive techniques is assumed.The report brings comparative results concerning the physiological and biochemical properties as well as the ultrastructure of the yeastlike and mycelial forms. Neither were found any differences in respiration intensity nor in respiration quotients during the development of both growth forms. Slight dissimilarities resulted from the incorporation experiments (using ¹⁴C labeled adenine, leucine and especially glycine). The mycelial cell walls were found to contain twice as much chitin as the yeastlike form.Some suggestion for further biochemical elucidation of dimorphism in Candida albicans and fungal morphogenesis generally are presented.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

Electron microscopy of the transitional conversion cell of Histoplasma capsulatum

Aspects of the fine structure of the transitional conversion cell formed during the early stages of the yeast to mold morphogenesis ofHistoplasma capsulatum as seen in ultrathin sections are described and illustrated by electron micrographs. Formation of the transitional cell was observed to occur with the highest degree of frequency between the 18th and 24th hr following induction of the conversional stimulus, although many yeastlike cells were observed to undergo degeneration or to initiate conversion only to abort the process. Cytoplasmic streaming and organelle migration from the parent yeast to the transitional cell was observed to occur prior to septation. The cell wall of the transitional form is thinner than that of the yeast and appears to arise from the inner portion of the laminated cell wall adjacent to the plasma membrane of the converting yeastlike cell. Interseptal or Woronin bodies were observed in association with the septal pore of the completed septum and were observed in the cytoplasm of both the yeastlike and transitional cell. The presence of these structures support strongly the pre-hyphal character of the converting cell complex.     

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.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Molecular and cellular events during the germination of conidia of Sporothrix schenckii

Hyaline, non pigmented microconidia of Sporothrix schenckii were harvested and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 °C. These conditions supported only the development of the mycelial form of Sporothrix schenckii in a reproducible, synchronized manner which allowed further analysis of the early cellular events ocurring during the germination of the conidia. The relationship between macromolecular synthesis (DNA, RNA and protein synthesis) and nuclear division, hyphal growth and septum formation were established. Following inoculation, protein synthesis was observed after 10 minutes followed by RNA synthesis, after 1 h and DNA synthesis after 2 h. The first nuclear division was observed during the 9 to 12 h interval after inoculation. Germ tube formation slightly preceeded nuclear division and was first evidenced 9 h after the induction of germination but was not completed until 12 h after inoculation. Septation was first observed in the germ tubes 0.25 m from the mother cell-germ tube function 9 h after induction of germination.     

Cytoplasmic alkalinization during germ tube formation in Candida albicans

Weak acids were used to measure the internal pH of yeast cells of Candida albicans that had been induced to form buds or germ tubes. Under conditions that supported germ tube formation the internal pH rose from around 6.8 to over 8.0 after 30 min in two different induction media. Internal pH measured by 31P NMR confirmed this pattern and also showed that the internal pH fell to around 7.0 prior to the outgrowth of germ tubes. Conditions which led to budding induced less cytoplasmic alkalinization. This alkalinization was brought about when cells were inoculated into media of neutral pH and at an increased temperature. Increasing the temperature of the medium augmented the alkalinization of the cytoplasm induced by raising the external pH. Strains of C. albicans defective in the ability to produce germ tubes did not show this dramatic cytoplasmic alkalinization under conditions which normally supported filamentous growth. The raising of internal pH may be due to the activation of the plasma membrane proton-pumping ATPase since diethylstilboestrol inhibited the cytoplasmic alkalinization and germ tube formation without causing irreversible loss of cell viability. The results show that the induction of the dimorphic transition in this organism is accompanied by a steep rise in internal pH. It is not known whether these changes are the cause or consequence of morphogenesis.     

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.     

An analysis of the metabolism and cell wall composition of Candida albicans during germ-tube formation

The uptake of nutrients (glucose, glutamine, and N-acetylglucosamine), the intracellular concentrations of metabolites (glucose-6-phosphate, cyclic AMP, amino acids, trehalose, and glycogen) and cell wall composition were studied in Candida albicans. These analyses were carried out with exponential-phase, stationary-phase, and starved yeast cells, and during germ-tube formation. Germ tubes formed during a 3-h incubation of starved yeast cells (0.8 X 10(8) cells/mL) at 37 degrees C during which time the nutrients glucose plus glutamine or N-acetylglucosamine (2.5 mM of each) were completely utilized. Control incubations with these nutrients at 28 degrees C did not form germ tubes. Uptake of N-acetylglucosamine and glutamine was inhibited by cycloheximide which suggests that de novo protein synthesis was required for the induction of these uptake systems. The glucose-6-phosphate content varied from 0.4 nmol/mg dry weight for starved cells to 2-3 nmol/mg dry weight for growing yeast cells and germ tube forming cells. Trehalose content varied from 85 nmol/mg dry weight (growing yeast cells and germ tube forming cells) to 165 nmol/mg weight (stationary-phase cells). The glycogen content decreased during germ-tube formation (from 800 to 600 nmol glucose equivalent/mg dry weight) but increased (to 1000 nmol glucose equivalent/mg dry weight) in the control incubation of yeast cells. Cyclic AMP remained constant throughout germ-tube formation at 4-6 pmol/mg dry weight. The total amino acid pool was similar in exponential, starved, and germ tube forming cells but there were changes in the amounts of individual amino acids. The overall cell wall composition of yeast cells and germ tube forming cells were similar: lipid (2%, w/w); protein (3-6%), and carbohydrate (77-85%). The total carbohydrates were accounted for as the following fractions: alkali-soluble glucan (3-8%), mannan (20-23%), acid-soluble glucan (24-27%), and acid-insoluble glucan (18-26%). The relative amounts of the alkali-soluble and insoluble glucan changed during starvation of yeast cells, reinitiation of yeast-phase growth, and germ-tube formation. Analysis of the insoluble glucan fraction from cells labelled with [14C]glucose during germ-tube formation showed that the chitin content of the cell wall increased from 0.6% to 2.7% (w/w).     

Early development in an algal gametophyte: role of the cytoskeleton in germination and nuclear translocation

Summary Biflagellate zoospores from the giant kelpMacrocystis pyrifera underwent germination after adhering to a substrate and produced germ tubes that were approximately 13–15 m in length. Coincident with the germ tube elongation was organelle (other than the nucleus) translocation along the tube. Shortly after formation of the germ tube, the zoospore nucleus divided and one daughter nucleus translocated along the germ tube. The nucleus did not appear to undergo chromosomal condensation prior to division. The nuclear division and/or translocation of the daughter nucleus did not begin until well after germ tube elongation was complete, demonstrating that these are temporally distinct developmental events. The translocation of one daughter nucleus coincided with differentiation of the distal end of the germ tube into a bulbous structure. Following this, the first gametophytic cross wall was formed and, subsequently, the daughter nucleus remaining in the original zoospore body underwent repositioning, assuming a position in the germ tube near the cross wall. Cytochalasin D inhibited germ tube elongation suggesting that actin microfilaments are probably involved in this developmental process. In addition, when cytochalasin D was added to the culture after the germ tube elongation was complete, it did not affect either nuclear division or translocation, indicating that microfilaments were not directly involved in these nuclear events. Colchicine and the plant specific microtubule disrupting agent, amiprophos methyl blocked nuclear division and translocation without affecting germination or germ tube elongation. These data suggest that actin microfilaments are primarily responsible for complete germination, specifically germ tube elongation, while microtubules are involved in nuclear division and translocation. The present study demonstrates that germination (and germ tube elongation) and nuclear translocation inM. pyrifera gametophytes are temporally and mechanistically distinct developmental events.     

A mother's sacrifice: what is she keeping for herself?

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited life span and undergo a form of senescence termed replicative aging. Replicative life span is defined as the number of daughter cells produced by a yeast mother cell before she ceases dividing. Replicative aging is asymmetric: a mother cell ages but the age of her daughter cells is 'reset' to zero. Thus, one or more senescence factors have been proposed to accumulate asymmetrically between mother and daughter yeast cells and lead to mother-specific replicative senescence once a crucial threshold has been reached. Here we evaluate potential candidates for senescence factors and age-associated phenotypes and discuss potential mechanisms underlying the asymmetry of replicative aging in budding yeast.     

Induction of mycelial type of development inCandida albicans by the antibiotic monorden and N-acetyl-D-glucosamine

Two new effective methods for synchronous germ tube production inCandida albicans have been described. Both are based on the use of stationary grown cultures and their further incubation in an aerated simple mineral medium enriched with vitamins containing either high glucose concentration (100 mmol/l) and the antibiotic monorden being added, or N-acetyl-D-glucosamine (100 mmol/l) as the sole carbon source. On the other hand yeast morphology could be maintained in the medium with high glucose concentration.On the basis of the methods developed it was possible to compare respiration intensity, respiration quotients, and sensitivity against some metabolic inhibitors in both morphological forms. Labeling experiments showed slight differences in the time course of glycine incorporation. The mycelial cell walls contained more chitin than the yeastlike cells. Using light and electron microscopy the interrelationships between concentration of monorden, or N-acetyl-D-glucosamine, physiological state of inoculum and the germ tube frequency were determined.The results are discussed with regard to the induction of germ tubes by low glucose concentration inCandida albicans from the more general aspect of regulation of fungal morphogenesis.     

Effect of yeast growth conditions on yeast-mycelial transition in Candida albicans

When grown and induced to form germ tubes in liquid defined media, yeast cells of Candida albicans must reach stationary phase before acquiring ability to carry out the yeast-mycelial transition. This study examined the effect of the carbon source utilized for yeast growth on the inducibility of stationary phase yeast. When grown to the same stationary phase cell density as glucose cultures, cultures grown on citrate were fully inducible while cultures grown on galactose and mannose showed a small reduction. Cultures grown on ethanol were reduced 80% in morphological conversion. When glucose grown cells were induced in the presence of these carbon sources, hexoses supported full induction while ethanol reduced induction 80%. Induction in the presence of carboxylic acids was similar to induction in the absence of added carbon source. When induced on the same source used in yeast growth, germ tube formation was reduced for all carbon sources except hexoses. When induced in the absence of added carbon source, yeasts grown on citrate and ethanol were inhibited 80-100%. Cultures starved for glucose were more inhibited than cultures starved for NH4Cl when induced without added carbon source. These observations suggest that the metabolic state of the stationary phase cell is an important factor in the ability to respond to conditions inducing germ tube formation.     

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.     

The involvement of cell wall expansion in the two modes of mycelium formation of Candida albicans

When budding cells of Candida albicans are starved for 20 min and then diluted into fresh nutrient medium at 37 degrees C, pH 6.7, they form mycelia by two alternative modes. For cells with small buds, the bud expands apically, resulting in a transiently tapered daughter cell. With continued growth, the daughter cell tapers into an elongated mycelium. For cells with large buds, the bud completes expansion in the budding form, the mother cell and then the daughter bud evaginate, and the evaginations grow as mycelia. The present study investigates whether the temporal and spatial changes in the zones of wall expansion during bud growth are involved in the two modes of mycelium formation. Data are presented which demonstrate that the transition circumference which determines the two modes of mycelium formation and the transition circumference at which the active apical expansion zone shuts down are both 7 micron. This exact correlation suggests that starved cells with buds with a circumference of less than 7 micron form mycelia in the tapering mode due to the reactivation of the still present apical expansion zone, and that starved cells with buds with a circumference greater than 7 micron complete bud growth by general expansion due to the absence of the apical expansion zone at the time of starvation.     

Differential growth rates of Candida utilis mother and daughter cells under phased cultivation.

The yeast Candida utilis was continuously synchronized by the phased method of cultivation with the nitrogen source as the growth-limiting nutrient. The doubling time (phasing period) of cells was 6 h. Both cell number and deoxyribonucleic acid synthesis showed a characteristic stepwise increase during the phased growth. The time of bud emergence coincided with the time of initiation of deoxyribonucleic acid synthesis. Size distribution studies combined with microscopic analysis showed that the cells expanded only during the unbudded phase of growth. Usually the cells stopped increasing in size about 30 min before bud emergence, and the arrest of the increase in cell volume coincided with the exhaustion of nitron from the medium. There was no net change in the volume of cells during the bud expansion phase of growth, suggesting that as the bud expanded, the volume of the mother portion of the cell decreased. After division the cells expanded slightly. The postdivision expansion of cells, unlike the growth before bud initiation, occurred in the absence of the growth-limiting nutrient. The newly formed daughter cells were smaller than the mother cells and expanded at a faster rate, so that both types of cells reached maximum size at the same time. Possible reasons for the different rates of expansion of mother and daughter cells are discussed.     

Surface carbohydrates and cell wall structure of in vitro-induced uredospore infection structures ofUromyces viciae-fabae before and after treatment with enzymes and alkali

Summary Uredospores ofUromyces viciae-fabae differentiate to form germ tubes, appressoria, infection hyphae and haustorial mother cells on oil-containing collodion membranes. The cell walls of these infection structures were studied with the electron microscope and with FITC-labeled lectins before and after treatment with enzymes and inorganic solvents. Binding of the FITC-labeled lectins was measured with a microscope photometer. The enzymes pronase E, laminarinase, chitinase and lipase had different effects on each infection structure. Pronase treatment uncovered the chitin of germ tubes, appressoria and haustorial mother cells, but not of substomatal vesicles and infection hyphae. A mixture of - and -1,3-glucanase which also contained chitinase activity dissolved germ tubes and appressoria completely, but not infection pegs, substomatal vesicles, infection hyphae and haustorial mother cells. After treatment with laminarinase or lipase, an additional layer, which is especially obvious over the substomatal vesicle, infection hypha and haustorial mother cell, bound to LCA-FITC. In the wall of the haustorial mother cell, a ring, which surrounds the presumed infection peg, had strong affinity for WGA after protease and sodium hydroxide treatment. The infection structures have a fibrillar skeleton. The main constituent seems to be chitin. This skeleton is more dense or has a higher chitin content in the walls of appressoria and haustorial mother cells. The fibrils of the skeleton extend throughout the cell wall of the germ tube and appressorium. They are embedded within amorphous material of complex chemical composition (-1,3-glucan, -1,3-glucan, glycoprotein). The chitin of the infection peg, substomatal vesicle, infection hypha and haustorial mother cell is covered completely with this amorphous material. These results show, that each infection structure has distinct surface and wall characteristics. They may reflect the different tasks of the infection structures during host recognition and leaf penetration.Abbreviations AP appressorium - FITC fluorescein isothiocyanate - GT germ tube - HC haustorial mother cell - IH infection hypha - IP infection peg - LCA Lens culinaris agglutinin - n nucleus - neu neuramic acid - p pyranoside - R ring - s septum - SV substomatal vesicle - WGA wheat germ agglutinin