Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in Ceratocystis ulmi

We studied the inoculum size effect in Ceratocystis ulmi, the dimorphic fungus that causes Dutch elm disease. In a defined glucose-proline-salts medium, cells develop as budding yeasts when inoculated at ≥10⁶ spores per ml and as mycelia when inoculated at <10⁶ spores per ml. The inoculum size effect was not influenced by inoculum spore type, age of the spores, temperature, pH, oxygen availability, trace metals, sulfur source, phosphorous source, or the concentration of glucose or proline. Similarly, it was not influenced by added adenosine, reducing agents, methyl donors, amino sugars, fatty acids, or carbon dioxide. Instead, growing cells excreted an unknown quorum-sensing factor that caused a morphological shift from mycelia to budding yeasts. This yeast-promoting effect is abolished if it is extracted with an organic solvent such as ethyl acetate. The quorum-sensing activity acquired by the organic solvent could be added back to fresh medium in a dose-dependent fashion. The quorum-sensing activity in C. ulmi spent medium was specific for C. ulmi and had no effect on the dimorphic fungus Candida albicans or the photomorphogenic fungus Penicillium isariaeforme. In addition, farnesol, the quorum-sensing molecule produced by C. albicans, did not inhibit mycelial development of C. ulmi when present at concentrations of up to 100 μM. We conclude that the inoculum size effect is a manifestation of a quorum-sensing system that is mediated by an excreted extracellular molecule, and we suggest that quorum sensing is a general phenomenon in dimorphic fungi.     

Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol.

The inoculum size effect in the dimorphic fungus Candida albicans results from production of an extracellular quorum-sensing molecule (QSM). This molecule prevents mycelial development in both a growth morphology assay and a differentiation assay using three chemically distinct triggers for germ tube formation (GTF): L-proline, N-acetylglucosamine, and serum (either pig or fetal bovine). In all cases, the presence of QSM prevents the yeast-to-mycelium conversion, resulting in actively budding yeasts without influencing cellular growth rates. QSM exhibits general cross-reactivity within C. albicans in that supernatants from strain A72 are active on five other strains of C. albicans and vice versa. The QSM excreted by C. albicans is farnesol (C(15)H(26)O; molecular weight, 222.37). QSM is extracellular, and is produced continuously during growth and over a temperature range from 23 to 43 degrees C, in amounts roughly proportional to the CFU/milliliter. Production is not dependent on the type of carbon source nor nitrogen source or on the chemical nature of the growth medium. Both commercial mixed isomer and (E,E)-farnesol exhibited QSM activity (the ability to prevent GTF) at a level sufficient to account for all the QSM activity present in C. albicans supernatants, i.e., 50% GTF at ca. 30 to 35 microM. Nerolidol was ca. two times less active than farnesol. Neither geraniol (C(10)), geranylgeraniol (C(20)), nor farnesyl pyrophosphate had any QSM activity.     

Farnesol-induced apoptosis in Aspergillus nidulans reveals a possible mechanism for antagonistic interactions between fungi

The dimorphic fungus Candida albicans secretes farnesol, which acts as a quorum-sensing molecule and prevents the yeast to mycelium conversion. In this study we examined the effect of farnesol in the filamentous fungus Aspergillus nidulans. We show that externally added farnesol has no effect on hyphal morphogenesis; instead, it triggers morphological features characteristic of apoptosis. Additional experiments suggest that mitochondria and reactive oxygen species (ROS) participate in farnesol-induced apoptosis. Moreover, the effects of farnesol appear to be mediated by the FadA heterotrimeric G protein complex. Because A. nidulans does not secrete detectable amounts of farnesol, we propose that it responds to farnesol produced by other fungi. In agreement with this notion, growth and development were impaired in a farnesol-dependent manner when A. nidulans was co-cultivated with C. albicans. Taken together, our data suggest that farnesol, in addition to its quorum-sensing function that regulates morphogenesis, is also employed by C. albicans to reduce competition from other microbes.     

Influence of heterocyclic and oxime-containing farnesol analogs on quorum sensing and pathogenicity in Candida albicans

A series of synthetic molecules combining a geranyl backbone with a heterocyclic or oxime head group are quorum-sensing molecules that block the yeast to mycelium transition in the dimorphic fungus Candida albicans. A number of the analogs have an IC50 10 microM, a level of potency essentially identical to the natural quorum sensing signal, the sesquiterpene farnesol. Two of the most potent analogs, neither toxic toward healthy mice, display remarkably different effects when co-administered with C. albicans. While neither offers protection from candidiasis, one analog mimics farnesol in acting as a virulence factor, whereas the other has no effect. The results offer the first example of highly potent synthetic fungal quorum-sensing molecules, and provide the first evidence for the ability to decouple quorum sensing and virulence.     

Quorum sensing activity in Ophiostoma ulmi: effects of fusel oils and branched chain amino acids on yeast-mycelial dimorphism

Aims: For Ophiostoma (Ceratocystis) ulmi, the ability to undergo morphological change is a crucial factor for its virulence. To gain an understanding of quorum‐sensing activity in O. ulmi as it relates to yeast‐mycelium dimorphism control, this study examines the effects of branched‐chain amino acids as well as their fusel alcohols and fusel acids as quorum sensing molecules. Methods and Results: In a defined medium containing glucose, proline and salts, O. ulmi grew as yeasts when the culture was inoculated with a high density of spores (2 × 10⁷ CFU ml^?1) and as mycelia when inoculated with a low spore density (4 × 10⁵ CFU ml^?1). The cultures displaying yeast morphology secreted a quorum‐sensing factor that shifted the morphology from mycelia to yeast. This quorum‐sensing molecule was lipophilic and extractable by organic solvents from the spent medium. Using GC/MS analysis, it was determined that the major compound in the extract was 2‐methyl‐1‐butanol. A similar effect was observed when the branched‐chain amino acids (fusel alcohol precursors) were used as the nitrogen source. E, E‐farnesol had no effect on the morphology of O. ulmi. Conclusions: Addition of the branched‐chain amino acids or one of the compounds detected in the spent medium, 2‐methyl‐1‐butanol or 4‐hydroxyphenylacetic acid, or methylvaleric acid, decreased germ tube formation by more than 50%, thus demonstrating a quorum sensing molecule behaviour in O. ulmi cultures. Significance and impact of the study: This study presents advances in the investigation of dimorphism in O. ulmi, complementing the existing scientific basis, for studying, understanding and controlling this phenomenon.     

Kidney homogenate, a test medium to determine the inhibitory effect of antimycotics on yeasts.

The test procedure described in this paper aims at assessing the effectiveness of antimycotics against yeasts. The inhibitory effect on Candida albicans growth is determined in the presence of kidney homogenate, an in vitro test medium offering fungal growth conditions similar to physiological conditions. The inoculum consists of kidney material from Candida-infected mice. By dilution with kidney material from uninfected mice the initial number of viable particles is standardised. Specimens withdrawn from this inoculum are mixed with different concentrations of the test substance and incubated for 24 hours. The inhibition of fungal growth is assessed on the basis of the number of colony-forming units in comparison with controls to which no test substance was added. The growth phase of C. albicans during incubation in vitro resembles that observed in the kidney in vivo, i.e. besides yeast cells, the fungus produces a great number of pseudomycelia and mycelia. The results of the tests conducted with the top-ranking antimycotics give good evidence of their therapeutic efficacy. The testing of further active substances revealed advantages of the new test model over existing test methods.     

Characterization of Dimorphism in Cladosporium werneckii

Yeast forms of the dimorphic fungus Cladosporium werneckii grow by polar budding and yield a homogeneous yeast phase when cultured at 21 C in an agitated sucrose-salts medium (Czapek-Dox broth). Yeast extract enrichment of such a yeast phase consisting of 10⁴ yeasts per ml induces a quantitative conversion of the yeasts to true hyphae. This conversion is not mediated by a transition cell and is often attended by capsule formation. When 10⁵ or 10⁶ yeasts per ml receive enrichment, a nonquantitative conversion to moniliform hyphae is effected and no capsule formation is observed. Rapid agitation compared to slow agitation or stationary incubation of the nutritionally mediated conversion cultures greatly accelerates the production of lateral hyphal buds or their yeast progenies. These cells appear incapable of undergoing nutritional conversion to hyphae, but instead must grow for several generations in the unenriched sucrose-salts medium to restore conversion competence. Temperature shifts affect directly the morphology and morphogenesis of the yeast in unenriched medium; at 17 C yeasts are smaller and more ovoid than at 21 C, and at 30 C marked conversion of yeasts to moniliform hyphae occurs. A methodology employing the Coulter counter and Coulter channelizer provides evidence that direct correlations do not always exist between the optimum conditions for the growth of C. werneckii and the optimum conditions for its yeast-to-mold conversion.     

Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens.

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.     

Sialoglycoproteins in Morphological Distinct Stages of Mucor polymorphosporus and their Influence on Phagocytosis by Human Blood Phagocytes

The possible role of sialic acids in host cells–fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.     

The Sporulation of Penicillium notatum Westling in Submerged Liquid Culture: II. THE INITIAL SPORULATION PHASE

The initial sporulation time of Penicillium notatum Westlinggrown in shaken submerged culture inoculated with spores orvegetative mycelia is inversely proportional to the logarithmof the inoculum load, with a minimum time of 17.5 hours in theformer and 8 hours in the latter cultures. The development ofcultures is divisible into two stages. Firstly, there is a periodof vegetative growth, the duration of which depends on the inoculumload, after which the culture can be described as mature. Calciumis not required for the development of this maturity. Secondly,the cultures when mature develop phialides and spores if calciumis added to the medium. The development of phialides and thefirst spores takes 6 hours from the time of adding calcium andthis period is not influenced by the inoculum load of the culture.The medium from a mature culture promoted more rapid sporulationof vegetative mycelia placed in it than did a fresh medium,indicating the presence of a sporulation factor(s) in maturemedium. Similar activity was also demonstrated in media whichhad supported growth of any one of five other Penicillium speciesor Aspergillus niger. The nature of the sporulation factor isso far unknown.     

Cyclic Guanosine 3′,5′-Monophosphate in the Dimorphic Fungus Mucor racemosus

The dimorphic fungus Mucor racemosus was found to contain the cyclic nucleotide guanosine 3′,5′-monophosphate (cGMP). Approximately equivalent amounts of the compound were found in ungerminated spores, yeastlike cells, and mycelia. Germinating spores contained severalfold higher amounts of cGMP than the other cell forms. cGMP levels did not change significantly during the morphogenetic conversion of yeast to mycelia. Added exogenous cGMP or the dibutyryl derivative did not influence cell morphology in any way and did not alter the effect that cyclic adenosine 3′,5′-monophosphate has upon cell morphology.     

Filament ring formation in the dimorphic yeast Candida albicans

Stationary phase cultures of Candida albicans inoculated into fresh medium at 37 degrees C synchronously from buds at pH 4.5 and mycelia at pH 6.5. During bud formation, a filament ring forms just under the plasma membrane at the mother cell-bud junction at roughly the time of evagination. A filament ring also forms in mycelium-forming cells, but it appears later than in a budding cell and it is positioned along the elongating mycelium, on the average 2 microns from the mother cell-mycelium junction. Sections of filament rings in early and late budding cells and in mycelia appear similar. Each contains approximately 11 to 12 filaments equidistant from one another and closely associated with the plasma membrane. In both budding and mycelium-forming cells, the filament ring disappears when the primary septum grows inward. The close temporal and spatial association of the filament ring and the subsequent chitin-containing septum suggests a role for the filament ring in septum formation. In addition, a close temporal correlation is demonstrated between filament ring formation and the time at which cells become committed to bud formation at pH 4.5 and mycelium formation at pH 6.5. The temporal and spatial differences in filament ring formation between the two growth forms also suggest a simple model for the positioning of the filament ring.     

A comparison of volume growth during bud and mycelium formation in Candida albicans: a single cell analysis

When stationary phase cells of the dimorphic yeast Candida albicans are diluted into fresh medium at pH 4.5 (low pH), they synchronously form ellipsoidal buds, but when diluted into the same medium at pH 6.7 (high pH), they synchronously form elongate mycelia. Using a perfusion chamber to monitor single cells, we have compared the rates of volume growth between budding and mycelium-forming cells. Results are presented which demonstrate that: (1) after release from stationary phase into medium of low or high pH, each original sphere grows in volume to the time of initial evagination, but does not grow subsequently; (2) successive budding on the original mother cell occurs without interruption resulting in continuous volume growth; however, an interruption in volume growth of the initial bud (B1) occurs before it in turn evaginates; and (3) the rate of volume growth of the first bud at low pH is identical to the rate of volume growth of the mycelium at high pH even though the surface to volume ratios are quite different. The last result is unexpected and is therefore considered in relation to cell wall deposition.     

Cytodiagnosis of Candida organisms in cervical smears

Cervical smears and cervical scrapings cultured on Sabouraud agar from 31 women suspected of having Candida genital infections were examined in a study of the cytomorphology of this fungal infection in cervical smears. Of the 31 samples, 20 (64.5%) grew C. albicans in culture. One sample (3.2%) grew C. paratropicalis, 2 (6.4%) grew mixed C. albicans and Torulopsis glabrata and 2 (6.4%) grew T. glabrata alone. Of the 25 fungus-positive samples, 20 (80%) had fungus-positive cervical smears and 5 (20%) had fungus-negative smears. There was no instance in which the cervical smear was positive but the culture was negative. Among the cases positive for C. albicans, organisms occurred in two forms: pseudohyphae without blastospores (29.4%) and pseudohyphae with blastospores (70.6%). T. glabrata was present in the smears as budding and nonbudding yeasts. Although the sensitivity of the cervical smear in detecting fungus in culture-positive patients was only 80%, the cervical smear can still be a useful means of rapid identification of C. albicans when blastospores and pseudomycelium are present. The presence of budding or nonbudding yeast without pseudohyphae should strongly suggest a T. glabrata infection.     

Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.     

Septin function in Candida albicans morphogenesis

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Delta and cdc11Delta mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Delta mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.     

Differences of asymmetrical division between the pseudomycelial and yeast forms of Candida albicans and their effect on multiplication

Unlike asymmetrical division of budding yeast, the daughter cell in the pseudomycelial form of Candida albicans at division was nearly equal in size to the mother cell, it had a larger amount of protein, RNA and active protoplasm (cell size minus vacuolar volume) than the mother cell, and it budded earlier than the mother cell. Results presented here suggest that the cell size control over bud initiation found in budding yeasts is also applicable to the pseudomycelial cells of C. albicans if vacuolar volume is omitted from cell size.     

Aspergillus SteA (sterile12-like) is a homeodomain-C2/H2-Zn+2 finger transcription factor required for sexual reproduction

Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell-specific or morphogenesis-specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63-75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12alphap. SteAp and Ste12alphap lack the pheromone induction domain found in budding yeast Ste12p, but have C-terminal C2/H2-Zn+2 finger domains not present in the other Ste12 proteins. A DeltasteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH-like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.     

Pbhyd1 and Pbhyd2: two mycelium-specific hydrophobin genes from the dimorphic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 degrees C and as yeasts (Y) at 37 degrees C, or after the invasion of host tissues. Although the dimorphic transition in P. brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood. Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P. brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2). Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence. These two hydrophobin genes are present as single copy in P. brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition.