Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response

A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.     

Gratuitous induction by N-acetylmannosamine of germ tube formation and enzymes for N-acetylglucosamine utilization in Candida albicans.

N-Acetylmannosamine did not support the growth of Candida albicans, and this sugar was not accumulated by cells. Incubation of starved yeast cells at 37 degrees C with N-acetylmannosamine plus glucose resulted in germ tube formation. Furthermore, N-acetylmannosamine alone induced the uptake system for N-acetylglucosamine and the enzymes of the N-acetylglucosamine catabolic pathway to the same extent as the natural substrate. Induction of the uptake system and the enzymes was observed at 28 degrees C without germ tube formation and at 37 degrees C with germ tube formation. N-Acetylmannosamine is thus a gratuitous inducer for enzymes of the N-acetylglucosamine pathway and germ tube formation in C. albicans.     

Effects of divalent cations and functionally related substances on the yeast to mycelium transition in Sporothrix schenckii

In a minimal basal medium with glucose at pH 4.0 and 25 degrees C, a lowering of the magnesium and zinc concentrations or increase in the calcium concentration of the medium favoured the yeast-mycelium transition in Sporothrix schenckii. Addition of zinc (1 and 10 mM) inhibited mycelial development and induced reversion to a yeast-like morphology. EDTA and EGTA also delayed germ tube formation, possibly by their calcium-chelating effects or by altering intracellular concentrations of this or other ions. Ionophore X537A also caused a delay in germ tube formation, possibly by interfering with magnesium metabolism in these cells.     

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.     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.     

Proline-induced germ-tube formation in Candida albicans: role of proline uptake and nitrogen metabolism

Proline-induced germ-tube formation and cell-cell aggregation in four strains of Candida albicans were completely inhibited when the pH of the medium was 5.0 or lower, whereas morphogenesis induced by N-acetylglucosamine (GlcNAc) was unaffected even at pH 4.5. The pH sensitivity of proline-induced germ-tube formation was not caused by a modulation of proline uptake, which was unchanged over the pH range 4.5-6.5. The proline uptake system was specific, constitutive and subject to ammonium repression, and only one permease was detected, with a Km of 179 microM. Cultures deprived of nitrogen in the presence of glucose were derepressed for proline uptake but the yeast-mycelial transition could not be mediated by either proline or GlcNAc. The inhibition of morphogenesis was reversed when the nitrogen starvation was relieved by the addition of ammonium ions, proline, or certain amino acids. These results indicate that the nitrogen status of the cells is critical for the morphogenesis of C. albicans.     

Induction of N-acetyl-D-glucosamine catabolic enzymes and germinative response in Candida albicans

The regulation of N-acetylglucosamine catabolic enzymes was studied in both yeast and germ tube forms of the dimorphic fungus Candida albicans. The induction pattern of these enzymes was the same for yeast cells incubated at 28 degrees C and in cells incubated at 37 degrees C which formed germ tubes. However, the level of activity of these enzymes in germ tube stage is lower as compared to yeast phase cells. A strain of C. albicans that did not form germ tubes was endowed with a pronounced ability for induction of N-acetylglucosamine catabolic enzymes. This result suggests that germ tube formation and N-acetylglucosamine metabolism are mutually exclusive events.     

The Candida albicans plasma membrane and H(+)-ATPase during yeast growth and germ tube formation.

PMA1 expression, plasma membrane H(+)-ATPase enzyme kinetics, and the distribution of the ATPase have been studied in carbon-starved Candida albicans induced with glucose for yeast growth at pH 4.5 and for germ tube formation at pH 6.7. PMA1 expression parallels expression of the constitutive ADE2 gene, increasing up to sixfold during yeast growth and twofold during germ tube formation. Starved cells contain about half the concentration of plasma membrane ATPase of growing cells. The amount of plasma membrane ATPase is normalized prior to either budding or germ tube emergence by the insertion of additional ATPase molecules, while ATPase antigen appears uniformly distributed over the entire plasma membrane surface during both growth phases. Glucose addition rapidly activates the ATPase twofold regardless of the pH of induction. The turnover of substrate molecules per second by the enzyme in membranes from budding cells quickly declines, but the enzyme from germ tube-forming cells maintains its turnover of substrate molecules per second and a higher affinity for Mg-ATP. The plasma membrane ATPase of C. albicans is therefore regulated at several levels; by glucose metabolism/starvation-related factors acting on gene expression, by signals generated through glucose metabolism/starvation which are thought to covalently modify the carboxyl-terminal domain of the enzyme, and possibly by additional signals which may be specific to germ tube formation. The extended period of intracellular alkalinization associated with germ tube formation may result from regulation of proton-pumping ATPase activity coupled with higher ratios of cell surface to effective cytosolic volume.     

Effect of tunicamycin on germ tube and yeast bud formation in Candida albicans

Tunicamycin is an antimicrobial agent which inhibits the first reaction of the dolichol pathway leading to N-glycosylation of proteins. The effect of tunicamycin on the growth of the dimorphic fungus Candida albicans differed depending on the growth phase of the organism. Addition of tunicamycin to stationary phase yeast cells inhibited the resumption of growth of those cells in either morphology, as cultures failed to initiate either yeast bud or germ tube formation. When tunicamycin was added to growing cells, growth was inhibited but not immediately. When it was added to germ tube cultures, nuclear division and septum formation continued for some time before ceasing. Addition of the drug to exponential phase yeast cultures resulted in an approximately 45% increase in cell number before cell division ceased and yeast accumulated in both budded and unbudded stages of the cell cycle. Accumulation of trichloroacetic acid precipitable radiolabelled protein and nucleic acid continued unchanged for some time following addition of tunicamycin; however, after a while a reduced rate of accumulation was noted.     

Ethanol-induced germ tube formation in Candida albicans

Ethanol is the first reported compound which can induce germ tube formation in Candida albicans without the addition of any nitrogen-containing nutrients. Conditions controlling induction of germ tubes in C. albicans by ethanol were investigated. Ethanol (17.1 mM) in buffered salts solution containing sodium bicarbonate induced 70 to 80% of yeast phase cells of C. albicans to form germ tubes. Germ tubes could be induced by ethanol (0.08 to 340 mM) at temperatures ranging from 29 to 41 degrees C (optimum 37 degrees C) and at pH values ranging from 3.0 to 8.0 (optimum 5.75). The germ tubes averaged 11 micron in length after 6 h at 37 degrees C. The percentage of cells forming germ tubes decreased as the concentration of cells in the induction solution was increased above 4 X 10(5) cells ml-1. Germ tubes first appeared 45 to 60 min after continuous exposure to ethanol at 37 degrees C and all cells which formed germ tubes did so by 2 h. Germ tube length decreased as the pH was increased but was independent of the concentration of ethanol. Oxygen was required for germ tube formation. In addition to ethanol, 1-propanol, 2-propanol, 1-butanol and acetic acid could induce germ tube formation, whereas methanol could not. These results indicate that the cells must mobilize their endogenous nitrogen and probably carbohydrate reserves in order to initiate formation of germ tubes. The evidence is inconclusive as to whether ethanol itself must be metabolized for germ tube induction to occur, although it is not thought to act by a nonspecific interaction with the cell membrane.     

The requirements for bicarbonate and metabolism of the inducer during germ tube formation by Candida albicans

Factors affecting germ tube formation in Candida albicans at suboptimal temperatures were investigated. Candida albicans formed germ tubes between 22 and 30 degrees C in solution when incubated without shaking, in the presence of bicarbonate (2 mg mL-1). Other conditions depended on the inducer used. Proline could induce germ tube formation optimally only when its concentration was between 200 and 400 mM. A concentration of 0.05 mM N-acetylglucosamine was sufficient to induce germ tube formation. N-Acetylglucosamine could induce germ tube formation at 30 but not at 25 degrees C. N-Acetylglucosamine induced germ tube formation was most reproducible when the cells were first starved by incubation in water for 16-24 h at 20 degrees C. Germ tubes induced by proline could be formed at pH values between 3.8 and 9.0 at 30 degrees C, but only between 7.0 and 7.5 at 25 degrees C. The addition of 0.05 to 5 mM glucose to a 5 mM proline induction solution allowed germ tube formation at 30 but not at 25 degrees C. Glucose (400 mM) did not suppress germ tube formation at 30 degrees C but only 5 mM was sufficient to cause a 65% suppression at 25 degrees C. The results show the importance of CO2 and (or) bicarbonate to the induction of germ tube formation and are consistent with the metabolism of the inducer.     

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.     

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.     

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.     

Alternate interactions of the d-galactose-specific yeast lectin Kb-CWL I with sensitive yeast strains

Abstract In vitro growth assays with a purified d -galactose-specific lectin Kb-CWL I extracted from Kluyveromyces bulgaricus showed marked antifungal effects on 9 of 12 test strains belonging to the genera Kluyveromyces, Saccharomyces, Pichia, Candida, Rhodotorula and Schizosaccharomyces . The inhibition of growth was proportional to the lectin concentration (0.04 and 0.08 mg ml⁻¹) in the culture medium. Under the test conditions, K. bulgaricus, K. lactis and S. bayanus were consistently agglutinated without growth inhibition. d -Galactose, the inhibitory sugar of the cell aggregation activity, did not abolish the antifungal effects of the lectin Kb-CWL I on other yeast strains tested. In C. albicans and C. tropicalis , the lectin influenced the dimorphism of these strains and stimulated germ tube formation.     

Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae

Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5-fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10(-8) M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10(-7) M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10(-5) M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth.     

The phosphonic acid analog of phosphatidylglycerol phosphate: influence on Escherichia coli growth and physiology.

At 20 microM, rac-3,4-dihydroxybutyl-1-phosphonate (DBP) has only a slight bacteriostatic effect on Escherichia coli. However, cells lose viability when the medium also contains either 20 mM magnesium or calcium ions. Magnesium ions stimulate the incorporation of DBP into (1,2-diacyl)-sn-glycerol-D-4'-phosphoryloxy-3'-hydroxybutyl-1'-pho sphonate, the phosphonate analog of phosphatidylglycerol phosphate. Much higher DBP concentrations are needed to block the growth of a pgsA3 mutant than to block the growth of an isogenic wild-type strain. The DBP-treated pgsA mutant also has a much higher survival rate when stored in the cold than does the DBP-treated wild-type strain. Furthermore, the pgsA3 mutant grows normally in the presence of DBP and magnesium ions. Treatment with DBP and magnesium ions does not appear to disrupt the cell's inner or outer membranes. However, it does block macromolecular and phosphoglyceride synthesis. A combination of 20 microM rac-DBP and 0.5 mM spermidine or 0.125 mM spermine is bacteriostatic. These studies indicate that the PGP analog contributes to DBP's bacteriostatic effect when the growth medium contains low concentrations of magnesium or calcium ions and is responsible for its bactericidal effect when the medium contains high concentrations of these ions.     

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).     

Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.     

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.