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.     

Analysis of wall glucans from yeast, hyphal and germ-tube forming cells of Candida albicans

Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained beta(1----3)- and beta(1----6)-chains but no mixed intra-chain beta(1----3)/(1----6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67.0% to 76.6%) of beta(1----6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29.6% to 38.9% beta(1----3) and 43.3% to 53.2% beta(1----6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67.0% beta(1----3) and 14% beta(1----6) linkages. Branch points accounted for 6.7%, 12.3% and 17.4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.     

Exo-(1----3)-beta-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans

Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.     

Changes in surface topography of Candida albicans during morphogenesis

Temporal studies of germ-tube forming yeast cells of Candida albicans by scanning and transmission electron microscopy indicate that extensive vacuolation and possibly also cell wall changes may cause walls of the parent yeast cell to collapse during specimen preparation. This collapse does not occur in cells which have been grown in conditions that suppress germ tube formation and have undergone the same preparative treatment.     

The dynamics of carbohydrate metabolism in Candida albicans

The total cellular concentrations of the intermediary metabolites and the carbohydrate end products were determined for starved Candida albicans yeast cells and cells forming germ tubes during a 60-min incubation in imidazole-HCl buffer in the absence and presence of 2.5 mM glucose, 2.5 mM glutamine, and 0.2% serum at 37°C. These cells were also incubated in the presence of tracer [U-¹⁴C]glucose and the specific radioactivities of the metabolites and end products determined. The labeling data indicated (1) a minimum of two metabolically independent pools of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, and uridine diphosphoglucose; (2) compartmentation of the pathways of catabolism and anabolism; (3) channeling of the exogenous tracer glucose into the anabolic pathway compartments of the starved cells; and (4) a significant rate of turnover of cell wall carbohydrates in cells incubated under nongrowth conditions and rapid turnover of these pools in germ tube forming cells. The labeling data will be used to construct kinetic models of carbohydrate metabolism in C. albicans.     

Role of nutritional status of the cell in pH regulated dimorphism of Candida albicans

Stationary phase cells of Candida albicans are under the control of glucose repression, as indicated by the inhibition of germ tube formation by glucose. This 'glucose effect' was absent in starved cells which were derived from similar stationary phase cells. Moreover, starved cells required glucose for germ tube formation, suggesting that it was depletion of energy reserves which was the main factor overriding the 'glucose repression machinery' during starvation. High concentration of phosphate in Lee's medium was the reason for the reduced ability of the starved cells to form germ tubes at pH 4.5 (20% of cells compared to 88% at pH 6.8). However, when phosphate was replaced or its concentration reduced, germ tube formation occurred as frequently at pH 4.5 as at pH 6.8. This 'phosphate effect' was not observed in stationary phase cells, as they were already repressed by glucose.     

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.     

The secretion of N-acetylglucosaminidase during germ-tube formation in Candida albicans

N-Acetylglucosaminidase was induced by either N-acetylglucosamine or N-acetylmannosamine in several strains of Candida albicans. Enzyme activity was not induced in a N-acetylglucosamine non-utilizing mutant which is unable to express the first three steps in the N-acetylglucosamine catabolic pathway. The enzyme, purified 500-fold, had a specific activity of 36.8 units (mg protein)-1 and catalysed the hydrolysis of p-nitrophenyl-beta-n-acetylglucosamine, N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose. No activity was observed toward colloidal chitin, hyaluronic acid or mucin. The cellular distribution of N-acetylglucosaminidase was determined by measuring in situ enzyme activity before and after acid treatment of intact cells. N-Acetylglucosaminidase (80-88% of the total cellular activity) was rapidly secreted to the periplasm when the enzyme was induced either during yeast growth at 28 degrees C or germ-tube formation at 37 degrees C. Export of the enzyme from the periplasm into the medium was fourfold greater during germ-tube formation, and after 6 h incubation the amount of enzyme released into the medium represented 70% of cell-associated enzyme activity.     

The in situ assay of Candida albicans enzymes during yeast growth and germ-tube formation

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (beta-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming 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.     

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.     

On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, X-ray diffraction and chemical study.

The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(1 leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was found de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.     

Analysis of alkali-soluble glucan produced by<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> wild-type and mutants

The alkali-soluble glucan of the yeast cell wall contains beta-(1,3)- and (1,6)-D-linkages and systemically enhances the immune system. To isolate Saccharomyces cerevisiae mutants producing glucan with a high degree of beta-(1,6)-D-glycosidic bonds, a wild-type strain was mutagenized with ultraviolet light. The mutants were then selected by treatment with 1.0 mg laminarinase, endo-beta-(1,3)-D-glucanase/ml. The alkali-soluble glucan was extracted by modified alkalysis followed by the Cetavlon method and concanavalin-A chromatography. The prepared alkali-soluble glucans from the wild-type and the mutants were compared with respect to yield and polymer structure using gas chromatography, 13C-NMR spectrometry, high performance liquid, and multi-angle laser light scattering and refractive index detectors. The results indicated that the S. cerevisiae mutants had ten-fold more alkali-soluble glucan than the wild-type. Structural analysis revealed that the alkali-soluble glucan from the mutants also had a higher degree of beta-(1,6)-D-linkage than that from the wild-type.     

Polysaccharide composition of the cell wall of baker's yeast with special reference to cell walls obtained from large- and small-sized cells

In order to obtain further information on the mode of cell wallformation during the growth process, the compositions of cellwall polysaccharides were compared in detail using cell wallsamples prepared from large and small cells obtained by fractionationof baker's yeast cells. Gas liquid chromatography of the methanolyzed specimen gavestable, reproducible results, when the sample contained bothglucose and mannose. Much mannan was liberated from the cellwall during its preparation and this must be taken into consideration. Total glucose and mannose accounted for about 40% each of dryweight of cell walls obtained from large and small cells. Glucanswere tentatively divided into alkali-soluble and alkali, acid-insolubleones. Alkaline extraction caused considerable degradation ofpolysaccharides. Nevertheless, a distinct difference existedbetween the glucan contents of the two cell walls. The cellwall sample of large cells contained a higher amount of insolubleglucan, whereas that of small cells contained a higher amountof alkali-soluble glucan. The mode of formation of cell wall polysaccharides during growthwas discussed on the basis of a small-to-large cell process. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan. (Received April 13, 1976; )     

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.     

Changes in the intracellular concentration of acetyl-CoA and malonyl-CoA in relation to the carbon and energy metabolism of Escherichia coli K12

Intracellular concentrations of acetyl-CoA and malonyl-CoA in Escherichia coli K12 were determined by a malonyl-CoA: acetyl-CoA cycling technique. Under aerobic growth conditions with glucose the acetyl-CoA and malonyl-CoA concentrations varied over a range of 0.05-1.5 nmol (mg dry wt)-1 (20-600 microM) and 0.01-0.23 nmol (mg dry wt)-1 (4-90 microM), respectively. The intracellular concentration of acetyl-CoA was highest in exponentially growing cells and it fell rapidly to less than 5% of the maximum level when the organism entered stationary phase after exhaustion of glucose. A linear relationship was observed between the intracellular concentration of total acyl-CoA and the logarithm of the concentration of glucose in the medium. Consequently, the acetyl-CoA/malonyl-CoA ratios also varied drastically, in a range of 0.6-41.7, under different conditions. Of several carbon sources tested, glucose was the most effective for promoting the synthesis of cellular acetyl-CoA. For cells grown on glycerol or acetate the maximum concentrations of total acyl-CoA were significantly lower. In cells incubated with citrate (not used as a carbon source by E. coli), the level was consistent with that in cells starved for exogenous carbon sources.     

Decreased synthesis of alkali-soluble glucan in a cell-wall mutant of Saccharomyces cerevisiae

In vivo studies and quantitative measurements of glucans provide evidence for a decreased rate of synthesis and a lower amount of alkali-soluble glucan in cells of the osmotically fragile VY1160 mutant of the yeast Saccharomyces cerevisiae. Combined genetic and biochemical analysis shows that the srb1 mutation is responsible for the reduction of alkali-soluble glucan. Data on beta(1----3) glucan synthase activity did not indicate the participation of the enzyme in the in vivo synthesis of alkali-soluble glucan and suggest the existence of other glucan synthases in Saccharomyces cerevisiae.     

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.     

Regulation of chitin synthesis during germ-tube formation in Candida albicans

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (K _i =1.2M) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.Non-Standard Abbreviations GlcNac N-acetyl glucosamine - UDP-GlcNac UDP-N-acetyl glucosamine - PMSF phenylmethylsulphonylfluoride     

Glucose oxidation by adherent and mechanically detached cultured cells.

This study examined the effect of mechanical detachment from the growth surface on energy metabolism of cultured cells. Oxidation of [1(-14)C]glucose measured by production of 14CO2 by adherent neuroblastoma (123 +/- 5 nmol/mg protein per minute), glioma (128 +/- 10 nmol/mg protein per minute), and fibroblast (137 +/- 5 nmol/mg protein per minute) cultures was similar. Removing cells from the culture flask by scraping reduced glucose oxidation by 62, 30, and 82% in neuroblastoma, glioma, and fibroblast cultures, respectively. Transferring cells from a culture flask to a test tube, to control for diffusional surface area, did not further reduce glucose oxidation. Detaching cells from the growth surface destroyed the extensive process formation and disrupted the normal spatial organization on the culture plate. These results indicate that it is essential to maintain these aspects of cellular architecture when evaluating metabolic properties of cultured cells.