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.     

Identification of envelope proteins of Candida albicans by vectorial iodination

Intact Candida albicans yeast cells were radiolabelled with 125I, and cell wall, mixed membrane and soluble fractions prepared. The majority (67%) of the 125I was detected in the protein of the cell wall fraction at a specific activity 70-fold higher than that in the membrane or soluble fractions. SDS treatment of the cell wall fraction released 52% of the total protein but only 3% of the wall-bound 125I, and the extract was shown to be severely contaminated with cytosolic and membrane proteins. Zymolyase digestion of SDS treated walls liberated material which contained 93% of the 125I and on electrophoresis migrated as a single diffuse zone (average Mr 260 kD) typical of heterogenous mannoproteins. Protein (1.5%), GlcN (0.08%) and hexose (98.4%) content was measured and amino acid analysis showed enrichment in Ser (15.9%) and Thr (20.2%). These results indicate that the major iodinated protein(s) in the cell envelope is a 260 kD mannoprotein fraction containing both O-linked and Asn-linked oligosaccharides.     

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).     

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.     

Age-Dependent Modifications and Further Localization of the CX 26-like Protein from Vicia faba L.

A strong age dependency together with alterations in the cellular distribution of CX 26 immunorelated protein(s) was found for differently developed leaves of Vicia faba L. With increasing age, an immunoreactive 40 kD band was observed in the soluble and microsomal fraction. In the cell wall protein preparation of young and fully differentiated leaves the 40 kD band was the minor constituent. A 33 kD polypeptide was dominantly localized in the microsomal fractions of all developmental stages and in SDS-extracts of total cell proteins of young leaves. A 21 kD protein together with a 16 kD polypeptide was associated with the cell wall fraction. The 21 kD protein, assumed to represent a plasmodesmatal constituent, was reduced with age. In SDS extracts, prepared from the different developmental stages of the leaves and of mesophyll protoplasts, the age-dependent appearance of the several immunostained bands was most obvious. A correlation of the 16, 33, and 40 kD bands to a turnover of the 21 kD protein is suggested. The reduced amount of the 21 kD protein with increasing age may be contemplated as an indication for a relative decrease of symplastic connections between cells of maturing leaves. This is in agreement with the results obtained by immunofluorescence studies using guard cell protoplasts. Here, observations pointed also to a reduction and final loss of CX 26-related protein at the protoplast surfaces.     

Examination of the Protein Composition of the Cell Envelope of Escherichia coli by Polyacrylamide Gel Electrophoresis

An envelope preparation containing the cell wall and cytoplasmic membrane of Escherichia coli was obtained by breaking the cells with a French pressure cell and sedimentating the envelope fraction by ultracentrifugation. This fraction was prepared for polyacrylamide gel electrophoresis by dissolving the protein in an acidified N,N'-dimethylformamide, removing lipids by gel filtration in the same organic solvent and removing the solvent by dialysis against aqueous urea solutions. More than 80% of the total protein of the envelope fraction was recovered in soluble form. Electrophoresis on sodium dodecyl sulfate-containing gels yielded from 20 to 30 well-resolved bands of protein. One major protein band was observed on the gels. This protein had a molecular weight of 44,000 and accounted for as much as 40% of the total protein of the envelope fraction. A double-labeling technique was used to examine the protein composition of the envelope fraction from cells grown under different sets of conditions which result in large changes in the levels of membrane-bound oxidative enzymes. These changes in growth conditions resulted in only minor alterations in the protein profiles observed on the gels, suggesting that this organism is able to adapt to changes in growth environment with only minor modifications of the major proteins of the cell envelope.     

Cellular localization of steroid hormone-regulated proteins during sexual development in Achlya

In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analysed [35S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. Since in a total cell extract, hormone-induced changes in specific proteins might not be apparent against a background of more abundant proteins, cells were fractionated prior to protein isolation. It was also necessary to establish a concentration of hormone carrier, in this case methanol, which by itself did not alter the pattern of protein synthesis. Using these approaches the addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell wall/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3 kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3 kD protein in the cell wall/cell membrane fraction. A marked increase in the labeling of 85, 63 and 47 kD proteins in the nuclear fraction from hormone-treated cells was also noted. The molecular weight (MW) and the behavior on 2D gels of the 85 kD hormone-induced protein appeared very similar to that of the 85 kD heat-shock protein reported in Achlya. Quantitive changes in the [35S]methionine labeling of several other proteins were noted in all three cell fractions.     

The role of glucose in the pH regulation of germ-tube formation in Candida albicans

It has been reported that Candida albicans can form germ-tubes only in the narrow pH range of 6-8, and that by changing only the pH one can regulate germ-tube formation. We found that the pH minimum for germ-tube formation could be dramatically lowered by eliminating the glucose present in many induction solutions. Lee's medium lacking glucose, ethanol, N-acetyl-D-glucosamine, and proline induced germ-tubes at pH values as low as 3 under most conditions. The presence of as little as 1 mM-glucose in these induction solutions was sufficient to cause the cells to grow either as yeasts with multiple buds or as pseudohyphae when the pH was 3.7. However, when C. albicans was grown in any of the above induction solutions (with the exception of ethanol), containing 200 mM-glucose buffered at pH 5.8, not only were germ-tubes formed, but their rate of formation and length were also increased. Preincubation of the cells in a solution buffered at pH 3.7 and containing 200 mM-glucose, before exposure to induction solutions lacking glucose at pH 3.7 or at pH 5.8, did not inhibit germ-tube formation. Likewise, addition of glucose after 45 min exposure to an induction solution was without effect. Theophylline and dibutyryl cAMP did not counteract the action of glucose. Other sugars which suppressed germ-tube formation at low pH were fructose, galactose, mannose, xylose, gluconic acid and the nonmetabolizable sugar 3-O-methylglucose. These results indicate that pH does not directly regulate dimorphism in C. albicans, and that glucose or its metabolites may play an important role.     

Effect of suramin on the human pathogen Candida albicans: implications on the fungal development and virulence

Candida albicans is an opportunistic pathogen that is of growing medical importance because it causes superficial, mucosal and systemic infections in susceptible individuals. Here, the effect of suramin, a polysulfonated naphthylurea derivative, on C. albicans development and virulence was evaluated. Firstly, it was demonstrated that suramin (500 microM) arrested its growth, showing a fungicidal action dependent on cell number. Suramin treatment caused profound changes in the yeast ultrastructure as shown by transmission electron microscopy. The more important changes were the enlargement of the fungi cytoplasmic vacuoles, the appearance of yeasts with an empty cytoplasm resembling ghost cells and a reduction in cell wall thickness. Suramin also blocked the transformation of yeast cells to the germ-tube and the interaction between C. albicans and epithelial cells. In order to ascertain that the action of suramin on C. albicans growth is a general feature instead of being strain-specific, the effects of suramin on 14 oral clinical strains isolated from healthy children and HIV-positive infants were analyzed. Interestingly, the strains of C. albicans isolated from HIV-positive patients were more resistant to suramin than strains isolated from healthy patients. Altogether, the results produced here show that suramin interfered with essential fungal processes, such as growth, differentiation and interaction with host cells.     

Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[³⁵S]methionine ([³⁵S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [³⁵S]met-labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water-soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane-rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water-soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1D and 2D-PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.     

Effects of culture conditions on the in vitro infection of fibroblasts by Candida albicans.

The effects of yeast culture age, carbon source, growth temperature, and germ-tube inducers on adherence to primary fibroblast cultures was studied in conjunction with the determination of adherence-mediated mammalian cell damage by measuring chromium-51 release from fibroblast monolayers. The results indicated that yeast culture age affected adherence only when the yeasts were grown at 37 degrees C, not after growth at 28 degrees C. At 37 degrees C, quantitatively fewer exponential-phase, glucose- or galactose-grown yeasts adhered to fibroblasts than did yeasts that were in lag or stationary phases. The reduced adherence correlated with less chromium-51 release and reduced germ-tube formation. The addition of germ-tube inducers, such as N-acetyl-D-glucosamine or serum, to exponential-phase yeasts caused an increase in germ-tube formation with a concomitant increase in yeast adherence and release of chromium-51 from the monolayers. Exponential-phase galactose-grown yeasts were more responsive to serum-induced germ-tube formation, germ-tube elongation, and fibroblast adherence than were exponential-phase glucose-grown yeasts. In addition, exponential-phase galactose-grown yeasts caused more chromium-51 release from monolayers in the presence of serum than did glucose-grown yeasts. Overall, conditions that enhanced germ-tube formation and elongation resulted in greatest adherence-mediated damage to the monolayers.     

The cellular location of proteases in Candida albicans

Vacuoles prepared from yeast cells of Candida albicans were enriched in proteinase ycaB (EC 3.4.21.48) but not in aminopeptidase or beta-glucosidase. Proteinase ycaB, assayed in situ, increased 1.5-fold during starvation whereas aminopeptidase activity decreased by 25%. Proteinase ycaB increased a further 1.5-fold during germ-tube formation.     

Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport

To extend our understanding of the mechanism by which the nuclear pore complex (NPC) mediates macromolecular transport across the nuclear envelope we have focused on defining the composition and molecular organization of the yeast NPC. Peptide sequence analysis of a polypeptide with a M(r) of approximately 100,000 present in a highly enriched yeast NPC fraction identified a novel yeast nucleoporin we term Nup120p. Nup120p corresponds to the open reading frame (ORF) YKL057c identified by the yeast genome sequencing project. The ORF predicts a protein with a calculated molecular mass of 120.5 kD containing two leucine zipper motifs, a short coiled-coil region and limited primary sequence similarity to Nup133p. Nup120p was localized to the NPC using a protein A-tagged chimera in situ by indirect immunofluorescence microscopy. Deletion of the NUP120 gene caused clustering of NPCs at one side of the nuclear envelope, moderate nucleolar fragmentation and slower cell growth. Transfer of nup120 delta cells to 37 degrees C resulted in the nuclear accumulation of poly(A)+ mRNA, extensive fragmentation of the nucleolus, spindle defects, and cell death.     

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.     

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.     

Antimorphogenic effects of 2-deoxy-D-glucose in Candida albicans

Abstract 2-Deoxy- d -glucose (dGlc) is able to inhibit both N -acetyl-hexosamine-induced chlamydosporogenesis and N -acetyl-glucosamine- or proline-induced germ-tube formation in Candida albicans . This inhibition is exerted also at dGlc concentrations which do not affect growth in the yeast form and do not reduce either the uptake or the incorporation of N-acetyl- d -glucosamine (GlcNAc) into yeast or hyphal cell material. Inhibition of germtube formation by dGlc does not occur in serum and is fully reversed by glucose. It is suggested that dGlc acts as a potent antimorphogenic effector in C. albicans .     

Binding of plasma proteins to Candida species in vitro

The ability of purified human albumin, fibrinogen and transferrin to bind to Candida species was measured by immunofluorescence. The proteins all bound with high avidity to germ-tubes formed by Candida albicans, but did not bind to blastospores of C. albicans or other pathogenic Candida species, not even to parent blastospores bearing germ-tubes. The extent of binding of the proteins to C. albicans germ-tubes varied between growth media and from germ-tube to germ-tube. Strains of C. albicans that did not form germ-tubes were incapable of binding any of the proteins. There was evidence that purified fibrinogen bound to germ-tubes with higher avidity than albumin and transferrin. When germ-tubes were treated with whole human plasma or serum, indirect immunofluorescence revealed that proteins were bound all over the surface of C. albicans blastospore-germ-tube units, indicating behaviour different from that seen with the purified proteins tested alone or in mixtures. C. albicans cells grown in the presence of azole antifungal agents bound purified plasma proteins in the same way as cells untreated with the drugs. The results of this study suggest that binding of host proteins to the surface of C. albicans may not be a property related directly to virulence of the fungus in vivo.     

Protein synthesis and amino acid pool during yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans

Protein synthesis at different stages of yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans was evaluated by following incorporation of radioactive amino acids into the acid-insoluble cellular material. In passing from the early germ-tube formation (60-90 min) to the mature hyphal cell (240-270 min) there was a marked decrease in the capacity for protein synthesis. Apparently, this decrease was not due to a decreased amino acid uptake into the soluble cellular pool or to exhaustion of carbon/energy source in the inducing medium with consequent arrest of growth. Protein synthesis, however, did not decay when amino acids at high concentration were added to the medium fostering the yeast-mycelial transition and this effect was potentiated by glucose. Analysis of the intracellular amino acid pool showed that both germ-tubes and hyphal cells were relatively depleted of several amino acids as compared to the yeast-form cells, whereas in the hyphae there was a higher concentration of glutamic acid/glutamine, the latter being the predominant component. These modulations in amino acid pool composition were not seen when yeasts were converted to hyphae in an amino acid-rich induction medium. This study emphasizes that yeast-form cells of C. albicans may efficiently convert to the mycelial form even under a progressively lowered rate of protein synthesis, and suggests that initiation of hyphal morphogenesis in the presence of N-acetyl-D-glucosamine is somehow separated from cellular growth.     

Effects of alkali metal ions on some virulence traits of<Emphasis Type="Italic">Candida albicans</Emphasis>

The effects of the alkali metal ions (Li+, Na+ and K+) on the growth and on certain virulence factors (adhesion, cell-surface hydrophobicity and germinating ability) of Candida albicans were determined. High concentrations of these ions displayed an inhibitory effect on the growth of the Candida cells; preincubation in their presence showed a negative effect on all virulence factors studied. The changes induced during the preincubation remained there even when high concentration of the ions was removed from the cell suspension. In contrast, a considerable growth was found at high Na+ and K+ concentrations. Although alkali metal ions significantly decreased certain virulence traits of the fungus they did not totally inhibit adhesion and germ-tube formation. This suggests that C. albicans may represent a health hazard even at a high salt concentration.