Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls

Wall mannoproteins of the two (yeast and mycelial) cellular forms of Candida albicans were solubilized by different agents. Boiling in 2% (w/v) SDS was the best method, as more than 70% of the total mannoprotein was extracted. Over 40 different bands (from 15 to 80 kDal) were detected on SDS-polyacrylamide gel electrophoresis of this material. The residual wall mannoproteins were released after enzymic (Zymolyase and endogenous wall beta-glucanases) degradation of wall glucan, suggesting that they are covalently linked to this structural polymer. Four bands (of 160 kDal, 205 kDal and higher molecular mass) were observed in the material released from yeast walls but only the two smaller components were detected in the material obtained from mycelial walls. Moreover, the mannoproteins of high molecular mass, which are covalently linked in walls of normal cells, were not incorporated into walls of regenerating protoplasts, but non-covalently linked mannoproteins were retained from the beginning of the process.     

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species.

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species.     

Structure of the Saccharomyces cerevisiae cell wall: Mannoproteins released by zymolyase and their contribution to wall architecture

Purified zymolyase containing β-glucanase activity preferentially released a 29 kDa mannoprotein from isolated yeast cell walls and a high-molecular-mass (greater than 120 kDa) material. Endo-β-N-acetylglucosaminidase H digestion indicated that the 29 kDa mannoprotein contains a unique core coligosaccharide N-glycosidically linked to a 26 kDa peptide moiety. Cells grown in the presence of tunicamycin incorporated the nonglycosylated 26 kDa peptide into the wall, but not the large mannoprotein molecules. Treatment of isolated walls with SDS solubilized more than 30 different mannoproteins, one of tehm being the 29 kDa species, but the large-size molecules were not affected. Regenerating protoplasts incorporated into the forming walls most of the SDS-solubilizable species seen in mature cell walls, but the zymolyase-solubilizable mannoproteins were absent. Wall mannoproteins have also been compared with those of the periplasmic space, most of the species being commonly present at both compartments. Turnover of individual species has been studied by pulse and chase experiments. While mannoproteins from the walls remain stable for long periods, periplasmic molecules exhibit a rapid turnover rate.     

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.     

Purification of Phosphomannanase and Its Action on the Yeast Cell Wall

An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.     

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.     

Altered extent of cross-linking of beta1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall beta1,3-glucan content.

The yeast cell wall contains beta1,3-glucanase-extractable and beta1,3-glucanase-resistant mannoproteins. The beta1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a beta1,6-glucan moiety, which in its turn is linked to beta1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The beta1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of beta1,6-glucosylated proteins, including Cwp1p, as the B1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the beta1,3-glucanase-resistant fraction through a beta1,6-glucan moiety. In wild-type cell walls, the beta1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less beta1,3-glucan but more chitin, beta1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased cross-linking of cell wall proteins via beta1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.     

Laminarinase from Penicillium funiculosum and its role in release of beta-glucosidase

An extracellular laminarinase (1----3)-beta-glucan glucohydrolase (EC 3.2.1.6) was purified from culture filtrates of Penicillium funiculosum. It was homogeneous on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. It had a Mr of 14,000 and isoelectric point of pH 4.2. The apparent Km value for lamimarinase was 8.3 mg/ml and Vmax was 8 mumol/min/mg. The distribution of beta-glucosidase activity in two different species of Penicillium showed that P. funiculosum had a higher ratio of extracellular to cell wall bound activity than Penicillium janthinellum. Treatment of mycelia of both species with NaCl, EDTA, Triton X-100, or proteolytic enzymes did not release the cell wall bound beta-glucosidase. Incubation of the mycelia with the laminarinase released 2-4 times more beta-glucosidase than the estimated cell bound activity in P. janthinellum and P. funiculosum.     

Interaction of Concanavalin A with the Cell Wall of Bacillus subtilis

Interactions between concanavalin A and cell wall digests of Bacillus subtilis 168 resulted in insoluble complexes as observed by double gel diffusion, turbidity, and analysis of the precipitate. The macromolecular constituent of the cell walls complexing with concanavalin A was the polyglucosylglycerol phosphate teichoic acid. The complex exhibited two pH optima: 3.1 and 7.4. The complex could be dissociated by saccharides which bind to concanavalin A. In contrast to concanavalin A-neutral polysaccharide complexes, formation of the concanavalin A-wall complex was inhibited by salts. It was subsequently shown that salts induce conformational changes in cell wall digests. The data suggested that for complex formation to occur a rigid rod conformation in the glucosylated teichoic acid is probably necessary. Concanavalin A can be used as a probe to study structural features of bacterial cell walls.     

Evidence for covalent attachment of diphytanylglyceryl phosphate to the cell-surface glycoprotein of Halobacterium halobium.

In a previous study, we demonstrated the occurrence of novel proteins modified with a diphytanylglyceryl group in thioether linkage in Halobacterium halobium (Sagami, H., Kikuchi, A., and Ogura, K. (1995) J. Biol. Chem. 270, 14851-14854). In this study, we further investigated protein isoprenoid modification in this halobacterium using several radioactive tracers such as [3H]geranylgeranyl diphosphate. One of the radioactive bands observed on SDS-polyacrylamide gel electrophoresis corresponded to a periodic acid-Schiff stain-positive protein (200 kDa). Radioactive and periodic acid-Schiff stain-positive peptides (28 kDa) were obtained by trypsin digestion of the labeled proteins. The radioactive materials released by acid treatment of the peptides showed a similar mobility to dolichyl (C55) phosphate on a normal-phase thin-layer plate. However, radioactive hydrolysates obtained by acid phosphatase treatment co-migrated not with dolichol (C55-65), but with diphytanylglycerol on both reverse- and normal-phase thin-layer plates. The mass spectrum of the hydrolysate was also coincident with that of diphytanylglycerol. The partial amino acid sequences of the 28-kDa peptides were found in a fragment (amino acids 731-816) obtainable by trypsin cleavage of the known cell-surface glycoprotein of this halobacterium. These results indicate that the cell-surface glycoprotein (200 kDa) is modified with diphytanylglyceryl phosphate.     

Autolytic Enzyme System of Streptococcus faecalis III. Localization of the Autolysin at the Sites of Cell Wall Synthesis

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 autolyzed in dilute buffers. Walls were isolated from cultures grown in the presence of (14)C-lysine for about 10 generations and then on (12)C-lysine for 0.1 to 0.8 of a generation (prelabeled). These walls released (14)C to the soluble fraction more slowly than they lost turbidity during the initial stages of autolysis. Walls isolated from cultures grown in the presence of (14)C-lysine for only the last 0.1 to 0.4 of a generation (postlabeled) released (14)C to the supernatant fluid more rapidly than they lost turbidity. Autolysin in both pre- and postlabeled walls was inactivated, and such walls were then incubated in the presence of unlabeled walls containing active autolysin. The inactivated walls lost their (14)C label only very slowly until autolysis of the unlabeled walls was virtually complete and release of soluble autolysin was expected. When this experiment was done in the presence of trypsin, a fourfold increase in the autolysis rate resulted, but the same pattern of (14)C release was observed. A parallel release of (14)C and loss of turbidity from pre- or postlabeled walls was observed upon trypsin "activation" and by addition of isolated soluble autolysin to inactivated walls. We conclude that the wall-bound autolysin acts first on the more recently synthesized portion of the wall. Trypsin appears to speed wall autolysis by activating additional latent autolysin in situ at sites in the older portion of the wall.     

Dolichyl phosphate linked sugars as intermediates in the synthesis of yeast mannoproteins: an in vivo study

Freshly prepared protoplasts of Saccharomyces cerevisiae X 2180 incorporate [³H]mannose and [¹⁴C]glucose for about 30 min into glycolipids and mannoproteins. Among the radioactive glycolipids formed dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides have been identified. The oligosaccharides released by weak acid from the dolichyl pyrophosphate were treated with endo-N-acetylglucosaminidase H and separated by gel filtration on Bio-Gel P-4. The largest oligosaccharide obtained corresponded exactly in size to Glc₃Man₉GlcNAc₁ the compound formed also in animal tissues. Other oligosaccharides released from dolichyl pyrophosphate in addition to the glucose containing ones were mainly Man₉GlcNAc₁ and Man₈GlcNAc₁. No mannosyl oligosaccharide corresponding in size to the total inner core region found in native mannoproteins could be detected in a lipid-bound form.The radioactive dolichyl pyrophosphate oligosaccharides were formed transiently; after 40 min only about 40% of the maximal radioactivity was observed in this fraction. In the presence of cycloheximide this decrease did not take place.It is concluded that the dolichol pathway of N-glycosylation of glycoproteins in yeast cells is very similar, if not identical, to the reaction sequence worked out for animal cells.Dedicated to Professor Dr. Otto Kandler on his 60th birthday     

Tunicamycin inhibition of epispore formation in Saccharomyces cerevisiae.

The ascopore wall of Saccharomyces cerevisiae was found to contain more protein, polymeric glucosamine, and beta-glucan than the vegetative cell wall, which was enriched in mannoprotein relative to ascospore walls. Tunicamycin inhibited sporulation, as judged by the absence of refractile ascospores visible by phase-contrast microscopy, but cells completed meiosis, as demonstrated by the presence of multinucleate asci. Such spores lacked the dense outer layer characteristic of normal spores. Thus, the tunicamycin effect was similar to that of glucosamine auxotrophy (W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975).     

Two homologous genes,DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.     

Protein Composition of the Cell Wall and Cytoplasmic Membrane of Escherichia coli

Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions. The fraction with the higher density was enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes, and the similar morphology of the fragments and intact cell walls. The less-dense fraction was enriched in vesicles derived from the cytoplasmic membrane, as indicated by the enrichment of cytochromes, the enzymes lactic and succinic dehydrogenase and nitrate reductase, and the morphological similarity of the vesicles to intact cytoplasmic membrane. Both fractions were rich in phospholipid. The protein composition was compared by mixing the cytoplasmic membrane-enriched fraction from a (3)H-labeled culture with the cell wall-enriched fraction from a (14)C-labeled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved; of these, 27 were increased two- to fourfold in the cytoplasmic membrane-enriched fraction, whereas 6 were similarly increased in the cell wall-enriched fraction. One of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope. This protein accounted for 70% of the total protein of the cell wall, and its occurrence in the envelope from spheroplasts suggests that it is a structural protein of the outer membranous component of the cell wall.     

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, α-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of α-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them.     

Lysis of Staphylococcus aureus Cell Walls by a Soluble Staphylococcal Enzyme

Enzyme preparations of Staphylococcus aureus were examined for their ability to solubilize (32)P-labeled cell walls of the parent organism. Enzymatic activity was observed in the growth medium, in soluble fractions, and associated with native cell walls. Enzyme associated with isolated cell walls could be inactivated with formaldehyde without reducing the susceptibility of the walls to the action of added enzyme. When cells are frozen and thawed, 50 to 75% of the intracellular enzyme is released along with 2% of the intracellular protein. This freeze-thaw extracted enzyme has little, if any, activity on intact S. aureus cells. It appears that the enzyme resides near the cell wall and acts on the cell-wall inner surface.     

Lysis of cell walls ofMucor ramannianus möller by aStreptomyces sp.

A study has been made of some chemical and ultrastructural changes that occur in the hyphal, arthrospore and sporangiospore walls ofMucor ramannianus during lysis by a soil streptomycete.Arthrospore and hyphal walls, which were shown to contain chitin, chitosan, other polysaccharides and phosphate (principally as polyphosphate), were lysed by culture fluid of the streptomycete after this organism had been grown on the same material. Alcohol-insoluble material found in the supernatants of the incubation mixtures gave on hydrolysis glucosamine, galactose, mannose and fucose. No laminarinase activity was detected in these culture fluids. Culture fluids of the streptomycete after growth on chitin and chitosan were also found to lyse the walls of arthrospores and hyphae.Despite the chemical similarities the walls were very different in thin section.A major component in the sporangiospore walls was glucan and an active laminarinase was shown to be present in the culture fluids of the streptomycete after growth on them. Further, ultrathin sections showed that an inner fibrillar layer of the sporangiospore wall was lysed leaving an outer electron-dense layer.