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The walls of both, yeast and mycelial cells of Candida albicans possess a species-specific antigen that is recognized by a monoclonal antibody (MAb 3H8). This antigen can be extracted in the form of a very high Mr complex, close or over 106 Da, by treatment, with β-1,3-glucanase, β mercaptoethanol or dithothreitol, or mild alkali, but not by saturated hydrogen fluoride (HF) in pyridine, suggesting that the complex is bound to wall β-1,3 glucans, and to proteins by disulfide bonds, but not to β-1,6 glucans. Through its sensitivity to trypsin and different deglycosylation procedures, it was concluded that the epitope is associated to a glycoprotein containing N-glycosidic, but not O-glycosidic mannan moieties. By means of electrophoresis in polycrylamide gradient gels, followed by mass spectrometric analysis, the epitope was pinpointed to a very high MW complex containing Agglutinin-Like Sequence (ALS) family proteins, and other cytoplasmic, membrane and secreted proteins. The components of this complex are bound by unknown covalent bonds. The material extracted with β mercaptoethanol or dilute alkali appeared under the electron microscope as large aggregates in the form of spheroidal and mostly web-like structures of large sizes. These, and additional data, suggest that this protein complex may constitute an important part of the basic glycoprotein structure of C. albicans. The possibility that similar complexes exist in the wall of other fungi is an attractive, although yet untested possibility.

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.

The effect of immobilization on the cell wall of Saccharomyces cerevisiae cells was investigated in comparison with freely suspended batch grown cells. The pattern of mannoproteins released from the cell wall after Zymolyase digestion showed the presence of new mannoprotein species when cell growth takes place in a state of immobilization. The same result was obtained by exposure of freely suspended cells to a stressful concentration of ethanol, showing that two different adverse culture conditions induce a similar molecular response at the cell wall level.

A total of 6047 open reading frames in theCandida albicansgenome were screened forZn(II)2C6-type zinc cluster proteins (or binuclear cluster proteins) involved in DNA recognition. These fungal proteins are transcription regulators of genes involved in a wide range of cellular processes, including metabolism of different compounds such as sugars or amino acids, as well as multi-drug resistance, control of meiosis, cell wall architecture, etc. The selection criteria used in the sequence analysis were the presence of theCysX2CysX6CysX5-16CysX2CysX6-8Cysmotif and a putative nuclear localization signal. Using this approach, 70 putativeZn(II)2C6transcription factors have been found in the genome ofC. albicans.

After screening of aCandida albicansgenome database, the product of an ORF (IPF 3054) that has 62 % homology withSaccharomyces cerevisiaeSsr1p, an internal cell-wall protein, was identified and named CaSsr1p. The deduced amino acid sequence shows that CaSsr1p contains an N-terminal hydrophobic signal peptide, is rich in Ser and Thr amino acids and has a potential glycosylphosphatidylinositol-attachment signal. CaSsr1p is released following degradation of isolated cell walls by zymolyase (mainly a 1,3-β-glucanase) and therefore seems to be covalently linked to theβ-glucan of the cell walls. Both disruption and overexpression of theCaSSR1gene caused an increased sensitivity to calcofluor white, Congo red and zymolyase digestion. These results suggest that CaSsr1p has a structural role associated with the cell-wallβ-glucan.

Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by β-mercaptoethanol (β-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with β-1,3-glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys214-12aa-Cys227-COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site-directed mutation of two other cysteine amino acids (Cys130Ser or Cys197Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2-unprocessed Pir4. Therefore, it seems that deletion or mutation of individual cysteine molecules does not seem enough to inhibit incorporation of Pir4 by disulphide bridges. In fks1Δ and gsc2/fks2Δ cells, defective in β-1,3-glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by β-ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the β-1,3-glucan of the cell wall continued. Deletion of the repetitive sequence (QIGDGQVQA) resulted in the secretion and incorporation by disulphide bridges of Pir4 in reduced amounts together with substantial quantities of the Kex2-unprocessed Pir4 form. Pir4 failed to be incorporated in alkali-sensitive linkages involving β-1,3-glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the β-1,3-glucan. Copyright © 2003 John Wiley & Sons, Ltd.

A collection of transposon-mutagenized strains of Yarrowia lipolytica was screened for wall defects by determination of their sensitivity to calcofluor white. A number of strains were hypersensitive, whereas others were resistant. Different non-allelic mutants displayed increased sensitivity to autolysis and lytic enzymes, independently of whether they were sensitive or resistant to calcofluor white. A thorough analysis of their cell walls revealed minor quantitative alterations, and no significant changes in chitin content. Electrophoretic analysis of wall-bound and excreted proteins proved to be a sensitive method that revealed defects in the cell wall structure of the mutants. Important alterations in the patterns of the wall proteins extracted by SDS or by enzymatic treatments were noticed for the mutants, as compared to the parental strain. Mutants released to the growth medium a larger number of protein species than the parental strain, suggesting impairment in wall assembly of certain polypeptides. Patterns of wall-bound and excreted proteins, as well as alterations in wall chemical composition were not diagnostic of calcofluor white sensitivity or resistance, but were specific for each mutant. Our data show that an increase in either sensitivity or resistance of Y. lipolytica to certain levels of calcofluor is equally indicative of alterations in cell wall structure, independent of chitin levels.

Yarrowia lipolytica is an ascomycete with biotechnological potential. In common media, the fungus grows as a mixture of yeast-like and short mycelial cells. The environmental factors that affect dimorphism in the wild-type strain, W29, and its auxotrophic derivative, PO1a, were analyzed. In both strains, pH was the most important factor regulating the dimorphic transition. Mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to become almost null at pH 3. Carbon and nitrogen sources, namely glucose and ammonium, were also important for mycelium formation; and their effect was antagonized by some alternative carbon and nitrogen sources. Citrate was an important positive effector of mycelium growth. Anaerobic stress induced formation of mycelial cells. The importance of the protein kinase A pathway was suggested by the inhibition of mycelium growth by cAMP. We propose that the interplay of these factors regulates the adaptation of the fungus, to better exploit its natural ecological niches.

Linkages of Ywp1 to other components of the Yarrowia lipolytica mycelial cell wall were studied by extraction with beta-mercaptoethanol and zymolyase (a beta-glucanase complex) and by the use of rabbit polyclonal antibody preparation raised against Ywp1. Ywp1 complexed with an N-glycosylated cell wall protein(s) to form supramolecular complexes through disulphide bridges (extractable with beta-mercaptoethanol) or bonded to beta-1,3-glucan (extractable with zymolyase). The lack of a specific morphological phenotype when YWP1 was knocked out by gene disruption might indicate that other proteins present in the cell wall of Y. lipolytica compensated for its loss. In this mutant, the electrophoretic pattern of proteins, detected with polyclonal antibodies against the entire cell wall, was different from that obtained with the parental strain, but sensitivity to calcofluor white, zymolyase and chitinase did not change. Quantitative analysis of fluorescence emitted by cells in the presence of fluorescent wheat germ agglutinin (FITC-WGA) indicated that chitin was organized in the cell wall of the mutant cells in a form different from that in the parental strain.

Urea or hot sodium dodecyl sulphate extracted a significant amount of the same proteins from the matrix of the cell wall of the yeast form and mycelial cells of Candida albicans. Gel filtration analysis of the urea-extracted proteins revealed that they occurred in the form of large complexes which were unaffected by up to 8 M urea. Among them, proteins en route to becoming covalently associated within the wall scaffold were identified by their reaction with specific antibodies. When urea was removed by dialysis, some of these proteins specifically reassociated into large aggregates which bound strongly with ConA, whereas others remained soluble in smaller associated products. The ability of some of these proteins to bind to the insoluble wall polysaccharides was also assessed. No self-assembling proteins were able to bind to glucans and/or chitin. Specificity of the binding to polysaccharides made of beta-bound glucosyl or N-acetylglucosaminyl residues was determined by the competitive effect of several disaccharides. Whereas laminaribiose and diacetylchitobiose were strong inhibitors of protein binding to both glucan and chitin, lactose, maltose and sucrose were ineffective.

Summary Cell wall regeneration of individual Candida albicans yeast and mycelial protoplasts was studied with confocal and electron microscopy using polyclonal antibodies and leetins. Quantitative measurements of the fluorescence emitted by individual protoplasts during the process of regeneration indicate that chitin is the first polymer to be laid down, whereas β(1,3)- and β(1,6)glucan are incorporated at a later stage. Mannoproteins were found on the surface of fresh protoplasts and those newly synthesized were then deposited with time. During the first steps of wall regeneration, the proteins that interacted covalently with chitin or glucan were different, but the same species were found linked to each polymer in yeast and mycelial regenerating forms. The aggregates formed by regenerating protoplasts were shown to be due to the chitin and mannoprotein network initially laid.

Screening cDNA libraries with polyclonal antibody preparations against Candida albicans yeast or mycelial cell walls resulted in isolation of several positive clones. Some of them encoded enolase; others encoded a protein of the 70 kDa heat-shock protein family (Hsp70p), etc. The presence of these cytosolic proteins in the cell wall of actively growing C. albicans was discovered by analytical (SDS-PAGE and Western blot) and cytological (indirect immunofluorescence) experiments. Supplementation of cell cultures with papulacandin B, an antibiotic that inhibits formation of the -glucan skeleton, resulted in the release of enolase to the supernatant fluids; this release was prevented when 0.6 M KCI was present as an osmotic stabilizer. The cell wall of C. albicans incorporated exogenously added proteins (enolase and Escherichia coli and C albicans cytosolic proteins). The presence in the C. albicans cell wall of enolase, Hsp70p, and probably other intracellular proteins that are highly immunogenic might help the fungal cells to evade the host defences, and consequently could represent a survival mechanism for C. albicans in vivo.

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a beta-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology.

Following synthesis of its individual components, the cell wall of Candida albicans is assembled extracellularly in two steps. First, a viscoelastic composite is formed by noncovalent interactions between mannoproteins and other wall components. Second, the initial network is consolidated by formation of covalent cross-linkages among the wall polymers. In both processes, specific proteins may regulate the final yeast or mycelial morphology. These proteins might carry out part of what could be called a morphogenetic code. Experimental results have shown that some mannoproteins form supramolecular complexes. They are secreted independently, but released together from cell walls by hydrolases. In C. albicans cell walls a transglutaminase activity has been detected that could be responsible for the formation of covalent bonds between structural proteins. Key words: fungal cell wall, construction, morphogenesis, protein interactions, noncovalent linkages, covalent linkages.

The use of a novel monoclonal antibody (mAb) that reacts with (1,6)-beta-glucan has permitted the study of the different covalent linkages between glucan and mannoproteins in the cell wall of Candida albicans. The mAb JRR1 was originally raised by immunization with Zymolyase extracts from C. albicans cell walls, but it soon became apparent that it reacted with a (1,6)-beta-glucan epitope. By using this antibody, we show the existence of glucan-mannoprotein complexes between the (1,6)-beta-glucan epitope recognized by the antibody and cell wall mannoproteins. The topology of the (1,6)-beta-glucan in the cell wall of C. albicans has also been studied.

Candida albicans Gca1p is a putative glucoamylase enzyme which contains 946 amino acids, 11 putative sites for N -glycosylation and 9 for O -glycosylation. Gca1p was identified in β-mercaptoethanol extracts from isolated cell walls of strain C. albicans SC5314 and it is involved in carbohydrate metabolism. The significance and the role of this protein within the cell wall structure were studied in the corresponding mutants. The homozygous mutant showed that GCA1 was not an essential gene for cell viability. Subsequent phenotypic analysis performed in the mutants obtained did not show significant difference in the behavior of mutant when compared with the wild strain SC5314. Zymoliase, Calcofluor White, Congo red, SDS, caffeine or inorganic compounds did not affect the integrity of the cell wall. No differences were observed when hyphal formation assays were carried out. However, an enzyme assay in the presence of substrate p -nitrophenyl-α-D-glucopyranoside enabled us to detect a significant decrease in glycosidase activity in the mutants compared with the parental strain, revealing the function of Gca1.

Electrophoretic mobility of secreted invertase (E.C. 3.2.1.26) from gelatin-immobilized yeast cells was analysed and compared with that of secreted invertase from freely suspended batch-grown cells. Invertase from immobilized cells showed a tower mobility after 24 h of incubation, in medium containing either glucose or raffinose as carbon source. Changes in invertase mobility were also followed in a time course both for immobilized and for freely suspended batch-grown cells. Mobility of invertase from free cells increased after approximately 15 h of incubation, independently of the carbon source, whilst that of invertase from immobilized cells remained constant. The differences observed were attributed to a different level of glycosylation of the protein moiety in free and immobilized cells. The amount of mannoproteins in the cell walls of immobilized cells was also investigated by ConA-ferritin labelling and quantification of ferritin particle density in ultrathin sections; the results of this experiment showed a higher content of mannoproteins in the walls of immobilized cells when compared with free cells. As a whole, these results are indicative of physiological changes that can be ascribed to the peculiar microenvironment of gel-immobilized cells.

Mild alkaline solutions (β-elimination), after removing the non-covalently bonded wall materials by hot SDS, released 13% and 26% of remaining wall proteins from mycelial and yeast cells of Candida albicans, respectively. When the β-elimination was carried out after digestion of the walls with chitinase, four-fold more proteinaceous materials were released from mycelium and a similar amount in yeast walls. The solubilized materials were shown to be highly polydisperse, and endo-glycosidase H reduced their polydispersity and molecular masses, revealing different electrophoretic patterns in yeast and mycelial cell walls. The solubilized mycelial proteins carried N-glycosidic sugar chains and the epitopes recognized by two monoclonal antibodies were preserved, although showing a different behaviour in yeast walls. These results are consistent with the idea that significant amounts of intrinsic O-glycosylated mannoproteins are interconnected in the walls of C. albicans.

The kinectics of incorporation of two precursor mannoproteins into the regenerating cell wall of Candida albicans protoplasts have been followed at 28°C and 37°C using two monoclonal antibodies specific for protein epitopes (MAb 1B12 and 4C12) as probes. Both molecules were secreted from the beginning of the regeneration process, and their incorporation was retarded significantly. Analysis of the secreted materials by Western immunoblotting with MAb 1B12 allowed the identification of two closely migrating bands at apparent Mr higher than 170 kDa and significant amounts of a highly polydisperse material of even greater molecular mass. Some of these mannoproteinaceous species carried both N- and O-glycosidically linked mannose residues, as deduced from their drop in apparent Mr when synthesized in the presence of tunicamycin and by their reactivity with Concanavalin A. Following secretion, the molecules reacting with MAb 1B12 were incorporated into the regenerating walls by covalent binding. Then, when the regenerating walls by covalent binding. Then, when the antigen molecules were solubilized from partially regenerated walls, their mobility differed when regeneration took place at 28°C (blastoconidia) or 37°C (mycelial cells).

The use of Saccharomyces cerevisiae mnn mutants has facilitated the study of the epitopes recognized by antisera against several antigenic factors of the genus Candida (Candida Check; Iatron Laboratories, Tokyo, Japan). We have taken advantage of the very well characterized structure of the mannans of the different mnn mutants to compare their reactivities with the factor antisera used in the identification of different species of the genus Candida. The results of this study provide evidence that one of the antigenic determinants recognized by factor 1 antisera is the O-linked mannose chains of the cell wall mannoproteins, while that recognized by factor 9 antiserum is the alpha 1-6-linked mannose backbone of the outer chain of the N-linked oligosaccharide.

Aculeacin A and papulacandin B block cell wall regeneration in Candida albicans protoplasts at an intermediate step in which the protoplasts have not yet synthesized the rigid structure of the cell wall and are therefore still osmotically sensitive. In the presence of the antibiotics, total synthesis of glucan is not significantly lowered with respect to control cells, although most of it appears either in the culture medium or in the regenerating wall as alkali-soluble glucan. Thus, it is proposed that echinocandins (such as aculeacin A) and papulacandins may not inhibit glucan synthesis per se but instead inhibit its incorporation into the supramolecular organization of the cell wall.

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) from C. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen, in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processes in vivo and in vitro or, alternatively, that both enzyme activities (active in vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml-1 of digitonin and 64 micrograms ml-1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 micrograms ml-1 of protease, whereas activity strongly decreases in the presence of 64 micrograms ml-1 of trypsin. Digitonin does not produce zymogen activation per se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.

Development of new effective antifungal drugs is limited by the absence of specific target sites in the fungal cells. Knowledge of the fungal cell wall structure and biosynthesis is of interest in searching for a potential target site for new chemotherapeutic agents. Our group has demonstrated that the fungal cell wall is a metabolically active structure where interaction between distinct components occurs to give rise to the mature cell wall structure. Mannoproteins play an essential role in the cell wall organization, and there is evidence for the formation of covalent bonds between these molecules and the structural polymers (glucans and chitin) outside the plasma membrane. Such interactions, which specifically occur at the fungal cell wall, are of great interest in defining target sites for potential new chemotherapeutic agents, which may inhibit the interactions and, thus, lead to a defective cell wall formation and cell death.

The effect of a lipase activity (EC 3.1.1.3) on the chitin synthetase from Candida albicans has been studied, both on the active and the trypsin activated enzyme. Removal of fatty acids from acylglycerides by lipase has an inhibitory effect on the activity as well as on the ‘in vitro’ activation process by trypsin in the membrane-bound enzyme and in the chitosomes. This would indicate that an adequate lipid environment is required for both the activation process and proper function of the synthetase activity.

Chitin synthetase activity was detected in a partly zymogenic form in cell-free homogenates obtained from C. albicans yeast cells and protoplasts of the same type of cells. By isopycnic centrifugation on sucrose gradients of the cell-free homogenates two fractions with chitin synthetase activity were obtained: one was associated with the plasma membrane (labelled with [ 3 H]concanavalin A (con A) and buoyant density 1·195 g ml −1 ) in a partly active state, and the second was in the cytoplasm, where the enzyme was in a particulate fully zymogenic form, lacking affinity to con A. The buoyant density of the enzyme found in this location depended on the method of cell breakage. Lysis of partly regenerated protoplasts gave a value of 1·135 g ml −1 , while cells broken with glass beads gave a major peak of activity at a buoyant density of 1·085 g ml −1 and a second minor peak at the buoyant density obtained in protoplasts (1·135 g ml −1 ). Breakage with glass beads resulted in the release of chitin synthetase, mainly zymogenic, from the membrane fraction (MMF) (buoyant density 1·085 g ml −1 ) whereas the cytoplasmic enzyme activity decreased in parallel with an increase in the buoyant density (from 1·135 g ml −1 to 1·165 g ml −1 ). The extent of both events depended on the conditions of cell disruption. Finally a precursor/product relationship between the cytoplasmic and membrane-bound enzymes could not be demonstrated because neither the subcellular distribution nor the activation state of the enzyme was modified following inhibition of protein synthesis.

SUMMARY: Schizosaccharomyces pombe cell walls contain two major glycoprotein species, I and II, with molecular masses of 2 x 106 and 5 x 105 Da respectively, as determined by gel filtration chromatography and PAGE. The ratio of sugar to protein is higher in species I than in species II. Much of the sugar in both glycoproteins (about 85% in wild-type cells) is O-linked to the peptide moiety. The morphological sph1 mutant is resistant to papulacandin B, and its cell wall contains less glycoprotein II (but not less glycoprotein I) than the parental wild-type strain, although glycoprotein II is still synthesized and released into the growth medium. Papulacandin B largely reverses the morphological alteration of the mutant, and returns the ratio between species I and II to about that found in the parental strain, although the absolute amount of species II is still lower in the mutant. The results point to the importance of the relative amounts of the different wall polymers in determining cell morphology.

Hyphal development in Candida albicans was selectively blocked by the ornithine decarboxylase competitive inhibitor 1,4-diaminobutanone (DAB). Inhibition of hyphal development required DAB during both yeast inoculum growth and subsequent incubation at 37 degrees C to induce mycelial growth. This effect was not due to general growth inhibition since DAB did not inhibit yeast growth, and reduced protein synthesis by 30% at most. Moreover, protein synthesis was unaffected by DAB when cells were pre-grown in drug-containing media. Since DAB inhibited dimorphic transition at 37 degrees C, morphology- and temperature-dependent protein synthesis could be distinguished. DAB stimulated the synthesis of several yeast wall-proteins, irrespective of morphology or growth temperature, and two at 37 degrees C only, but it inhibited the synthesis of a single mycelial-specific glycoprotein species.

Most of the manosyl transferase activity in Phycomyces blakesleeanus was found associated with a crude membrane fraction sedimenting at 48,400 g ( R av ). Triton X-100 and Nonidet NP-40 inhibited 95% of the enzyme activity. Digitonin caused 47% of inhibition and when removed, the membrane-bound enzymatic activity increased by about 35%; no activity was detected in supernatant. The rate of mannosyl transfer increased in the presence of 4 or 8 m M Mg 2+ ions. Several compounds, including glycoproteins, mucoran, and mucoric acid, failed to act as acceptors of mannosyl residues. Guanosine diphosphate and guanosine monophosphate inhibited the transfer of mannosyl residues by 60 and 19%, respectively. Mannosyl transfer involves participation of lipid intermediates.β elimination of the product synthesized in vitro revealed the presence of mannose, mannobiose, and mannotriose, suggesting that they are bound to protein via O -glycosidic linkages. The alkaline-resistant carbohydrate part of the glycoproteins consisted mainly of mannose residues that were probably connected to the protein moiety through N -glycosidic bonds.

After screening a Candida albicans genome data base, the product of an open reading frame (IPF 19760/CA2574) with 41% identity to Saccharomyces cerevisiae vacuolar acid trehalase (Ath1p) was identified and named Atc1p. The deduced amino acid sequence shows that Atc1p contains an N-terminal hydrophobic signal peptide and 20 potential sites for N-glycosylation. C. albicans homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two ATC chromosomal alleles. Analysis of these null mutants shows that Atc1p is localized in the cell wall and is required for growth on trehalose as a carbon source. An Atc1p endowed with acid trehalase activity was obtained by an in vtro transcription-translation coupled system. These results strongly suggest that ATC1 is the structural gene encoding cell wall acid trehalase in C. albicans. Determinations of ATC1 mRNA expression as well as acid trehalase activity in the presence and absence of glucose point out that ATC1 gene is regulated by glucose repression.

Stability of the protein populations present in the cell wall of three ascomycetous fungi Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica was investigated. Cell wall proteins were either labeled with biotin or radiolabeled with amino acids, and chased for a period of time representing several generations. Proteins linked by non-covalent or covalent bonds were separated and their turnover was analyzed. No significant turnover took place during the chase period, and in fact radioactive proteins were accumulated in the wall during the period possibly by transfer through the secretory pathway. This transfer did not involve de novo protein synthesis; it was inhibited by azide, and by incubation of a sec1 mutant of S. cerevisiae at the non-permissive temperature. It is concluded that proteins bound to the cell wall are stable and that there is no precursor-product relationship among those linked by non-covalent bonds and the covalently bound ones.

The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls.

A cDNA encoding a 37 kDa protein was isolated from an expression library using antibodies raised against mycelial cell walls fromCandida albicans.The 37 kDa protein has over 60% sequence identity with the 37 kDa laminin-binding protein (LBP) from humans and over 80% identity with the Yst proteins ofSaccharomyces cerevisiae. TheC. albicansprotein was named CaYst1. It was found in membrane and ribosome fractions but surprisingly, was not found in cell walls. Unlike the human LBP, CaYst1p does not bind laminin. These data indicate that CaYst1p is not a cell-surface receptor for laminin as has been proposed for the human LBP. Instead, like theS. cerevisiaeYst proteins, it appears to be a ribosomal protein. This conclusion is supported by the finding thatCaYST1-cDNA complements the lethal phenotype linked to the disruption of bothYSTgenes inS. cerevisiae.

Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated PRA1 , for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that the PRA1 protein may have a role in the host-parasite interaction during candidal infection.

Cell wall extracts from the double-mutant mnn1 mnn9 strain were used as the immunogen to obtain a monoclonal antibody (MAb), SAC A6, that recognizes a specific mannoprotein--which we have named Icwp--in the walls of cells of Saccharomyces cerevisiae. Icwp runs as a polydisperse band of over 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Zymolyase extracts of cell walls, although an analysis of the secretory pattern of the mannoprotein shows that at the level of secretory vesicles, it behaves like a discrete band of 140 kDa. Immunofluorescence analysis with the MAb showed that Icwp lies at the inner layer of the cell wall, being accessible to the antibody only after the outer layer of mannoproteins is disturbed by treatment with tunicamycin. The screening of a lambda gt11 expression library enabled us to identify the open reading frame (ORF) coding for Icwp. ICWP (EMBL accession number YLR391w, frame +3) codes for 238 amino acids, of which over 40% are serine or threonine, and contains a putative N-glycosylation site and a putative glycosylphosphatidylinositol attachment signal. Both disruption and overexpression of the ORF caused increased sensitivities to calcofluor white and Congo red, while the disruption caused an increased sensitivity to Zymolyase digestion, suggesting for Icwp a structural role in association with glucan.

The topological distribution of two epitopes in the cell wall of Candida albicans, the kinetics of their incorporation into the regenerating protoplast wall, and the effect of different antibiotics upon their incorporation and localization have been studied. To do so, two monoclonal antibodies that react against an O-glycosylated mannoprotein (1B12) and against a 1,6-beta-glucan epitope (JRR1) were used. The results show that the JRR1 epitope is localized in an internal layer of the cell wall, in contrast to the 1B12 epitope, which is superficial, and that the incorporation of the JRR1 epitope into walls of regenerating protoplasts precedes that of the 1B12 epitope. The JRR1 epitope is normally found in the culture medium of control cells, but not in that of papulacandin-B-treated cells, and tunicamycin interferes with the incorporation of the 1B12 epitope into the cell walls. Finally, the results support the hypothesis that mannoproteins are not 1,6-beta-glycosylated before their secretion.

Summary Free and gelatin immobilized yeast cells from two different strains were analyzed by means of SDS-PAGE as regards: i) the glycosylation levels of external invertase before and after treatment with endo-β-N-acetylglycosaminidase H (Endo H), ii) the pattern of mannoproteins released from the cell wall after extraction with SDS, β-mercaptoethanol and Zymoliase digestion. The results obtained suggest that the composition and structure of the cell wall is modified when cell growth takes place in the state of immobilization.

Activity of the enzyme glutaminyl-peptide--glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N epsilon-(gamma-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms of Candida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.

The organization of the components of the cell wall from Candida albicans was studied by means of sequential treatment with hot SDS, anhydrous ethylenediamine (EDA) and lytic enzymes, followed by chemical and microscopic analyses of the different separated fractions. The EDA-insoluble fraction retained the original morphology of the wall, which was destroyed by beta-glucanase, but not by chitinase treatments. Staining with fluorescent lectins revealed distinct distributions of mannoproteins, glucans and chitin in the wall. Amino acid analysis of SDS-extracted walls, and the EDA-soluble and -resistant fractions gave similar results, with seven amino acids making up about 70% of the total protein weight. Treatment of the EDA-insoluble fraction with Zymolyase or chitinase released fragments of variable size whose susceptibility to these and other hydrolases suggests that they are made of glucan, chitin and mannan oligomers associated with proteins. Treatment of the Zymolyase-insoluble residue with chitinase released a series of low-molecular-mass oligomers made of neutral sugars, GlcNAc and amino acids, mainly lysine. It is suggested that they represent fragments of the core making up the scaffold of the cell wall of the fungus.

Cell walls play essential roles in growth, development, and in interactions of fungi with the environment and with other cells. Besides its primary protective role in shielding the cell against osmotic, chemical, and biological harm, the wall is involved in many other functions including morphogenesis, and some activities that may be denominated as “social”, such as morphological responses, antigenic expression, adhesion, and cell-cell interaction (Peberdy 1990; Ruiz-Herrera 1992; Sentandreu et al. 1991). There are many data supporting the idea that temporal and spatial regulation of wall polymer synthesis and assembly are critical for the properties of the walls, which thus do not exclusively depend on their chemical composition, but also on the way that different polymers interact.

SUMMARY: The mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with β-glucanase (Zymolyase) have higher molecular masses than their probable precursors present in the supernatant of regenerating protoplasts. It therefore appears that the mannoproteins are released from the walls as part of supramolecular complexes. Immunological analysis using both polyclonal and monoclonal antibodies has demonstrated the probable relationship between molecules found in a mixed membrane preparation, those secreted by regenerating protoplasts, and those present in yeast cell walls. Some mannoproteins secreted by protoplasts incubated in the presence of tunicamycin had significantly increased mobility on SDS-PAGE, whereas others were not affected by the treatment. It is therefore possible that two types of mannoproteins are secreted by protoplasts: one carrying N-glycosylated chains (mannan) and one lacking them. All the proteins secreted in the presence of tunicamycin stained with Concanavalin A-peroxidase, demonstrating that they all, including the N-glycosylated ones, carried O-glycosylated sugar residues. Both classes of mannoproteins, secreted independently of each other, were found in the molecular complexes rendered soluble from the wall by Zymolyase digestion. Data obtained with a monoclonal antibody demonstrated the presence of a repeated epitope within one wall protein(s) detectable in a mixed membrane preparation and in the wall complexes released by Zymolyase.

The shape of Candida albicans cells depends on their cell walls and some of their mannoproteins may act as modulators of the final molecular architecture. If that were the case, the wall mannoproteins might form part of what could be called a “morphogenetic code”.

In higher cells, glycoproteins play a variety of functions as surface receptors, cell-cell mediators, carriers of enzyme activities, components of the extracellular matrix, etc. In most glycoproteins, the protein moiety will be the functional part whereas the carbohydrate moiety would contribute to the attainment of an adequate tertiary structure, modify the glycoprotein molecule making it more resistant to degradation, and facilitate its secretion.

Most cells are covered with a complex network of interacting molecules that form the extracellular matrix. These molecules (proteins and polysaccharides) are secreted locally and interact among themselves to form an organized structure outside the cell plasma membrane. In unicellular eukaryotic organisms and plant cells, this structure is reinforced to withstand osmotic changes in the external environment, giving rise to the so-called cell wall.

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118-1125]. Zymolyase, chitinase and beta-mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (greater than 180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.

Aculeacin A is a lipopeptide that inhibits beta-glucan synthesis in yeasts. A number of Saccharomyces cerevisiae mutants resistant to this antibiotic were isolated, and four loci (ACR1, ACR2, ACR3, and ACR4) whose products are involved in the sensitivity to aculeacin A of yeast cells were defined. Mutants containing mutations in the four loci were also resistant to echinocandin B, another member of this lipopeptide family of antibiotics. In contrast, acr1, acr3, and acr4 mutants were resistant to papulacandin B (an antibiotic containing a disaccharide linked to two fatty acid chains that also inhibits beta-glucan synthesis), but acr2 mutants were susceptible to this antibiotic. This result defines common and specific steps in the entry and action of aculeacin A and papulacandin B. The analysis of double mutants revealed an epistatic effect of the acr2 mutation on the other three mutations. Cell walls of the four different mutants did not show significant alterations in composition with respect to the parental strain, and in vitro glucan synthase activity was also unaffected. However, cell surface hydrophobicity in three of the mutants was considerably decreased with respect to the parental strain.

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.

Inhibition of the synthesis of alkali-insoluble glucan by aculeacin A in Saccharomyces cerevisiae cells caused a decrease in the incorporation of a high molecular weight heterogeneous mannoprotein material and of a 33,000 mannoprotein into the wall network. This was concomitant with the excretion of the latter molecule into the growth medium. Regenerating yeast protoplasts liberated considerable amounts of the heterogeneous material to the medium independently of the presence of aculeacin. The protoplast walls did lack this component and contained only minor amounts of the 33,000 molecule, which was also completely absent from walls of aculeacin-treated protoplasts. Considerable levels of the 33,000 species were immunodetected in the supernatants from treated and untreated protoplasts. These results point to the existence of specific interactions between the glucan network of the yeast cell surface and some of the wall mannoproteins. On the other hand, the presence of a population of SDS-solubilizable mannoproteins in the wall was independent of glucan levels.

Protein fractions that overcome m7GMP inhibition of mRNA translation have been purified from the yeast S. cerevisiae . An active fraction isolated from polysomes contains two polypeptides of 220- and 190-kDa. The active fraction isolated from postribosomal supernatant contains a major polypeptide of 28-kDa and other species of 32-, 24-, 22- and 21-kDa, and sediments in sucrose gradients as a high molecular weight complex of about 200000. This fraction restored yeast mRNA translation in reticulocyte lysates under conditions of yeast and globin mRNA competition; however, this effect was not observed with the 220- and 190-kDa polypeptides from polysomes. Nevertheless, translation of yeast mRNA was stimulated by a partially purified fraction containing a 28-kDa polypeptide from polysomes.

The zymogenic form of the chitin synthase present in mixed membrane preparations was extracted by digitonin treatment. The residual extracted membranes exclusively retained the basal activity. Trypsin activation of the zymogenic form of the enzyme did not modify the digitonin solubilization characteristics of the original zymogenic form, suggesting significant differences between ‘in vivo’ activation of chitin synthase and that carried out by trypsin ‘in vitro’.