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.     

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.     

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.     

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.     

Dynamic changes of the cell wall surface of Candida albicans associated with germination and adherence

The distribution of mannoproteins at the cell wall surface of Candida albicans was analyzed during the process of germination in conditions favoring adherence of germ tubes to a plastic matrix. Three cytochemical methods allowing the detection of concanavalin A binding sites, anionic sites and the enzyme acid phosphatase, respectively were used. All three methods gave similar results, indicating a spatial and temporal reorganization of some cell wall mannoproteins: a strong labeling was observed on blastoconidia; in contrast, as soon as the emergence of germ tubes took place, these reactions decreased dramatically at the surface of mother cells, whereas the germ tube surface was strongly stained. Some new components with multiple biological activities were detected at the germ-tube surface. Indeed, among mannoproteins responsible for an enhanced adhesion to plastic surfaces, two components with molecular weights of 68 and 60 to 62 kDa were shown to interact with laminin, fibrinogen, and C3d. This study therefore indicates that germination, and then adherence of germ tubes, imply a degradation of surface mannoproteins, and a simultaneous presentation of new molecules which can interact with their nonbiological materials or host proteins.     

FTIR spectroscopic analysis of Saccharomyces cerevisiae cell walls: study of an anomalous strain exhibiting a pink-colored cell phenotype

A new strain, exhibiting an intriguing pink-colored cell phenotype, was obtained after an encoding alpha-glucosidase gene from an archaebacteria Thermococcus hydrothermalis was cloned by functional complementation of a mal11 Saccharomyces cerevisiae mutant TCY70. The possible implications of the alpha-glucosidase on the cell wall were evaluated by infrared spectroscopy and data indicate a 30% decrease in mannoproteins and an increase in beta-glucans. The loss of mannoproteins was confirmed by experiments on cells deprived of peptidomannans. Modifications in the major components of the cell wall did not jeopardize cell viability. Such rapid optical spectroscopic method can be used to screen a wide range of yeast mutants.     

Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

Metabolism of Saccharomyces cerevisiae envelope mannoproteins

By pulse and chase labeling experiments, two independent mannoprotein pools have been found associated with the Saccharomyces cerevisiae envelope. One of them probably corresponds to mannoproteins localized in the periplasmic space. These molecules showed a high turnover rate at 28° C. The second pool is formed by intrinsic wall mannoproteins which are apparently stable for long periods of time, after a small initial turnover. These results suggest that at least part of the mannoproteins initially found in the periplasmic space may move into the wall.The time lag between the addition of the radioactive precursors and their incorporation in the cell envelope (20–30 min for amino acids and about 10 min for carbohydrate) indicates that protein formation and carbohydrate incorporation take place in succession. Moreover, bulk glycosylation of mannoproteins seems to occur close in time to the moment of secretion into the periplasmic space.     

Incorporation of mannoproteins into the walls of aculeacin A-treated yeast cells

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 33000 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 33000 molecule, which was also completely absent from walls of aculeacin-treated protoplasts. Considerable levels of the 33000 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.Abbreviations SDS sodium dodecyl sulphate - YNB Yeast nitrogen base     

Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7% protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 31.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34, 30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.     

Structural mannoproteins released by β-elimination from Candida albicans cell walls

Abstract 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 .     

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA

Hyphal development in Candida albicans was blocked by EDTA. This effect was not due to a general growth inhibition since the chelator did not affect protein and DNA synthesis. Recovery of mycelial growth was observed when EDTA-grown cells were incubated at 37°C in EDTA-free medium. High-molecular-weight mannoproteins (HMWM) that are mycelium-specific wall components, and particularly a 260-kDa species (HMWM-260), were absent in the wall of cells grown under germination conditions in the presence of EDTA. Synthesis of the HMWM-260 species was not inhibited but its incorporation (secretion) into the wall structure seemed to be blocked in EDTA-treated cells.     

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.     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

The cell wall structure: developments in diagnosis and treatment of candidiasis.

Candidiasis are among the fungal infections the most difficult to diagnose and treat. Research focused on specific fungal components which are absent in the host, such as the cell wall has lead to a better understanding of Candida albicans pathogenicity and clinical impact. The cell wall is responsible for antigenic expression and primary interaction with the host. It is composed mainly of beta-glucans, chitin and mannoproteins, which account for the rigidity of the wall and for the fungal morphology. Of these components, mannoproteins might carry a "morphogenetic code" which might modulate the molecular architecture of the cell wall. The features of specific cell wall proteins as part of building blocks to form this structure is revised, and the usefulness of monoclonal antibodies obtained against cell wall components to study those processes, together with their clinical applicability, is discussed.     

Deletion of New Covalently Linked Cell Wall Glycoproteins Alters the Electrophoretic Mobility of Phosphorylated Wall Components of Saccharomyces cerevisiae

The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.     

Effect of papulacandin B and calcofluor white on the incorporation of mannoproteins in the wall of Candida albicans blastospores

Incorporation of mannoproteins into the walls of Candida albicans blastospores (yeast phase) was followed by continuous labelling and pulse-chase experiments. The effect in the process of compounds that interfere with synthesis (papulacandin B) or assembly (calcofluor white) of structural polymers was also assessed. Mannoproteins which are kept in place by non-covalent bonds (mainly hydrogen bonds) were incorporated rapidly after their release into the periplasmic space, this process being blocked by calcofluor white. The stain had no effect on the incorporation of covalently linked mannoproteins. Papulacandin B inhibited formation of beta-glucans and incorporation of covalently linked mannoprotein molecules, whereas incorporation of hydrogen-bonded species took place normally. The results suggest that the formation of the non-covalent bonds between the mannoproteins occurs once they are secreted into the periplasmic space, whereas the formation of covalent connections between mannoproteins and wall glucan takes place at the level of the plasma membrane.     

The molecular basis for the immunogenicity of Cryptococcus neoformans mannoproteins

T-cell-mediated immunity is necessary for effective host defenses against infections caused by Cryptococcus neoformans. Clinical and experimental studies have identified a heterogeneous family of mannoproteins as critical cryptococcal antigens responsible for stimulating T-cell responses. The archetypal mannoprotein has a signal sequence, a functional domain, a serine/threonine-rich region and a site for attachment of a glycosylphosphatidylinositol anchor. Extensive O-mannosylation, which occurs at the serine/threonine region, facilitates recognition by mannose receptors on antigen-presenting cells, particularly dendritic cells. This results in efficient antigen uptake, processing and presentation to T cells. Inhibition of mannose receptors or deglycosylation of mannoproteins profoundly inhibits T-cell responses, demonstrating the crucial contribution of mannosylation to immunogenicity.