Candida Albicans Cell Wall Research Articles

Crystallization of the exo(1,3)-β-glucanase from Candida albicans

An exoglucanase, with specificity for β(1,3) linkages, from the cell wall of Candida albicans has been crystallized by the hanging drop method in the presence of polyethylene glycol 8000. The crystals, which diffract to better than 1.9 Å resolution, belong to the orthorhombic space group P2 12 12 1 with cell constants a = 60.2 A ̊ , b = 65.2 A ̊ , c = 96.5 A ̊ and with one molecule in the asymmetric unit.

Functional effects of a monoclonal antibody directed against a distinct epitope on 4F2 molecular complex in human peripheral blood mononuclear cell activation

The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1β but not IL-2 or interferon-γ were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43 + cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43 + cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation.

In Vitro production of tumor necrosis factor by murine splenic macrophages stimulated with mannoprotein constituents of Candida albicans cell wall

Mannoprotein components from Candida albicans were investigated for their ability to induce production of tumor necrosis factor (TNF) by cultured splenocytes from naive or Candida-infected mice. Two chromatographically separated mannoproteins preparations, designated F1 and F2, were as able as the heat-inactivated Candida cells to induce the production of TNF from splenocytes of naive animals. In addition, they caused a significant augmentation of basic TNF secretion by splenocytes of Candida-infected animals. Experiments using plastic and/or nylon wool adherence, as well as treatments with antibodies depleting T or NK cells, consistently indicated that most if not all TNF was produced by splenic macrophages. In cultures of splenocytes from Candida-infected mice. mannoprotein addition also stimulated interferon-γ (IFN-γ) production by Thy 1.2 positive cells. Depletion of these cells or addition of anti-IFN-γ antibodies abolished IFN production and reduced TNF secretion by adherent cells to the levels found in the cultures of mannoprotein-stimulated spleen cells from naive mice. These data add further evidence to the immunomodulatory properties possessed by some cell wall constituents of the human commensal microorganism C. albicans and suggest that IFN-γ is endowed with a regulatory role in TNF production by mouse macrophages in vitro.

Phosphate-containing proteins and glycoproteins of the cell wall of Candida albicans

The distribution of phosphate, carbohydrate, and protein in the cell wall components extracted from intact yeast cells of Candida albicans by beta-mercaptoethanol (beta ME) at pH 8.6 was examined by analysis of the material separated by DEAE-cellulose chromatography. All protein peaks did not coincide with peaks of both carbohydrate and phosphate. Subsequent analysis was performed on material obtained from yeast cells and germ tubes which were grown in medium containing [32P]phosphate. Two extracts were obtained by treating cells with beta ME or with zymolyase following beta ME. The extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. beta ME-extracted material contained high-molecular-mass (HMM), greater than or equal to 200 kDa, polydisperse material and a major and minor band of 19 to 20 kDa, Zymolyase extracts contained (i) three components of less than or equal to 40 kDa, one of which may correspond to the major beta ME band; (ii) four bands within the HMM region which may correspond to previously reported bands; and (iii) one band of 100 to 120 kDa. After longer exposures, additional midrange bands were detected in the zymolyase extract. In extracts treated with endo-beta-N-acetylglucosaminidase H, the HMM polydisperse material increased in mobility although retaining sufficient radiolabel for detection. Western immunoblot analysis of extracts with germ tube-specific antiserum and a germ tube-specific monoclonal antibody and concanavalin A showed that not all components contained detectable phosphate, not all glycoproteins contained detectable phosphate, and at least one 19- to 20-kDa protein may be phosphorylated in the absence of carbohydrate.

Interferon induction in rabbits after intraduodenal administration of a phosphorylated glucomannan-protein fraction of the cell wall of Candida albicans

The aim of this work was to demonstrate whether a glucomannan protein fraction (GMP) of Candida albicans cell wall could induce interferon after intraduodenal administration in normal rabbits and rabbits immunized against C. albicans. For this purpose we collected simultaneously plasma and abdominal lymph for 10 h after the administration of the inducer. We observed a peak of antiviral activity in the lymph 4 h after intraduodenal administration of 20 mg GMP dissolved in saline to 6 normal rabbits. Immunized rabbits (anti-GMP titres greater than 1024) responded earlier (peak after 2 h) and more intensely; analysis of the values of the areas under the curve indicated that the IFN response in the lymph of immunized rabbits was significantly higher (P less than 0.0025) than in normal rabbits. Antiviral activity was absent in plasma in all cases. Preliminary characterization of the IFN activity has shown it to be trypsin-sensitive, acid and heat stable, and species-specific.

Influence of Surrounding Media on Preservation of Cell Wall Ultrastructure of <italic>Candida albicans</italic> Revealed by Low Temperature Scanning Electron Microscopy

Influence of surrounding media on the surface structures of the cell wall of Candida albicans was discussed with respect to the preservation of ultrastructure during the specimen preparation for scanning electron microscopy. The fibrillar structure of the cell surface was distinctly identified by the rapid-freezing technique. It was difficult, however, to observe this structure by the conventional specimen preparation technique. The reason for the difference between these two preparation techniques was studied using a low temperature SEM. Through investigating the influence of each step of the conventional technique on the fibrillar structure, it was found that the fibrils were drastically deformed and disappeared during the dehydration step in ethanol above 80% in concentration. In order to study which physicochemical properties participated in this disappearance phenomenon, yeast cells were treated with various media: solutions in different pH ranges and at different salt concentrations, ionic solutions, surfactants, formamide, dimethyl sulfoxide, acetone and Fehling's solution. As a result, the fibrillar structure was found well preserved when the medium had an affinity for the constituent molecules of the fibrils. When without affinity, the fibrils suffered a remarkable deformation. The mechanism of this deformation is discussed in terms of molecular interaction of solute and solvent.

Lymphoproliferative and cytotoxic responses of human peripheral blood mononuclear cells to mannoprotein constituents of Candida albicans

Two major proteoglycan constituents (designated F1 and F2) of the cell wall of Candida albicans were separated by ion-exchange chromatography from a crude carbohydrate-rich extract (GMP), and investigated for their chemical and molecular composition, antigenicity and immunomodulatory properties in cultures of human peripheral blood mononuclear cells (PBMC). Both fractions consisted predominantly of Periodic acid-Schiff (PAS) and concanavalin A (Con A)-reactive material consisting of greater than 90% mannose, 3-5% protein and small amounts of phosphorus; each was recognized by an anti-Candida rabbit serum as well as by a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope present on the fungal cell surface. When F1 and F2 were subjected to SDS-PAGE, transblotted and stained with enzyme-conjugated mAb AF1 or Con A, most of the antibody or lectin bound to high molecular mass (greater than 200 kDa) polydisperse material, some of which was present in F2 (as in the starting GMP extract) but absent in F1. This difference was also observed in PAS-stained gels of the two fractions. The F2, but not the F1, constituent was as active as the unfractionated GMP extract in inducing lymphoproliferation, production of the cytokines interleukin-2 and interferon-gamma, and generation of cytotoxicity against a natural-killer-sensitive target cell line (K562). These immunomodulatory properties were, like those possessed by GMP, protease-sensitive and heat-stable. Treatment of PMBC cultures with a modulatory anti-T-cell receptor antibody abolished the lymphoproliferation induced by GMP and F2 but not that induced by phytohaemagglutinin, showing that the mannoprotein materials of C. albicans acted through interaction with the antigen receptor complex.

Immunoreactivity of neoglycolipids constructed from oligomannosidic residues of the Candida albicans cell wall

To establish a model to study the immunoreactivity of oligosaccharidic structures from the Candida albicans cell wall, we attempted to construct neoglycolipids with these residues by using oligomannosides released after mild acid hydrolysis of the phosphopeptidomannans isolated from yeast forms. From a mixture of manno-oligosaccharides ranging from mannobiose to mannononaose, the structure of a quantitatively major component (mannotriose) was determined to be Man (beta 1-2) Man (beta 1-2) Man alpha by 1H nuclear magnetic resonance analysis. After coupling of the pool of oligosaccharides to a lipid (4-hexadecylaniline), the synthesized molecules were injected into mice and rats. Antibody responses were detected on enzyme-linked immunosorbent assay plates coated with either phosphopeptidomannans or neoglycolipids. The hybrid molecules exhibited both immunogenicity and antigenicity. The kinetics of antibody responses as well as immunofluorescence patterns observed on whole C. albicans cells strongly mimicked results from the immunization of animals with natural antigens. Construction of neoglycolipids could therefore provide an interesting approach to the study of specific oligosaccharides of C. albicans and their recognition by the host immune system.

Pathogenesis of candidiasis. Immunosuppression by cell wall mannan catabolites.

Candida albicans cell wall mannan polysaccharide has an ability to negatively influence cell-mediated immune function. We have attempted to identify the mechanism of this phenomenon by testing the modulatory effects of isolated mannan and the chemical catabolites of mannan on cell-mediated immune function in vitro. We have determined that mannan isolated by complexation with cetyltrimethylammonium bromide (CTAB) is more antigenic than mannan isolated by precipitation with copper and that CTAB mannan does not inhibit lymphoproliferation stimulated by another antigen. We have also determined that oligosaccharides of three sizes, derived by chemical catabolism of CTAB mannan, are not antigenic, but instead are immunoinhibitory. Immunoinhibition does not involve interference with the mitogenic activity of interleukin 2. A similar occurrence of oligosaccharides may be produced by catabolism of mannan in vivo as evidenced by the presence of oligosaccharides of similar size in cell-free supernatant fluids derived from mononuclear leukocytes incubated with tritiated mannan. We propose that catabolites of fungal mannan may contribute significantly to suppression of cell-mediated immunity in candidiasis.

Detection and characterization of two antigens specific for cell walls ofCandida albicans mycelial growth phase

Antiserum againstCandida albicans ATCC 10231 mycelium growth phase absorbed with yeast cells and intracellular material of mycelium cells showed a positive reaction with mycelium cells in immunofluorescence assay, whereas with yeast cells the reaction was negative. Mycelium and blastospore cell wall were extracted with dithiothreitol (DTTMy- and DTTB-extract). When DTTMy was separated in gel-electrophoresis, two glycoprotein bands of 87 and 67 Kd could be detected. In immunoblot these bands showed a strong reaction with mycelium cell wall-specific antiserum, but also a weak reaction with the blastospore antiserum. Whereas pronase treatment destroyed antigenicity, mannanase treatment did not. After enzymatic digestion with endoglycosidase H, four major enzymatic digestion products were found at 37, 35, 30, and 27 Kd when protein staining was performed. The digestion products at 37 and 35 Kd could be made visible through glycoprotein staining. Antibodies of yeast-phase-immunized animals reacted only with the 37 and 30 Kd bands, whereas the digestion products at 35 and 27 Kd were also detected by mycelium cell wall-specific antiserum. This proves the existence of two mycelium cell wall-specific antigens (35 Kd and 27 Kd) that can be isolated from the DTT-mycelium cell wall extract by endoglycosidase H treatment.

Cytotoxic effectors in human peripheral blood mononuclear cells induced by a mannoprotein complex of Candida albicans: A comparison with interleukin 2-activated killer cells

In this study, the major histocompatibility complex-unrestricted cytotoxic effectors elicited in human peripheral blood mononuclear cells (PBMC) by a mannoprotein (MP) component from the cell wall of the human indigenous microrganism Candida albicans have been compared with those obtained by stimulation with interleukin 2. (Interleukin 2-activated killer cells: LAK). It has been found that MP-induced lytic effectors were substantially similar to LAK in potency, target specificity, and type of precursor/effector cells. In both cases, natural killer (NK)-susceptible and NK-resistant targets as well as fresh tumor (glioma) cells were efficiently killed by a population of effectors showing a predominant CD3−, CD16+ phenotype. However, the precursors of MP-induced killers were highly sensitive to the lysosomotropic toxic drug l-leucine methyl ester (Leu-OME) whereas the generation of LAK cells was unaffected by this drug. The Leu-OME sensitivity of MP-induced cytotoxicity generation was not due to a nonspecific effect on antigen-presenting cells or inhibition of cell proliferation. In addition, the generation of MP-induced killer cells was totally abrogated by treatment with CD 16 antibodies and complement, whereas a minor but significant fraction of LAK precursors was not susceptible to the above treatment. These results indicate that a defined component(s) of the cell wall of C. albicans has some properties of biological response modifiers in cultures of human PBMC in vitro.

Cell wall of Candida albicans: its functions and its impact on the host.

The purpose of this chapter is to summarize and critically compare current ideas about the cell wall of Candida albicans, its functions in the microorganism, and its impact on the host. Candida albicans is an important human commensal that has received much attention as a model for studies of cell biology and microbial opportunism (164). This review deals mostly with the second aspect, as it is of great relevance for medical mycologists. Thus the role of cell wall components in morphogenesis, pathogencity, and immunity receives the focus here, although at times more basic questions concerning cell wall chemistry and organization of wall constituents are addressed, especially when the answers to these questions are pertinent to understanding the pathogenicity of the microorganism and the host response. Those who wish to know more about the basic aspects of cell biology and wall biosynthesis may consult a number of excellent reviews elsewhere (13,33,55,84,86,100,165,168,183,192,196,201,202,230). The growing interest in C. albicans and related pathogens has led to a dramatic increase in publications on Candida biopathology, and when writing this review selection was inevitably based on personal assessments and ideas.

Evidence for a glycosidic linkage between chitin and glucan in the cell wall of Candida albicans.

The alkali-insoluble glucan was isolated from regenerating spheroplasts and intact cells of Candida albicans. Sequential enzymic hydrolysis of this fraction by Zymolyase 100T and purified chitinase and subsequent gel filtration produced a fraction which was enriched in glycosaminoglycans. This fraction was analysed by partial acid hydrolysis, TLC and GLC-MS. The GLC-MS peaks identified included 2,3,4,6-tetra-O-methylglucitol acetate and 2,3,4-tri-O-methylglucitol acetate of beta-1,6-glucan and the 3,6-di-O-methyl-2-N-methylglucosaminitol acetate of chitin. In addition, 3-O-methyl-2-N-methylglucosaminitol acetate was identified, which indicated a branch point in chitin. These data provide evidence for a covalent linkage between chitin and beta-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.

Effect of Candida albicans cell wall components on the adhesion of the fungus to human and murine vaginal mucosa.

In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance. Fractionation of CSE yielded two fractions: FI and FII, of which only FI exhibited inhibitory activity. Chemical analysis of CSE and its two fractions revealed that CSE contains over 70% of proteins, most of which were found in the non-active fraction. In addition, 3% of amino-sugars were found in the FI active fraction. Lipids were also detected in the unfractionated CSE and in both fractions. Experiments to further characterize the component(s) in the CSE inhibiting the attachment of C. albicans are in progress in our laboratory.

Variable expression of a surface determinant during proliferation of Candida albicans.

The surface expression of an antigenic determinant that is present in the cell wall of Candida albicans was investigated with monoclonal antibody 24 (MAb24), an immunoglobulin M MAb. The proportion of the cell population that expressed the epitope under different growing conditions was determined by indirect immunofluorescence microscopy. More than 90% of stationary-phase yeast cells of strain B311 grown at 28 degrees C expressed the antigen. Less than 50% of yeast cells grown exponentially at 28 degrees C or either growing or stationary-phase yeast cells cultivated at 37 degrees C expressed the epitope. Germ tubes, which were induced at 37 degrees C from stationary-phase yeast cells grown at 28 degrees C, expressed the determinant on the parent yeast but not the hyphal portion of the germ tube. The change in antigen expression by stationary-phase cells grown at 28 degrees C, when they resumed growth by bud formation, suggested that antigen expression was lost by cells in the inoculum prior to the first cell division. By using the same assay, strong positive reactions were observed in stationary-phase cultures of other isolates of C. albicans, C. guilliermondii, C. stellatoidea, and C. tropicalis, but not with isolates of C. krusei, C. parapsilosis, or Torulopsis glabrata. The identification of the antigenic determinant as a carbohydrate was based on three observations: (i) interaction with a mannan preparation from the same organism, (ii) sensitivity of the antigen to periodate but not proteases, and (iii) coincidence of the migration of antigen during electrophoresis with material which stained intensely with carbohydrate but not with protein reagents. These observations suggest that the expression of the antigenic determinant of MAb24 is dependent on the growth conditions, growth state, and morphology of the cell and that the topography of the cell surface is dynamic.

The effect of RU 41740 on the in vitro maturation of human B-cells

We have tested the effect of a Klebsiella pneumoniae extract, RU 41740, and its lipopolysaccharidic fraction (LPS-LAP) on the in vitro activation of human B-cells. Two models have been used: the polyclonal activation induced by pokeweed mitogen and the specific antibody production to mannan, a polysacharide extracted from the cell wall of Candida albicans. We have shown that RU 41740, and especially its lipopolysacharidic fraction, significantly increases the production of immunoglobulins and specific antibodies. This effect is mediated by T-lymphocytes, since preincubation of isolated T-cells, but not of non-T-cells, resulted in the same effect. Together with the known enhancing effect of LPS-LAP on T-cell proliferation, these data strongly suggest that LPS-LAP increases the production of B-cell-activating lymphokines by T-cells.

Enhancement of non-Candida antibody responses by Candida albicans cell wall glycoprotein.

Two cell wall glycoprotein extracts from Candida albicans (glycoprotein [GP] and peptidoglucomannan [PGM]) were tested for their influence on antibody responses to type III pneumococcal polysaccharide and sheep erythrocytes. GP was isolated from lipid-extracted cell walls with ethylenediamine, whereas PGM was extracted with dilute sodium hydroxide. Both glycoproteins increased the number of antibody-producing plaque-forming cells in the spleens of mice immunized with type III polysaccharide or sheep erythrocytes, although PGM appeared to be about 10 times more effective. PGM could be administered up to 3 days prior to immunization with sheep erythrocytes to elicit enhancement; it did not have to be administered by the same route as the immunogen to cause significant enhancement. Enhancement did not appear to be the result of a direct mitogenic effect of GP and PGM on lymphocytes, nor did these glycoproteins appear to stimulate the production of B-cell growth factors or interleukin 2.

Restriction of the in vitro anti-mannan antibody response by HLA-DQ molecules

In humans, the in vitro antibody response directed towards mannan, a polysaccharide extracted from the cell wall of Candida albicans, has been previously shown to be dependent on the presence of T lymphocytes and monocytes. Evidence is now given for the existence of a genetic restriction governing this response since antibody production is achieved provided that monocytes and T lymphocytes on one side and monocytes and B lymphocytes on the other side are of the same origin. In order to delineate the restriction element governing these interactions, blocking experiments have been designed using well-defined monoclonal antibody, anti-HLA class II molecules. The results clearly indicate that the restriction element belongs to the HLA-DQ molecular series, as shown in T-cell proliferation and antibody production assays in the presence of either T cells or T-cell supernatants. Incubation of isolated cell populations (T, B lymphocytes and monocytes) with the monoclonal antibody have indicated that DQ determinants are involved in the mannan presentation by monocytes to T and B cells. The HLA-DQ mediated restriction of the in vitro immune response to mannan has been observed in all the subjects tested, suggesting that mannan epitopes are preferentially, or even only, recognized in association with an unique group of HLA-class II molecules, namely HLA-DQ.

Use of a method of lipid extraction from Candida albicans cell walls blastospores. Ultrastructural implications concerning their localization and parietal microfibril organization

A chemical method for lipid extraction has been applied to Candida albicans blastospores previously to their examination by transmission electron microscopy. The results led to the concept of a superficial location of lipids bounded to the peptidopolysaccharidic matrix of the cell wall. This lipid extraction also allowed us to describe cell wall microfibrillar structures. Two types of microfibrills have been particularly identified: microfibrils of approximately 50 A in diameter, involved in a network of the cell wall intermediate layers and supposed to correspond to beta 1-3 glucans; fibrillar structures of 120 A in diameter, similar to chitin microfibrils, observed in the bud scar septum.

Enzymatic release of germ tube-specific antigens from cell walls of Candida albicans

The differences between blastospores and germ tubes of Candida albicans, as previously shown by immunofluorescence, were further studied by comparing digests of cell walls of both growth forms. Organisms were surface labeled with 125I, and cell walls were digested enzymatically. When Zymolase digests were treated with polyclonal, polyspecific antiserum to C. albicans 441B, which stains only germ tubes in immunofluorescence assays, components of molecular weights 200,000 and 155,000 were immunoprecipitated from digests of germ tubes of strain B311, but nothing was recovered from blastospores. Whereas the 200,000-molecular-weight component was found in the three strains tested, the 155,000-molecular-weight component was found only in strain B311. When Zymolase digests were treated with unadsorbed antiserum, which stains both blastospores and germ tubes in immunofluorescence assays, an additional component was precipitated from digests of both growth forms with a molecular weight greater than the 200,000-molecular-weight marker. All three antigens were mannoproteins, as was shown by their abilities to bind concanavalin A and to be labeled by 125I. Also, all antigens were located on the cell surface, as was shown by the following criteria: adsorption of antisera with live organisms removed antibody to these components, and antibody eluted from surfaces of whole organisms precipitated all components. Components common to both growth forms, as well as germ tube-specific components, were detected in trypsin and chymotrypsin digests, but their molecular weights differed from those of Zymolase digests. Thus, germ tube-specific surface determinants as well as determinants common to both growth forms were detected on enzymatically released cell wall components.