A monoclonal antibody to a cell wall component of Candida albicans.

A heterologous fusion between mouse myeloma cells and rat lymphocytes resulted in the isolation of a rat immunoglobulin M monoclonal antibody with both agglutinating and precipitating activity. Indirect immunofluorescence and direct agglutination tests showed that the corresponding antigen was present in the cell wall of the three Candida species considered to be the most pathogenic, C. albicans, C. tropicalis, and C. glabrata, and also in the cell wall of C. guilliermondii. The antigen appeared to be predominantly polysaccharide in nature. Precipitation by counterimmunoelectrophoresis suggested that the epitope is shared by at least two separate molecules with different electrophoretic mobilities. Presence of this epitope varied from strain to strain within a given species and may be related to the morphological stage in the cell cycle. Antigen was shown to be present in the cytoplasm, in the periplasmic space, and at the cell surface of C. albicans. Indirect immunofluorescence also suggested that antigen is excreted from the cell.     

Enhanced antibody responses induced by Candida albicans in mice.

Candida albicans may immunopotentiate antibody responses in mice to antigens unrelated to the fungus. This effect occurred best with cell-associated, rather than soluble, antigens. When dead yeasts, cell walls, or a water-soluble candidal polysaccharide were used, immunopotentiation was most dramatic when the antigen and fungal materials were given concomitantly via an intraperitoneal injection. However, mice infected with viable yeasts several days before antigen administration also developed heightened responses to the antigen. The mechanism of the C. albicans-induced adjuvanticity was not defined, but the effect seemed to correlate with induction of inflammation. The presence of C. albicans or other inflammatory agents in the peritoneal cavity caused a more rapid uptake of particulate antigen by the liver. The relationship between this event and immunopotentiation is not known. These studies demonstrate that C. albicans may have profound effects on host immune responses. Because immunological aberrations are commonly found in patients with candidiasis it may be important to determine whether some of these aberrations result from, rather than precede candidiasis.     

Immunoregulation in experimental murine candidiasis: specific suppression induced by Candida albicans cell wall glycoprotein.

Immune regulation in candidiasis is inferred from studies of both human and animal infection, with a suppressive role suggested for cell wall polysaccharide. To study the immunosuppressive potential of Candida albicans in a murine model, whole blastoconidia or purified cell wall components of C. albicans were tested for their effects on the development of acquired immune responses by superimposing a pretreatment regimen upon an established immunization protocol. CBA/J or BALB/cByJ mice were pretreated twice intravenously with 100 micrograms of mannan (MAN), 100 or 200 micrograms of glycoprotein (GP), or 5 X 10(7) heat-killed C. albicans blastoconidia, followed 1 week later by an immunization protocol of two cutaneous inoculations of viable C. albicans blastoconidia given 2 weeks apart. Delayed hypersensitivity (DTH) to GP or to a membrane-derived antigen, B-HEX, was tested 7 days after the second inoculation, and lymphocyte stimulation was tested with mitogens and Candida antigens after 12 days. To assess protection, mice were challenged intravenously with viable C. albicans blastoconidia 14 days after the second cutaneous inoculation and sacrificed 28 days later for quantitative culture of kidneys and brains. Sera were obtained for enzyme-linked immunosorbent assays at selected intervals. Pretreatment with GP resulted in specific in vivo suppression of DTH to GP but not to B-HEX antigen and specific in vitro suppression of lymphocyte stimulation to GP but not to other Candida antigens or mitogens. MAN and heat-killed C. albicans blastoconidia had no such effects. GP pretreatment also diminished the protective effect of immunization against challenge, demonstrable in the brain, while not altering significantly the production of antibody in response to infection. Contrary to clinical evidence, MAN was not immunosuppressive in this model, and in fact, the immunosuppressive potential of GP, which is composed largely of MAN, was found to be dependent upon the presence of its heat-labile protein moiety.     

A monoclonal antibody directed against a Candida albicans cell wall mannoprotein exerts three anti-C. albicans activities

Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.     

Identification of two germ-tube-specific cell wall antigens of Candida albicans.

Outer cell wall layers of intact yeast- and mycelial-phase Candida albicans B311 were extracted with dithiothreitol. Antisera against mycelial-phase organisms were absorbed with yeast-phase organisms or yeast-phase extract and used to stain Western blots of sodium dodecyl sulfate-polyacrylamide gels loaded with yeast- and mycelial-phase extracts. Autoradiography of gels loaded with extracts from organisms surface labeled with 125I was used to detect surface antigens containing proteins. Antigen bands of interest identified in Western blots were cut from the blots and used to immunize rabbits. Two antigens were identified in the mycelial-phase extract which were not present in the yeast-phase extract. The first was a 19-kilodalton protein that was present in the cell walls of germ tubes but was not expressed on their surfaces. The second was a polysaccharide-rich high-molecular-weight antigen which was expressed on the surface of the germ tube. Treatment of mycelial-phase extract with protease and endo-beta-N-acetylglucosaminidase H demonstrated that this antigen was composed of polysaccharides linked through di-N-acetylchitobiose groups to proteins.     

Characterization of Candida albicans cell wall antigens with monoclonal antibodies.

The antigenic composition of Candida albicans is very complex. In order to study the antigenic relationship between blastoconidia and germ tubes of C. albicans, we produced several monoclonal antibodies and analyzed their reactivity against cell wall antigens either in intact cells or in cells treated with dithiothreitol. Overall, four types of reactivity were found. Monoclonal antibodies 3D9 and 15C9 stained the germ tubes only when tested by indirect immunofluorescence. However, they showed a different reactivity by immunoblotting. Monoclonal antibody 3D9 reacted with antigens with molecular masses of > 200 and 180 kDa specifically expressed in the germ tube. Monoclonal antibody 15C9 reacted with antigens of 87, 50, and 34 kDa present in the germ tube extract and with antigens of 92, 50, 34, and 32 kDa present in the blastoconidium extract. The reactivity of blastoconidia treated for different times with dithiothreitol with these monoclonal antibodies was also studied by enzyme-linked immunosorbent assay. The reactivity of monoclonal antibody 3D9 did not significantly change during the cell wall extraction. However, the reactivity of monoclonal antibody 15C9 was increased for blastoconidia extracted for 60 min and decreased markedly for blastocondia extracted for 120 min. Monoclonal antibody G3B was nonreactive by indirect immunofluoresence but reacted with antigens of 47 and 38 kDa present in the germ tube extract and with an antigen of 47 kDa present in the blastoconidium extract. Monoclonal antibody B9E stained both morphological phases by indirect immunofluorescence. By immunoblotting, it reacted with antigens of > 70 kDa present in the germ tube extract and with antigens of > 63, 56, 47, and 38 kDa present in the blastoconidium extract. Based on the results presented in this study, four types of antigens are described. Type I antigens are expressed on the outermost layers of the germ tube cell wall only. Type II antigens are expressed both on the germ tube cell wall surface and within the blastoconidium cell wall. Type III antigens are found within the cell wall of both blastoconidia and germ tubes. Type IV antigens are expressed on both the blastoconidium and germ tube surface. Two types more can be hypothesized for antigens expressed on the blastoconidium cell surface and within the germ tube cell wall (type V) and for those expressed on the blastoconidium surface only (type VI).     

Candida albicans cell wall antigens for serological diagnosis of candidemia.

Serological tests for diagnosis of disseminated fungal infections in the immunocompromised host are used with varying results. In the present study, the relative ability of antibodies to specifically recognize Candida albicans cell wall components was evaluated in order to find antigenic markers for serological diagnosis of candidemia. Native C. albicans cell wall fragments (CW), periodate- (CWIO4) and proteinase-K- (CWP) treated CW, a mildly extracted phosphopeptidomannan (PPM), and beta(1-3)(1-6)-glucan were used as antigens in ELISA with sera from rabbits immunized with C. albicans (n = 10), patients with culture proven candidemia (n = 8) and healthy individuals (n = 8). The antibody response in rabbits consisted predominantly of anti-PPM antibodies, a finding that was substantiated by inhibition-ELISA. Consistently, periodate treatment (CW104) destroyed a major proportion of the antigenic epitopes. Low rabbit antibody levels were found against glucan, the major Candida cell wall component. These results supported the conclusion that glucan is localized mainly in the inner part of the C. albicans cell wall. In contrast to rabbits' serum IgG antibody response against PPM, which was at least tenfold higher than that raised against CW, patients with candidemia had similar IgG antibody levels against both antigens. These levels were significantly higher than those seen in healthy controls (CW, P = 0.0005 and PPM, P < 0.0001). Although the human anti-glucan and anti-CWIO4 IgG antibody levels were low overall, they were nonetheless significantly increased in the patient group (P = 0.0159 for antiglucan and P = 0.0491 for anti-CWIO4). In addition, a correlation was noticed between levels of these antibodies. No significant differences were found between patients and controls for IgM antibodies when CW, CWIO4, PPM and Glu were used as antigens. In conclusion, IgG antibodies to PPM and native cell wall fragments (CW) were highly discriminatory for recognition of candidemia and these antigens are thus promising candidates for use in serodiagnosis.     

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.     

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.     

Signaling the glycoshield: maintenance of the Candida albicans cell wall

In fungi, the cell wall is a scaffold, an armor and an environmental gate. Sugar polymers including protein-O- or N-linked glycosyl chains or polysaccharides such as glucan or chitin are essential components to maintain cell wall functions. We describe mechanisms in the human fungal pathogen Candida albicans, by which the integrity of glycostructures are sensed and regulated. The results stress the importance of membrane sensors and MAP kinase pathways in the maintenance of cell wall structure and function.     

Enhancement of MHC class II antigen expression by exposure to Candida albicans.

In oral infections with the yeast Candida albicans, the expression of MHC class II antigens on keratinocytes has been reported to be enhanced. In the present experiments, exposure to C. albicans or its products in vitro was found to increase the expression of class II MHC antigens on thioglycollate-induced mouse macrophages, and on LK cells (an antigen-presenting cell line). The implications of this finding for the understanding of immunoregulation and susceptibility to C. albicans infection are discussed.     

Detection by immunofluorescent anti-idiotypic antibodies of yeast killer toxin cell wall receptors of Candida albicans

Yeast killer toxin cell wall receptors of Candida albicans were observed by indirect immunofluorescence using an affinity purified rabbit anti-idiotypic antiserum. The antiserum had been raised against a monoclonal antibody neutralizing the in vitro activity of a killer toxin produced by a selected strain of Hansenula anomala UCSC 25F. This simple procedure permitted the location of killer toxin cell wall receptors in various morphological phases of the yeast cells. The use of the indirect immunofluorescence technique with anti-idiotypic antibodies may have potential value in determining the occurrence of killer toxin receptors in other microbial systems.     

Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans.

Cell surface hydrophobicity (CSH) of blastoconidia and blastoconidia bearing germ tubes of Candida albicans ATCC 26555 was monitored by assessing attachment of polystyrene microspheres to the cell surface, and we found that mature hyphae were significantly hydrophobic. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) or proteases abolished or significantly reduced attachment of latex beads to hyphae. This effect paralleled an obvious reduction in CSH of the entire cell population, as measured by an aqueous-hydrocarbon biphasic partitioning assay. Analysis of the cell wall material released by Zymolyase and adsorbed on polystyrene microspheres indicated that germ tube-specific cell wall proteins and mannoproteins with apparent molecular masses of 20 to 67 kDa may be responsible for the hydrophobicity of hyphae. Zymolyase released from blastoconidia cell walls a different set of proteins and mannoproteins that were able to adsorb to polystyrene microbeads. Such molecular species might in turn be responsible for the CSH exhibited by blastoconidium populations as determined by the biphasic partitioning assay, although these probably hydrophobic components can be masked on the surface of blastoconidia, as the latter had no or very few latex microspheres attached to their surfaces. Treatment of cells of both C. albicans morphologies with 2-mercaptoethanol released qualitatively distinct species of polystyrene-adsorbed proteins and mannoproteins from yeast and mycelial cells. These observations suggested that hydrophobic proteins and mannoproteins that could be associated with CSH are bound to the cell wall structure through diverse types of linkages.     

A comparison of specific IgG antibody levels to the cell wall mannan of Candida albicans in normal individuals and in patients with primary antibody deficiency.

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure specific IgG antibody to the polysaccharide, cell wall mannan of Candida albicans (mannan). The results were expressed as arbitrary units/ml, with an inter- and intra-assay coefficient of variation of 7-11%. In establishing normal ranges we found that specific IgG to the mannan increased with age, with 18% of healthy children aged 3-10, 48% of healthy children aged 11-19 and 76% of an adult donor population having specific IgG antibody to mannan (greater than 30 U/ml). We have compared these normal ranges, with a group of patients with primary antibody deficiency (PAD). None of the 23 patients with PAD, which included common variable immunodeficiency, IgG subclass deficiency, and selective IgA deficiency, had titres greater than 30 U/ml. The patients with PAD had significantly lower levels of specific IgG anti-mannan antibody (median 9 U/ml) compared to healthy children aged 11-19 (median 26 U/ml) or adults (median 58 U/ml) (p = less than 0.001) but not children aged 3-10, (median 1 U/ml) (p = 0.08).     

Analysis of cell wall extracts of Candida albicans by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques.

Cell walls of intact yeast- and mycelial-phase Candida albicans B311 were extracted with different compounds: dithiothreitol, dithiothreitol with protease, dithiothreitol with lyticase, and dithiothreitol with protease followed by beta-glucuronidase with chitinase. Extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques. Dithiothreitol extracts contained the most satisfactory array of components for study. Analysis of these extracts demonstrated that the outer cell wall layers of Candida blastoconidia and germ tubes contained a complex array of polysaccharides, glycoproteins, and proteins. The proteins contributed to a latticework stabilized by covalent bonds that was important in determining the porosity of the outer cell wall layers. When equivalent weights were analyzed, mycelial-phase extract contained a more varied array of proteins than did yeast-phase extract. Only a portion of proteins in mycelial-phase extract elicited antibody responses in hyperimmunized rabbits or infected humans. A polysaccharide-rich, high-molecular-weight component (migrating at a position that would correspond to proteins having molecular weights of 235,000 to 250,000) and a protein component (molecular weight, 19,000) were readily demonstrable in the mycelial-phase extract but could not be identified in the yeast-phase extract.     

Cell wall of Candida albicans and host response

Modulation in chemistry and organization of cell wall macromolecules play decisive roles in the morphogenic processes and virulence of Candida albicans. Cell wall components also have a diversified range of effects on the host's immune system, including immunopotentiating or immunodepressing activities. Mannan, mannan-protein, and glucan fractions have been especially studied in this context. In in vitro cultured human peripheral blood mononuclear cells, a mannan-protein fraction (GMP) from the cell wall of the yeast form acted as a strong antigenic activator by stimulating lymphokine production and lymphocyte proliferation. Cytolytic effectors active against several tumor targets were also generated. In the mouse, GMP was a strong inducer of natural killer lymphocytes. Other cell wall components, mostly the insoluble beta-glucan, modulated the activity of macrophages and monocyte precursors. Some of the immunomodulating properties of artificially extracted components were shared, even with greater potency, by antigens which were released from C. albicans during its growth and hyphal morphogenesis. Altogether, the range of the immunoresponses elicited and the intensity of the observed effects are such as to individuate in this human indigenous fungus a microorganism capable of profoundly affecting the host's immune system.     

Proliferation of human peripheral blood mononuclear cells induced by Candida albicans and its cell wall fractions

Glutaraldehyde-inactivated cells and cell-wall fractions of Candida albicans were studied for their capacity to induce or inhibit the in-vitro proliferation of human peripheral blood mononuclear cells (PBMC), as measured by 3H-thymidine incorporation. Both the intact cells (CA) and a phosphorylated gluco-mannan-protein complex of the cell wall (GMP), in microgram doses, were strong inducers of PBMC proliferation, with a peak of activity at 6-9 days of culture and varying with the PBMC donor. A significant but much lower proliferation was observed on exposure of PBMC to a low-protein (less than 3% by weight) mannan component (M) of the cell wall. Both a hot-alkali extracted mannan-protein complex (M-alk), comparable to GMP in crude chemical composition, and an alkali-insoluble cell-wall glucan (GG) were inactive. None of the Candida fractions induced a lymphoproliferation of umbilical cord blood cells and all fractions, except GG, were equally effective in binding human anti-Candida antibodies as shown by a sensitive ELISA-inhibition assay. Moreover, a monoclonal antibody against the class II determinant of the HLA complex inhibited PBMC proliferation irrespective of the Candida antigen used. Taken together, the data shows that in inducing lymphoproliferation, Candida fractions act as specific antigens rather than as non-specific mitogens. Use of intact Candida cells and chemically-defined cell-wall components appears preferable to use of undefined antigenic mixtures as stimulators of PBMC proliferation.     

Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis.

One strategy for improving resistance to opportunistic pathogens is to determine host cellular responses during the invasion process and upregulate those responses that are relevant to host defense mechanisms. Within this context, we have shown previously that invasion of endothelial cells by Candida albicans in vitro causes increased production of prostaglandins. As a prerequisite for modulating endothelial cell prostaglandin production, we now characterize the mechanisms through which this process occurs. Endothelial cell invasion by C. albicans appeared to stimulate the conversion of arachidonic acid into prostaglandins by upregulating the synthesis of endothelial cell cyclooxygenase and increasing the activity of the endothelial cell phospholipase. The enhanced activities of these two enzymes were independent of calphostin C-sensitive protein kinase C and resulted in the increased production and extracellular secretion of prostaglandin I2 (PGI2), PGF2 alpha, and PGE2. The secretion of these prostaglandins had no effect on the amount of endothelial cell injury induced by C. albicans. The role of the increased prostaglandin secretion by endothelial cells is likely related to modulation of the leukocyte response at the candida-leukocyte-endothelial cell interface.     

Specific and common antigenic determinants of Candida albicans isolates detected by monoclonal antibody.

We isolated a hybridoma cell line which produced monoclonal antibody to one determinant of an exoantigen of Candida albicans. The immunoglobulin G antibody product was characterized by using a Western blot technique and was used for a serological analysis of numerous homologous and heterologous yeast isolates. Based on specific immunologic determinants, C. albicans strains were identified and clustered into five groups. The monoclonal antibodies were effective reagents for identifying and serotyping our C. albicans isolates; they have potential application in the epidemiology of yeast infections.