Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans.

Hydrophobic yeast cells of Candida albicans are more virulent than hydrophilic yeast cells in mice. Results of experiments performed in vitro suggest that surface hydrophobicity contributes to virulence in multiple ways. Before definitive studies in vivo concerning the contribution of fungal surface hydrophobicity to pathogenesis can be performed, biochemical, physiological, and immunochemical characterization of the macromolecules responsible for surface hydrophobicity must be accomplished. This report describes our initial progress toward this goal. When hydrophobic and hydrophilic yeast cells of C. albicans were exposed to various enzymes, only proteases caused any change in surface hydrophobicity. Hydrophobic cell surfaces were sensitive to trypsin, chymotrypsin, pronase E, and pepsin. This indicates that surface hydrophobicity is due to protein. Papain, however, had no significant effect. The hydrophobicity of hydrophilic cells was altered only by papain. The proteins responsible for surface hydrophobicity could be removed by exposure to lyticase, a beta 1-3 glucanase, for 30 to 60 min. When 60-min lyticase digests of hydrophobic and hydrophilic cell walls were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12.5% resolving gel, each protein population contained a single unique protein that was not evident in the other protein population. However, when the cell wall surface proteins of hydrophobic and hydrophilic cells were first labeled with 125I and then removed by lyticase and analyzed by SDS-PAGE, at least four low-molecular-mass (less than 65 kilodaltons) proteins associated with hydrophobic cells were either absent or much less abundant in the hydrophilic cell digests. This result was seen for both C. albicans strains that we tested. When late-exponential-phase hydrophilic cells were treated with tunicamycin, high levels of surface hydrophobicity were obtained by stationary phase. These results indicate that the surface hydrophobicity of C. albicans reflects changes in external surface protein exposure and that protein mannosylation may influence exposure of hydrophobic surface proteins.     

Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants.

Strains of Candida albicans, selected on the basis of their reduced agglutination with a polyclonal anti-Candida antiserum, were tested for their adherence to human buccal epithelial cells (BEC). Of four strains, one (A9V2) had reduced binding to BEC in vitro. Adherence of wild type (wt) yeast cells (A9), as measured by the percentage of BEC with adhering Candida cells, was 73.4% +/- 3.8% compared with 49.3% +/- 3.1% for A9V2 (P less than 0.01). From yeast cells of A9 and A9V2, whole-cell extracts and dithiothreitol-, Zymolyase-, or beta-mercaptoethanol-solubilized cell extracts were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). From dithiothreitol-solubilized cell extracts, proteins with molecular masses of 55 to 60, 80 to 84, 115, and 165 kDa were observed from wt (A9) cells but were highly reduced in amount or absent from A9V2 cells. Western blot profiles of Zymolyase-solubilized extracts from both A9 and A9V2 were similar in appearance, while 55-, 80- to 84-, 115-, and 165-kDa proteins were observed only in A9 cells extracted with beta-mercaptoethanol. Strain A9V4, also selected by reduced agglutination but which adhered as well as strain A9, lacked the 80- to 84-kDa and 115-kDa proteins but otherwise was similar to strain A9. These results indicate that the 55- to 60- and 165-kDa proteins may be related to an adhesin function in C. albicans. The differences observed in the protein profiles of the wt adhering strain and its derived nonadhering mutant are similar to those described for another matched pair of C. albicans strains.     

Isolation and characterization of an avirulent Candida albicans yeast monomorphic mutant.

Mutagenesis of Candida albicans strain ATCC 26555 with N-methyl-nitro-N-nitrosoguanidine followed by plating on solid yeast nitrogen base-N-acetylglucosamine medium at 37 degrees C yielded colony morphology variants that were characterized as forming smooth colonies, in contrast to the rough colonies formed by the parental strain. One yeast monomorphic mutant, CAL4, was studied in detail. Strain CAL4 is defective in filamentous growth, unable to form hyphae or pseudohyphae in vivo and in vitro. These filamentous structures are not elicited by commonly used external stimuli such as serum. The mutant had no obvious alterations in its mannan, glucan or chitin content. The total quantity of non-covalently linked wall proteins was reduced in the mutant strain, but the electrophoretic pattern shown by these proteins was identical to that of proteins from the parental strain. CAL4 showed major differences from the parental strain in its formation of covalently linked wall proteins. An important aspect of these differences lay in the practical absence of proteins recognized by two monoclonal antibodies, 1B12 and 3H8, which are considered valuable tools in the diagnosis of candidiasis in part because they normally react strongly with all strains. The C. albicans mutant, blocked in yeast-mycelium transition, was avirulent in a mouse model, although it was able to grow in animal tissues.     

Physiological and morphological characterization of tert -butylhydroperoxide tolerant Candida albicans mutants

tert -Butylhydroperoxide (t BOOH) tolerant Candida albicans mutants developed from clinical isolates were characterized with increased tolerance of the oxidative stress generating agents t BOOH and H2O2, continuous induction of the antioxidative defence system, reduced pseudohypha and hypha-forming capabilities, decreased phospholipase secretion and delayed growth in Sabouraud dextrose agar and broth media. Changes in antimycotic (fluconazole, voriconazole, amphotericin B, 5-fluorocytosine) tolerances as well as in total and cytochrome c-dependent respirations showed versatile patterns, meanwhile the intensified alternative oxidase-dependent respiration of the mutants indicated that this respiratory pathway was an important element of the antioxidative defence in general. Because the phenotypes of increased oxidative stress tolerance and reduced virulence attribute production always emerged concomitantly in t BOOH-tolerant mutants the natural selection of C. albicans strains more tolerant of oxidative stress is unlikely. Not surprisingly, a screening study failed to detect any C. albicans strains with increased oxidative stress tolerance among 46 randomly selected clinical isolates.     

Isolation and partial characterization of Hsp90 from Candida albicans

Hsp90 is a stress-induced protein involved in many cellular processes including the regulation of signal transduction and steroid hormone response pathways in higher eukaryotic cells. Candida albicans hsp90 has a mass of 82 -Da and has previously been implicated as a virulence factor. A 47-kDa C-terminal fragment of Candida hsp90 is a target for an immune response to C. albicans infections. A C. albicans hsp90 specific polyclonal antibody was developed against a synthetic peptide containing a previously defined epitope of the 47-kDa fragment. This antibody was used to investigate the cellular localization and induction of hsp90 in the fungus. By means of cell surface protein extraction, hsp90 is shown to be localized on the cell surface as well as in the cytoplasm. On the cell surface, it appears only as an 82-kDa protein. In the cytoplasm, anti-hsp90 detected the 82-kDa protein as well as 72-kDa and 47-kDa bands on SDS-PAGE gels. The cytoplasmic protein bands were heat inducible and appeared to be estrogen induced as well, suggesting that C. albicans modulates hsp90 expression in response to environmental changes. Since the 82-kDa protein is also found on the surface of the cells, hsp90 may be directly involved in sensing environmental changes. It may also be important for recognition of its host or elements of the host immune system and antibody responses to the molecule and may therefore be useful for diagnostic or prognostic evaluation.     

Characterization of cerulenin-resistant mutants of Candida albicans.

Cerulenin, an inhibitor of fatty acid biosynthesis, has been used to study the role of the plasma membrane in germination of Candida albicans. To further elucidate this association, spontaneous, cerulenin-resistant mutants of C. albicans were isolated. Two of the mutants, 4918-2 and 4918-10, were compared biochemically with wild-type cells (4918). All strains grew equally well at 37 degrees C and synthesized fatty acids at comparable rates in the absence of the drug. In the presence of cerulenin, wild-type cells did not proceed through a logarithmic growth stage and exhibited a significantly impaired ability to incorporate [3H]acetate into newly synthesized lipid material. All strains were examined ultrastructurally. Alterations were observed in the membranous structures of cerulenin-treated wild-type cells. Such changes were not observed in cerulenin-treated mutant strains. Further examination of mutant strains revealed differences in cell wall protein and polysaccharide compositions when compared with those of wild-type cells. These apparent alterations in cell surface components may be correlated with the reduced abilities of mutant strains to adhere, in vitro, to mammalian cells.     

Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures.     

Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata.

The effect of cultural conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata was tested. C. albicans cells grown at room temperature were more hydrophobic than cells grown at 37 degrees C. No consistent pattern was observed with C. glabrata. Relative hydrophobicity was found to vary with the growth phase and growth medium for both species. The implications for pathogenesis studies are discussed.     

Isolation and biochemical characterization of the iC3b receptor of Candida albicans.

In an effort to identify the protein structure on Candida albicans, pseudohyphal forms which had been shown earlier to bind human iC3b, a protein of about 42 kDa (p42), were obtained from lysates of pseudohyphal forms by absorption with C3(H2O)-Sepharose. An antiserum raised in rabbits against this protein effectively inhibited adherence of sheep erythrocytes carrying iC3b (EAC3bi) to pseudohyphal forms. p42 cross-reacted with OKM-1, a monoclonal antibody directed against the human complement receptor type 3 (CR3, CD11b). This protein, p42, was designated p42-CR3. The antiserum against p42-CR3 was used for further purification of lysates by affinity chromatography. Three proteins of 66, 55, and 42 kDa were isolated. All were recognized by OKM-1 in immunoblots (p66-, p55-, and p42-CR3). The different proteins were separated and treated with neuraminidase and endoglycosidase F. Almost complete deglycosylation of the p66-CR3 protein was obtained after treatment with neuraminidase, indicating a high degree of glycosylation. Neuraminidase also had an effect on p55-CR3, but not on p42-CR3. Endoglycosidase F did not alter any of the three proteins. In ligand blots, p42-CR3 bound C3(H2O), C3b, and iC3b but not C3d; p55-CR3 clearly reacted with C3(H2O) and weakly reacted with C3b and iC3b. p66-CR3 never showed reactivity. It is suggested that p55 and p66 represent glycosylated forms of p42-CR3. Although C. albicans CR3 and human CR3 cross-react and bind identical ligands, the two receptors differ in structure.     

Reduced virulence of Candida albicans PHR1 mutants.

Candida albicans mutants lacking PHR1 exhibit a pH-dependent morphogenic defect which is expressed at pH 7.5, a pH comparable to that of mammalian blood (S. M. Saporito-Irwin, C. E. Birse, P. S. Sypherd, and W. A. Fonzi, Mol. Cell. Biol. 15:601-613, 1995). The in vivo relevance of this expression pattern was tested in a mouse model of systemic candidiasis. A phr1/phr1 mutant was found to be less virulent than an isogenic Phr1+ strain and exhibited altered morphological development in vivo. These results indicate that PHR1 contributes to the virulence of C. albicans.     

Isolation and purification of chiamydospores of Candida albicans.

Two methods were investigated for their efficiency in isolating and purifying chlamydospores of Candida albicans. Chlamydospores were disconnected from pseudomycelial cells either enzymatically using beta-glucuronidase or mechanically by ultrasonic treatment. Free chlamydospores were separated from other cell material by sucrose gradient centrifugation. The resulting preparations were inspected by light-microscopy and electron-microscopy. Both methods yielded preparations with a level of over 90% chlamydospore cells. Ultrasonic treatment caused little change to the ultrastructure of the chlamydospores, whereas the enzyme treatment profoundly affected the cell wall. It is concluded that ultrasonic treatment is an efficient method for obtaining pure preparations of chlamydospores.     

Isolation of a variant of Candida albicans.

During the course of Candida albicans antigen production, a variant of this organism was encountered which did not produce hyphae at 37 degrees C. Presented here are some of the characteristics of this variant. It produces hyphae at 25 degrees C on cornmeal agar and synthetic medium plus N-acetylglucosamine and Tween 80. At 37 degrees C, it does not produce hyphae on these media, although C. albicans normally does produce hyphae under these circumstances. In liquid synthetic medium, this variant does not produce hyphae at 37 degrees C. The variant strain was analyzed for DNA, RNA, protein content, and particle size. After 50 to 70 h in balanced exponential-phase growth, particle size distribution was narrow, and there were no differences in the DNA, RNA, or protein content per particle in the two strains. When balanced exponential-phase cultures were brought into stationary phase, both strains contained the same amount of DNA per cell.     

New assay for measuring cell surface hydrophobicities of Candida dubliniensis and Candida albicans

Hydrophobic interactions, based on cell surface hydrophobicity (CSH), are among the many and varied mechanisms of adherence deployed by the pathogenic yeast Candida albicans. Recently it was shown that, unlike C. albicans, C. dubliniensis is a species that exhibits an outer fibrillar layer consistent with constant CSH. Previously, C. dubliniensis grown at 25 or 37 degrees C was shown to coaggregate with the oral anaerobic bacterium Fusobacterium nucleatum. C. albicans, however, demonstrated similar coaggregation only when hydrophobic or grown at 25 degrees C. This observation implied that coaggregation of Candida cells with F. nucleatum is associated with a hydrophobic yeast cell surface. To test this hypothesis, 42 C. albicans and 40 C. dubliniensis clinical isolates, including a C. albicans hydrophobic variant, were grown at 25 and 37 degrees C and tested with the established hydrophobicity microsphere assay, which determines CSH levels based on the number of microspheres attached to the yeast cells. The coaggregation assay was performed in parallel experiments. All C. dubliniensis isolates grown at either temperature, hydrophobic 25 degrees C-grown C. albicans isolates, and the C. albicans hydrophobic variant, unlike the 37 degrees C-hydrophilic C. albicans isolates, exhibited hydrophobic CSH levels with the microsphere assay and simultaneously showed maximum, 4+, coaggregation with F. nucleatum. The parallel results obtained for C. dubliniensis using both assays support the use of the CoAg assay both as a rapid assay to determine CSH and to differentiate between C. dubliniensis and C. albicans.     

Interactions of monospecific antisera with cell surface determinants of Candida albicans.

Flow cytometric analysis of indirect immunofluorescence showed that surface determinants recognized by antisera (Candida Check; Iatron Laboratories, Tokyo, Japan) for factors 1, 4, 5, and 6 were expressed to the same extent by all cells of Candida albicans under each growth condition and for each morphology examined. Fluorescence intensity increased with increasing cell size.     

Pathogenicity of Candida albicans auxotrophic mutants in experimental infections.

Auxotrophic and prototrophic control strain pairs of Candida albicans constructed by molecular biology methodologies were evaluated for pathogenicity in a systemic mouse model. Mutants that were auxotrophic for adenine, uracil, and heme each showed a lowered level of pathogenicity relative to control strains. It can be concluded from these experiments that decreased pathogenicity in each case is due to the auxotrophic mutation, because mutant and control strains were constructed so as to differ at a single locus. These observations suggest that new therapeutic agents for Candida infections might be designed based upon the inhibition of biosynthetic pathways that, in some cases, might be absent from the host.     

Effect of glutaraldehyde fixation on cell surface binding capacity of Candida albicans.

The ability of viable and glutaraldehyde-fixed, stationary-phase yeast cells of Candida albicans to bind concanavalin A and monospecific antiserum for antigenic factor 1 was examined. Both fluorescence flow cytometric analysis and transmission electron microscopy indicated that glutaraldehyde-fixed cells bound less of the two reagents than did unfixed viable cells.     

Variability in expression of cell surface antigens of Candida albicans during morphogenesis.

The location and expression of two different cell surface antigens on germinating and nongerminating Candida albicans cells was examined by using transmission electron microscopy after labeling with monoclonal antibodies (H9 or C6) and immunocolloidal gold. Immunodeterminant expression of the two carbohydrate antigens was followed from early germination events through 20 h of development. The determinant detected by H9 antibody, which was initially lost from the mother cell surface and preferentially expressed only on hyphae during the first 4 h of germination, reappeared on the mother cell by 20 h, whereas the antigen detected by antibody C6 was continually expressed on mother cells and germ tubes throughout germination.     

Relationship between cell surface composition, adherence, and virulence of Candida albicans.

A comparison was made of the adherence to acrylic and to human buccal epithelial cells of seven strains of Candida albicans isolated from active infections (I strains) and two strains obtained from asymptomatic carriers (C strains). After growth in defined medium containing a relatively low concentration (50 mM) of glucose as the carbon source, the adherence of I and C strains to either surface was similar and all strains were sensitive to spheroplast formation with Zymolyase 5000. Growth in medium containing a high concentration (500 mM) of sucrose or galactose enhanced the adherence of I strains up to 5- and 11-fold, respectively, and there were corresponding increases in resistance to spheroplast formation. Sucrose- or galactose-grown C strains showed only small increases in adherence and remained relatively sensitive to spheroplast formation. When inoculated intravenously into mice, I strains grown in 500 mM sucrose were up to five times more virulent than organisms grown in 50 mM glucose, while I strains grown in 500 mM galactose showed a 5- to 24-fold increase in virulence. Fifty percent lethal doses obtained for C strains were similar after growth on all three carbon sources. We conclude that I strains are able to modify their surface composition in response to high concentrations of certain sugars in the growth environment. Such modification can enhance both their ability to adhere to surfaces and their virulence. C strains lack this capability, or possess it to a lower degree, and may therefore have a lower pathogenic potential.     

Identification of a 58-kilodalton cell surface fibrinogen-binding mannoprotein from Candida albicans.

Treatment of both yeast (blastoconidia) and hyphal (blastoconidia with germ tubes) cells of Candida albicans with beta-mercaptoethanol (beta ME) releases a complex array of cell wall-bound proteins and glycoproteins. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting with fibrinogen-anti-fibrinogen antibody allowed the identification of a 58-kDa mannoprotein (mp58) in both extracts which specifically interacts with human fibrinogen. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) for short periods or with beta ME abolished or significantly reduced binding of fibrinogen. A rabbit polyclonal antiserum was raised against the purified mp58 species released by beta ME from germinated blastoconidia (PAb anti-mp58). By Western blotting, the antiserum cross-reacted with the homologous 58-kDa fibrinogen-binding mannoprotein present in beta ME extracts from blastoconidia, and by indirect immunofluorescence, the antiserum labelled both yeast cells and hyphae, yet reactivity was found primarily on the cell surface of filamentous forms. Immunostaining of human infected tissue sections with PAb anti-mp58 showed that the mp58 species is also expressed in vivo; in this case, the species is in the forms of both yeast and hyphal elements similarly labelled by the antiserum. Purified immunoglobulin G fraction from the antiserum did not alter the binding of fibrinogen as determined by a modified enzyme-linked immunosorbent assay and Western blotting. The N- and O-glycosidically linked carbohydrates represent 18 to 20% and 3 to 4%, respectively, of the molecular mass of the mp58. O-linked sugar residues may be involved in the interaction of the molecule with fibrinogen.