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.     

Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans.

Ultrastructural and biochemical analyses of hydrophobic and hydrophilic yeast cell surface proteins of Candida albicans were performed. Hydrophobic and hydrophilic yeast cells were obtained by growth at 23 and 37 degrees C, respectively. In addition, hydrophilic yeast cells were converted to surface hydrophobicity by treatment with tunicamycin and dithiothreitol. When freeze-etched cells were examined, the temperature-induced hydrophilic cells had long (0.198 micron), compact, evenly distributed fibrils while temperature-induced hydrophobic cells had short (0.085 micron), blunt fibrils. Hydrophobic microsphere attachment to the hydrophobic cells occurred at the basement of and within the short fibril layer. Dithiothreitol-induced hydrophobic cells had the long fibrils removed; tunicamycin-induced hydrophobic cells retained some of the long fibrils, but the fibrils were less compact and more aggregated than the untreated controls. These results suggest that the long fibrils prevent hydrophobic microsphere attachment to the hydrophobic area of the cell surface. This was confirmed by assessing the hydrophobic avidity of hydrophobic yeast cell populations differing in fibril density and arrangement. 125I-labelled surface proteins from hydrophobic and hydrophilic cells were compared after separation by hydrophobic interaction chromatography-high-performance liquid chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The yeast cell populations had hydrophilic proteins of similar molecular masses (greater than 200 kDa), but the hydrophilic cells possessed at least two additional proteins (ca. 63 and 69 to 71 kDa). Hydrophobic surface proteins appeared to be similar. However, the amount of total radiolabelled hydrophobic proteins was approximately 10-fold higher for the hydrophobic cells than for the hydrophilic cells. This result agrees with the ultrastructural observations which showed that yeast cell surface hydrophobic proteins are masked by hydrophilic high-molecular-mass surface fibrils. Taken together, the data indicate that yeast cell hydrophobicity is not determined by differences in surface hydrophobic proteins but by the presence of hydrophilic, surface fibrils.     

Comparative study of the C3d receptor and 58-kilodalton fibrinogen-binding mannoproteins of Candida albicans.

Using polyclonal antibodies (PAbs) raised against the Candida albicans C3d receptor (CR2; PAb anti-CR2) and the 58-kDa fibrinogen-binding mannoprotein (mp58; PAb anti-mp58) as well as ligand interactions, we have studied the relationship between these two receptors. In an indirect immunofluorescence assay with germ tubes, greater intensity was observed on the mother blastoconidium when PAb anti-CR2 was used, whereas greater intensity was localized to the hyphal extension when PAb anti-mp58 or binding of soluble fibrinogen was used. No competition or change in the fluorescence pattern was observed in dual-labeling experiments with PAb anti-CR2 and either fibrinogen or PAb anti-mp58. Binding competition also was not observed in an enzyme-linked immunosorbent assay using the components present in a beta-mercaptoethanol extract from the cell wall of germ tubes. In immunoblots, PAb anti-CR2 recognized three different discrete bands with apparent molecular masses of 21, 40, and 66 kDa in the beta-mercaptoethanol extracts from the cell wall, whereas a different, single, broader band with an apparent molecular mass of 58 kDa was detected with PAb anti-mp58. However, when nondenaturing conditions were used to separate the materials present in the cell wall extracts, no reactivity could be detected on Western blots (immunoblots) with PAb anti-mp58. When PAb anti-CR2 was used for analysis, a single band migrating in the area corresponding to approximately 40 kDa was detected. These observations suggest a higher molecular weight for mp58 and one or more of the components detected with PAb anti-CR2 in their native state.     

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.     

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.     

Purification of actin from Candida albicans and comparison with the Candida 48,000-Mr protein.

Actin was purified from Candida albicans cells by affinity chromatography by DNase-Sepharose and was recognized by immunoblotting with monoclonal antibody directed against chick muscle actin. The C. albicans 48-kilodalton protein recognized by sera from patients with invasive candidiasis was shown by DEAE chromatography and immunoblotting not to be identical with the purified C. albicans actin.     

Inhibitors of protein phosphorylation including the retinoblastoma protein induce germination of Candida albicans.

It has been previously shown that the induction of germination in Candida albicans occurs following its cessation of growth as a yeast. Similarly, mammalian cells undergo a differentiation process that is preceded by a growth cessation associated with a hypophosphorylation of proteins of the retinoblastoma gene family. It is postulated that a similar type of mechanism may be operative in C. albicans and protein phosphorylation inhibitors: forskolin (stimulates cyclic adenosine monophosphate production), okadaic acid (phosphatase inhibitor) and D-erythro-sphingosine (retinoblastoma protein phosphorylation inhibitor) have been used to further strengthen this hypothesis. Okadaic acid (1-1000 nM) and D-erythro-sphingosine (100 microM) significantly inhibited the growth of yeast cells of C. albicans. D-Erythro-sphingosine at 1000 microM was candidicidal. Forskolin did not significantly affect growth. Exponentially grown C. albicans pretreated with forskolin (10 microM), okadaic acid (1000 nM) or D-erythro-sphingosine (100 microM) readily germinated. In comparison, when these inhibitors were incorporated in the same medium, germination of exponentially grown cells did not occur. These results suggest that protein dephosphorylation may be necessary at an early stage of the yeast-hyphae transition in C. albicans.     

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.     

Isolation and characterization of cell surface mutants of Candida albicans.

Mutant strains of Candida albicans were obtained by selecting for cells that escaped agglutination by a polyclonal antiserum raised against standard C. albicans serotype A isolate B311. Mutants were obtained from strains B311 and B792 and from four strains isolated from patients with acquired immunodeficiency syndrome. All 15 tested mutants retained characteristic sugar assimilation patterns. All but one of the mutants retained the ability to form germ tubes and chlamydospores. Two mutants from an acquired immunodeficiency syndrome-derived isolate were deficient in binding complement ligands iC3b and C3d, whereas another mutant was deficient in binding ligand iC3b but not C3d. The hyphae of these three mutants lacked antigens when examined by Western immunoblotting with monoclonal antibody Ca-A, which detects several glycoproteins, including C3d-binding proteins. One of the complement-binding-deficient mutants was tested for its ability to colonize the gastrointestinal tract of rabbits but did not differ from the wild-type parent in site or degree of colonization. The proton magnetic resonance spectra of bulk mannan carbohydrate extracted from tested mutants showed the loss of a signal characteristic of the mannosyl alpha-PO4 linkage; each mutant also had a distinct pattern of other changes.     

Expression of surface hydrophobic proteins by Candida albicans in vivo.

Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The polyclonal antiserum distinguished between hydrophobic and hydrophilic cell surfaces in vitro and gave similar staining patterns and intensities for C. albicans cells in vivo. Of the yeast forms present within tissue lesions, approximately half exhibited moderate to intense immunofluorescence with the antiserum. Immunoblot analysis indicated that antigens recognized by the antiserum are predominantly low-molecular-mass hydrophobic proteins that are expressed by different C. albicans isolates and are expressed regardless of growth temperature. Taken together, the immunofluorescence and immunoblot analyses of antigens indicate that C. albicans displays surface hydrophobic proteins during pathogenesis and these proteins are available for hydrophobic interactions with host tissues. The effect of hydrophobic protein exposure on the virulence of C. albicans is discussed.     

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.     

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.     

Adherence of Candida albicans to a cell surface polysaccharide receptor on Streptococcus gordonii.

Candida albicans ATCC 10261 and CA2 bound to cells of the oral bacteria Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguis when these bacteria were immobilized onto microtiter plate wells, but they did not bind to cells of Streptococcus mutans or Streptococcus salivarius. Cell wall polysaccharide was extracted with alkali from S. gordonii NCTC 7869, the streptococcal species to which C. albicans bound with highest affinity, and was effective in blocking the coaggregation of C. albicans and S. gordonii cells in the fluid phase. When fixed to microtiter plate wells, the S. gordonii polysaccharide was bound by all strains of C. albicans tested. The polysaccharide contained Rha, Glc, GalNAc, GlcNAc, and Gal and was related compositionally to previously characterized cell wall polysaccharides from strains of S. oralis and S. sanguis. The adherence of yeast cells to the immobilized polysaccharide was not inhibitable by a number of saccharides. Antiserum raised to the S. gordonii NCTC 7869 polysaccharide blocked adherence of C. albicans ATCC 10261 to the polysaccharide. The results identify a complex cell wall polysaccharide of S. gordonii as the coaggregation receptor for C. albicans. Adherent interactions of yeast cells with streptococci and other bacteria may be important for colonization of both hard and soft oral surfaces by C. albicans.     

Candida albicans strain delineation.

Candida albicans is a major opportunistic pathogen causing a wide spectrum of disease in human beings. Methods for strain delineation of this species to assess or predict virulence or to conduct epidemiologic or pathogenetic investigations have been developed. Although factors associated with virulence have been identified, there is no rapid system to quantitate them in a clinical laboratory. Therefore, many typing methods are based on variable phenotypic characteristics within this species including morphotyping, serotyping, antibiogram, resistogram typing, biotyping, biotyping based on commercial carbon assimilation patterns, enzyme profiles, sensitivity to yeast killer toxins, and typing based on protein variability. Phenotypically defined strains generally do not correlate with the pathogenic potential of a strain with the exception of morphotyping. However, these methods can be useful in epidemiologic investigations; for example, they have revealed that most individuals harbor one strain and that infections are frequently due to an endogenous strain. Problems with these methods usually relate to their discriminatory power. When this is maximized, reproducibility (especially between laboratories) suffers. Recently, methods based on differences in DNA structure (genotyping) for strain delineation have been developed, including electrophoretic karyotyping and restriction enzyme fragment length polymorphisms. The development of a computer-assisted data bank and analysis for these genotypic strain delineators will open investigations into the pathogenesis of this infection and permit epidemiologic studies previously not possible with this important human pathogen.     

Evidence for the presence of collagenous domains in Candida albicans cell surface proteins.

Rabbit polyclonal antibodies (PAbs) directed towards the amino-terminal cysteine-rich 7S domain (PAb anti-7S), the major internal collagenous domain (PAb anti-type IV), and the C-terminal noncollagenous region (PAb anti-NC1) of the type IV collagen molecule were probed by indirect immunofluorescence against Candida albicans blastoconidia and germinated blastoconidia. Most nongerminating cells and mother blastoconidia from which germ tubes originated showed strong fluorescence when PAb anti-7S was used, whereas with PAb anti-type IV, fluorescence was found almost exclusively on the surface of filamentous forms. A patched fluorescent pattern rather than a homogenous confluent fluorescence was observed in all cases. No fluorescent cells were observed with PAb anti-NC1. By Western immunoblotting, PAb anti-type IV cross-reacted primarily with a polypeptide of 37 kDa present in wall extracts obtained from intact cells of both growth forms by treatment with beta-mercaptoethanol, whereas PAb anti-7S recognized a major 58-kDa antigen also present in both extracts, along with some other high-molecular-mass (> 106-kDa) polydisperse species present only in the material released from blastoconidia with beta-mercaptoethanol. No reactive bands were observed when PAb anti-NC1 was used as a probe in Western immunoblotting experiments. The sensitivities or resistances to collagenase digestion of the different polypeptides that cross-reacted with PAbs anti-type IV and anti-7S suggest the existence of cell wall components in C. albicans that contain epitopes that mimic the collagenous domains of the type IV collagen molecule.     

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.     

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.     

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.     

Prevalence and intraoral distribution of Candida albicans in Sj?gren's syndrome.

An imprint culture technique has been employed to study the prevalence and intraoral distribution of Candida albicans in 16 patients with Sjögren's syndrome and in 16 healthy controls matched for age, sex, and dental status. The prevalence and intraoral density of C. albicans was found to be significantly higher at almost all sites in the Sjögren's patients than in the controls. The distribution of candida was also altered, being significantly higher in the floor of the mouth and anterior labial sulcus in the Sjögren's group. There was an approximate inverse relationship between candida populations and rate of salivary flow. Mean candida densities were found to be significantly higher in those Sjögren's patients with detectable serum rheumatoid factor in the serum. However, patients with primary Sjögren's syndrome had significantly higher mean candida densities compared with patients with secondary Sjögren's syndrome.