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Interactions between Candida albicans and the Human Extracellular Matrix Component Tenascin-C
Molecular Cell Biology Research Communications 2, 58 – 63 (1999) Article ID mcbr.1999.0152, available online at http://www.idealibrary.com on
Interactions between Candida albicans and the Human Extracellular Matrix Component Tenascin-C Jose´ L. Lo´pez-Ribot,* ,† Joseba Bikandi,* ,1 Rosario San Milla´n,* ,1 and W. LaJean Chaffin* ,2 *Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430; and †Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Received July 23, 1999
individual ECM components including laminin, fibronectin, fibrinogen, collagens, entactin, vitronectin, and thrombospondin (4 –14). The tenascins, a family of large multimeric glycoproteins with typical multidomain structures, are another component of ECM (15). The four members of this family are tenascin-C, tenascin-R, tenascin-X, and tenascin-Y (15, 16). Tenascin-C is found in a large number of developing tissues and is frequently overexpressed in tumor cells. It can have profound regulatory effects on cell adhesion since it displays both proadhesive and anti-adhesive properties, interacts with surface receptors (including integrins) on the surface of different cell types and also binds to other ECM proteins such as fibronectin (15, 17–25). Tenascin-C is secreted as a disulfide-linked hexameric protein. Each subunit consists of amino-terminal heptad repeats, followed by a domain of epidermal growth factor (EGF)type repeats, a variable number (due to alternative splicing) of fibronectin type-III repeats, and a terminal fibrinogen-like domain (15) (Fig. 1). In the present study we describe the binding of soluble human tenascin-C to intact C. albicans cells, as well as the interactions between fungal cell wall components and immobilized tenascin-C. The ability of C. albicans to interact with tenascin-C expands the number of ECM components to which the fungus is able to bind specifically and may be important in colonization and disseminated infection.
Tenascins are large multimeric proteins that contain repeated structural motifs that include epidermal growth factor (EGF)-like repeats, fibronectin type III repeats and a globular fibrinogen-like domain, and are involved in tissue and organ morphogenesis, as well as in adhesion and migration of cells. C. albicans germtubes, but not blastospores, were able to bind to soluble human tenascin-C as revealed by an indirect immunofluorescence assay. However, materials present in cell wall extracts from both morphologies attached to tenascin-C immobilized in wells of a microtiter plate. The binding specificity was demonstrated by the inhibitory effect of antibodies against C. albicans cell wall components and an anti-tenascin antibody, but not anti-laminin antibody. Fibronectin, but not fibrinogen, inhibited binding, thus indicating a role of the fibronectin type III repeats in the interaction between the fungus and tenascin-C. Binding of C. albicans cell wall materials to tenascin was RGD- and divalent cation-independent. © 1999 Academic Press Key Words: Candida albicans; tenascin-C; adhesion.
Candida albicans is a dimorphic fungus that is both a commensal and opportunistic pathogen of man. Depending on the underlying host defect, the fungus is able to cause a variety of infections that range from mucosal to life-threatening disseminated candidiasis. Adhesion of the fungus to host cells and tissues is considered the initial step leading to establishment of infection (1–3). Adherence is mediated by complementary molecules on the surface of the fungus and the host. Host structures supporting attachment of C. albicans include the extracellular matrix (ECM) and different groups have described binding of C. albicans to
MATERIALS AND METHODS Organism and culture conditions. C. albicans strain 3153A was maintained on Sabouraud medium containing 2% (w/v) agar. Yeast cells (blastoconidia or blastospores) were grown in suspension culture in the medium of Lee et al. (26) at 22°C in an orbital shaker at 180 –200 rpm. Germ tubes (germinated blastoconidia) were induced from stationary phase yeast cells that were resuspended at 5 3 10 7 cells per ml in fresh prewarmed medium and incubated at 37°C for 4 h with shaking.
1 Present address: Departamento de Microbiologı´a, Facultad de Medicina y Odontologı´a, Universidad del Paı´s Vasco, Campus de Leioa s/n 48080 Leioa, Bizkaia, Spain. 2 Corresponding author. Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas 79430. Fax: 806-743-2334. E-mail: [email protected].
1522-4724/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Structural model of a human tenascin-C subunit depicting the different domains in the molecule. The constant fibronectin type III repeats (white squares) are numbered, while the additional alternatively spliced repeats intercalated between repeats 5 and 6 are shaded and letter coded. The position of the RGD motif, present in the third fibronectin-type III repeat, is also indicated.
Indirect immunofluorescence (IIF). Blastoconidia and germinated blastoconidia were harvested, washed in water and resuspended at approximately 10 6 organisms per ml in phosphate buffered saline (PBS). Organisms were incubated with 0.5 mg per ml human tenascin-C (Chemicon International Inc., Temecula, CA) at 37°C with gentle agitation for 2 hr. After four washes with PBS they were incubated 1 hr incubation at 37°C in a 1:50 dilution of rabbit anti-tenascin immunoglobulin (Chemicon) in PBS containing 1% bovine serum albumin (PBSB). The organisms were washed as before and incubated with fluorescein-conjugated goat anti-rabbit immunoglobulin (Boehringer Mannheim, Indianapolis, IN) diluted (1:10) in PBSB. Cells were examined with a Nikon Labophot microscope equipped for epifluorescence and digital images were taken with a cooled charge-coupled device camera. Images were processed for brightness and contrast (Adobe Photoshop), and printed. Negative controls included the omission of tenascin-C or anti-tenascin antibody.
recovered and washed four times with 50 mM phosphate buffer (pH 6) and once with PBS. Binding of extracted cell wall components to immobilized tenascin-C. Wells of a microtiter plate (Immulon 2, Dynex Technologies, Inc., Chantilly, VA) were coated with tenascin-C diluted in 100 mM borate buffer, pH 8.2. The plates were incubated overnight at 4°C, washed with PBS and treated with 5% (wt/v) nonfat dry milk in PBS for 2 h at 37°C. Biotinylated components were diluted in PBS 1 0.05% Tween 20 (PBST) containing 1% BSA, 1 mM Ca 21 and 1 mM Mg 21 (binding buffer) and added to the appropriate wells (6 mg/well) and incubated for 1 h at 37°C. Following extensive washing of the plate with PBST, peroxidaseconjugated ExtrAvidin (Sigma Chemical Co., St. Louis, MO) diluted 1:3,000 in PBST containing 1% BSA was added to the wells for a 1 h incubation at 37°C. The plates were washed again. Reactivity was determined by standard colorimetric methods. Background values from uncoated wells were subtracted from experimental values. To determine RGDS inhibition, 2 mg of cell wall extract in binding buffer containing 200 mg/ml of RGDS peptide was incubated for 1 h at 37°C and then transferred to wells of a microtiter plate coated with 2 mg tenascin-C. The assay was completed as described above. For inhibition by antibodies, pooled rabbit polyclonal antiserum to cell wall proteins (11), rabbit polyclonal anti-tenascin antiserum (Chemicon) or rabbit polyclonal anti-laminin antiserum (Sigma) were diluted 1:50, 1:20 and 1:20 respectively in binding buffer and added to the appropriate wells and incubated for 1 hr at 37°C. The cell wall extract was added and the assay continued as described above. For competition with fibronectin and fibrinogen, 50 mg of the respective component was added to the wells of a microtiter plate coated with 2 mg of tenascin-C and the assay completed as described before. All experiments were performed in
Preparation of cell wall extracts. Cell wall extracts were prepared from collected intact cells (labeled with biotin or unlabeled) by treatment with b-mercaptoethanol (bME) (5, 27). Organisms were resuspended in 1/10 the original culture volume in ammonium carbonate buffer containing 1% (v/v) of bME and incubated with shaking for 30 min at 37°C. The supernatant fluid was recovered, dialyzed against distilled water (four changes) for 48 h at 4°C, and lyophilyzed. Quantitation of cell wall extract was based on total sugar content that was determined colorimetrically with mannose as standard (28). Intact cells were labeled with biotin as previously described (27). Organisms were washed with 100 mM phosphate buffer (pH 8), and resuspended (0.1 original culture volume) in buffer containing 1 mg/ml NHSbiotin (Sigma Chemical Co., St. Louis, MO). After incubation for 1 h at 22°C with shaking, the cells were 59
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Material present in the bME extracts from both C. albicans morphological phases bound to tenascin in a dose-dependent and saturable manner (Fig. 3). The levels of binding were similar for materials extracted from germ-tubes and yeast cells. This indicated that yeast cells of C. albicans possess the receptor for tenascin-C, but apparently it is not expressed at the surface as demonstrated by lack of fluorescence in IIF experiments. Effect of RGD and divalent cations on C. albicans binding to tenascin-C. The integrin recognition motif Arg-Gly-Asp (RGD) is present in the third fibronectin type-III repeat of tenascin-C, where it can support adhesion to different cell types (18, 21, 22, 31). Since RGD-mediated binding of C. albicans to other ECM components has been described previously (9, 11, 13, 32, 33), this motif could mediate binding of C. albicans to tenascin-C also. However, the presence of an RGDcontaining peptide (RGDS) enhanced rather than inhibited the binding of C. albicans cell wall extracts to immobilized tenascin-C (Fig. 4). This observation clearly indicates that the interactions between C. albicans and tenascin-C are not mediated by the RGD motif. The enhanced binding observed in the presence of RGDS may be due to a bridge effect of the peptide between tenascin-C and its receptor on C. albicans. However, a similar effect has been described for fibronectin binding to C. albicans, that appeared to be non-specific since irrelevant peptides at similar high concentrations also enhanced binding (12). The microtiter plate adhesion assay was performed in the presence of increasing concentrations (0-10 mM) of either Mg 21 or Ca 21. The interaction between C. albicans and tenascin-C does not require presence of
FIG. 2. Binding of soluble tenascin-C to whole cells of C. albicans as detected by IIF. Only germ-tubes, but not mother blastospores from which the tubes emerged, showed positive reactivity. Binding of the ECM component to isolated germ-tubes is shown in A and B, where arrows point to the mother blastospores that did not show fluorescence. Bars are 5 mm for all panels.
duplicate or triplicate wells and repeated with similar results. To assess the effect of divalent cations on binding, wells of a microtiter plate were coated with 1 mg of tenascin-C per well. Biotinylated cell wall extract (6 mg) in PBST with 1% BSA containing various concentrations of Mg 21 or Ca 21 were added to the wells and the assay completed as described before. RESULTS Binding of soluble human tenascin-C to intact C. albicans cells. Binding of human tenascin-C to the surface of C. albicans organisms was demonstrated by IIF (Fig. 2). Binding was restricted to the filamentous forms of the fungus. Blastospores growing at 28°C and mother blastospores from which the germ-tubes emanated showed no detectable levels of fluorescence (Fig. 2, arrows). Thus, as reported for other adhesive activities in C. albicans (4, 10, 11, 29, 30), binding of tenascin-C to C. albicans seemed to be dependent on morphology, and binding was heterogeneously distributed along the surface of germ-tubes.
FIG. 3. Binding of C. albicans extracted cell wall materials to immobilized tenascin-C in a solid-phase assay. bME cell wall extracts from biotinylated yeast cells (squares) or biotinylated germ tubes (circles) were reacted with immobilized tenascin-C as described in Materials and Methods. The wells on the plate were coated with increasing amounts of the respective ECM component, and incubated with biotinylated material from the correspondent morphological form. Values shown are the mean and standard deviation of duplicate samples. Levels of non-specific binding (binding to plastic in a non-coated well) were subtracted from the experimental values.
Binding of C. albicans extracted cell wall components to immobilized tenascin-C. The interaction was further analyzed using a modified solid phase adhesion assay in which biotinylated cell wall extracts of C. albicans were added to wells of a microtiter plate previously coated with different amounts of tenascin-C. 60
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antibody reduced binding approximately 40% at a 1:20 dilution, but more concentrated preparations of the antibody did not exhibit any additional effect on binding (not shown). An anti-laminin antiserum, used as a control, did not affect binding of C. albicans extracted materials to immobilized tenascin-C. DISCUSSION Adhesion of microorganisms to host cells and tissues represents a critical step in the infectious process (34). C. albicans is no exception as its survival in the host as a commensal and its pathogenic potential depend greatly on its ability to adhere to and invade host structures (35). Adhesion of C. albicans to host cells and tissues is mediated by a number of cell surface adhesins that specifically recognize complementary molecules present on host cells and tissues (reviewed in 3). The extended repertoire of adhesins displayed by C. albicans may reflect the range of sites that it can invade in the host, and the contribution of the different receptor-like molecules to adhesion may be differ depending on the type of cell or tissue in consideration. C. albicans interactions with ECM components have received special attention since they are likely to play an important role in the development of life-threatening disseminated disease (36). Binding of C. albicans to ECM components laminin, fibronectin, fibrinogen, collagens, entactin, vitronectin, and thrombospondin has been reported (4 –11, 13, 37). The enormous complexity of the interactions between this pathogenic fungus and ECM arises from four observations: (i) the existence of promiscuous receptors with the ability to interact with different ECM components (11, 38, 39), (ii) the presence of multiple receptor-like molecules that bind to the same ECM component (4, 6, 10), (iii) the existence of structural domains and recognition motifs shared by multiple ECM components (Fig. 1), and (iv) the fact that in vivo binding to ECM is likely to involve multiple receptors binding to different ligands (3). The strong fluorescence exhibited by hyphal elements of C. albicans after incubation with soluble tenascin-C was indicative of their ability to bind to this ECM component. This capacity expands the number of ECM components that the fungus is able to interact with. Tenascin-C also supports binding of certain pathogenic bacteria such as Streptococcus pyogenes (40). The fluorescence was patchy (Fig. 2) similar to that observed for other receptor-like molecules on the fungal surface (10, 11, 30). All mother blastoconidia from which germ-tubes emerged, as well as nongerminating yeast cells were not labeled. Contrary to these results, extracted cell wall materials from both morphologies were able to bind to immobilized tenascin-C to a similar extent and binding was dosedependent and saturable (Fig. 3). Thus, although both germ-tubes and blastospores posses the receptor(s) for
FIG. 4. Effect of RGDS, ECM components, and antibodies on binding of C. albicans cell wall extracts to tenascin-C. Wells of a microtiter plate were coated with 2 mg of tenascin-C and incubated with biotinylated cell wall extracts in adhesion buffer in the presence or absence of RGDS, fibronectin, fibrinogen, a 1:50 dilution of antiC.albicans cell wall antibodies (pooled-Pab), a 1:20 dilution of antitenascin-C antiserum (Pab anti-tenascin), and a 1:20 dilution of anti-laminin antiserum (Pab anti-laminin) (see Materials and Methods for details). Binding in the absence of the inhibitor was considered 100% (0% blocking). Values shown are mean 6 standard deviation of the percent inhibition of binding in the presence of inhibitor of duplicate samples in the same experiment.
divalent cations, since levels of binding were similar in the absence and in the presence of cations (data not shown). Effect of fibronectin and fibrinogen on binding of C. albicans cell wall components to immobilized tenascin-C. The tenascin-C molecule contains both fibronectin- and fibrinogen-like domains. Since C. albicans is able to bind to both ECM components, the interaction between human tenascin-C and C. albicans may be mediated by these domains. The addition of fibronectin markedly reduced adhesion of extracted cell wall components to immobilized tenascin-C (62% inhibition), whereas addition of fibrinogen to the adhesion mixtures had only a minor effect on binding (14% reduction) (Fig. 4). These results suggested that the interaction between human tenascin-C and C. albicans is likely to be mediated by the fibronectin-like domains in the ECM component, but not the terminal fibrinogen-like globular domain. Effect of antibodies on the interaction between C. albicans cell wall extracts and tenascin-C. The specificity of binding was supported by the fact that a pooled polyclonal antisera raised against materials present in different C. albicans cell wall extracts (11) and an anti-tenascin antiserum but not an anti-laminin antiserum were able to partially inhibit binding of biotinylated C. albicans extracts to immobilized tenascin-C (Fig. 4). The anti-cell wall antibody was the most effective inhibitor (approximately 50% at a 1:50 dilution), and almost completely abolished binding at a higher concentrations (not shown). The anti-tenascin 61
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groups have reported RGD- and divalent cationdependent binding of C. albicans to fibronectin, mediated mainly by the fibronectin cell-binding domain and consistent with an integrin-like type of interaction (7, 9, 13, 32, 33, 49), whereas Ne´gre and colleagues identified additional domains of fibronectin mediating binding to C. albicans, including the collagen-, heparin-, and fibrin-binding domains (12). The same group also identified an hemoglobin-induced receptor in C. albicans recognizing the cell binding domain of fibronectin which is composed of type III repeats similar to those found in tenascin-C, in an RGD- and divalent cation independent manner (12, 14, 37, 39). Thus, C. albicans interaction with tenascin-C resembles that of the hemoglobin-induced fibronectin receptor in that binding is mediated by fibronectin type III repeats, is inhibited by fibronectin, and does not require RGD or divalent cations. In summary, we have studied the characteristics of the interaction between C. albicans and human tenascin-C, thus expanding the number of ECM components with which the fungus interacts. As suggested for binding to other ECM components, C. albicans interaction with tenascin-C may facilitate adhesion to different tissue sites. Moreover, the regulatory properties of tenascin-C on adhesion may also modulate binding of C. albicans to other ECM components. Since the structure of the other members of the tenascin family is similar to tenascin-C, it is likely that the fungus can also interact with tenascin-R, tenascin-X and tenascin-Y, which have different spatio-temporal tissue distribution. Further studies will focus on the biochemical characterization of the molecules in the C. albicans cell wall responsible for tenascin binding and their uniqueness or homology with other candidal receptors for ECM components.
tenascin, it may be in a cryptic state in yeast cells and expressed only at the surface of germ-tubes. Similar differences in cell wall location have been reported previously for several binding proteins such as the C3d receptor (41, 42). Also, it is possible that the interactions between C. albicans blastospores and tenascin-C are dependent on conformational changes in the ECM component upon immobilization on the solid surface (43). The specificity of the interaction between C. albicans and tenascin-C was further demonstrated by the ability of an anti-tenascin antibody and antibodies against fungal cell wall components to partially inhibit binding (Fig. 4). RGD-dependent binding of C. albicans to a number of ECM components is well characterized (9, 11, 13, 32, 33) and this motif is present in the third fibronectin type III repeat of tenascin-C where it mediates interaction with a number of cell types mainly via integrins (21, 22, 31). However, the interactions between C. albicans and tenascin-C did not occur through this recognition motif since an RGD-bearing peptide (RGDS) failed to inhibit binding (Fig. 4). Moreover, these interactions were independent of the presence of divalent cations. Together these observations differed from that expected for an integrin-like receptor and suggested the putative “integrin-analogue” of C. albicans (44 – 46) does not mediate binding of the fungus to tenascin-C. The tenascin-C molecule exhibits a multidomain structure that includes fibronectin type III repeats and a globular fibrinogen-like terminal domain. Both domains display adhesive properties mediating the interaction between tenascin-C and different types of cells (18, 22–25, 31, 40, 47, 48). Also, the ability of C. albicans to bind fibrinogen and fibronectin is well established (5–9, 12, 14, 32, 33, 37, 39, 49). Inhibition experiments revealed that fibronectin, but not fibrinogen, was an effective competitor of the binding of C. albicans cell wall materials to immobilized tenascin-C. Since tenascin-C is also able to interact with fibronectin (17), it is possible that the residual binding observed in these competition experiments results from binding of C. albicans materials to tenascin-bound fibronectin, or possibly to the EGF-like repeats in the tenascin molecule (although there are no known reports of candidal binding to EGF motifs). Together, these observations suggested that the C. albicans-tenascin interactions are mediated by the fibronectin type-III repeats in the tenascin molecule and that fibronectin and tenascin-C share, at least to some extent, the same receptor(s) in C. albicans. This is also the case in Streptococcus pyogenes where fibronectin type III repeats in both ECM components showed affinity for the bacterium, also in an RGD-independent fashion (40). Discrepancies in studies of the candidal fibronectin receptor may reflect the presence of multiple adhesins on the surface of C. albicans with the ability to bind to fibronectin (reviewed in 3). Different
ACKNOWLEDGMENTS This work was supported by Public Health Service grants from the National Institutes of Health R29 AI42401 and AI23416 to J.L.L.-R. and W.L.C., respectively. R.S.M. acknowledges the receipt of a postdoctoral fellowship from the Direccio´n de Polı´tica Cientı´fica of the Basque Government (Spain).
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Interactions between Candida albicans and the Human Extracellular Matrix Component Tenascin-C Jose´ L. Lo´pez-Ribot,* ,† Joseba Bikandi,* ,1 Rosario San Milla´n,* ,1 and W. LaJean Chaffin* ,2 *Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430; and †Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Received July 23, 1999
individual ECM components including laminin, fibronectin, fibrinogen, collagens, entactin, vitronectin, and thrombospondin (4 –14). The tenascins, a family of large multimeric glycoproteins with typical multidomain structures, are another component of ECM (15). The four members of this family are tenascin-C, tenascin-R, tenascin-X, and tenascin-Y (15, 16). Tenascin-C is found in a large number of developing tissues and is frequently overexpressed in tumor cells. It can have profound regulatory effects on cell adhesion since it displays both proadhesive and anti-adhesive properties, interacts with surface receptors (including integrins) on the surface of different cell types and also binds to other ECM proteins such as fibronectin (15, 17–25). Tenascin-C is secreted as a disulfide-linked hexameric protein. Each subunit consists of amino-terminal heptad repeats, followed by a domain of epidermal growth factor (EGF)type repeats, a variable number (due to alternative splicing) of fibronectin type-III repeats, and a terminal fibrinogen-like domain (15) (Fig. 1). In the present study we describe the binding of soluble human tenascin-C to intact C. albicans cells, as well as the interactions between fungal cell wall components and immobilized tenascin-C. The ability of C. albicans to interact with tenascin-C expands the number of ECM components to which the fungus is able to bind specifically and may be important in colonization and disseminated infection.
Tenascins are large multimeric proteins that contain repeated structural motifs that include epidermal growth factor (EGF)-like repeats, fibronectin type III repeats and a globular fibrinogen-like domain, and are involved in tissue and organ morphogenesis, as well as in adhesion and migration of cells. C. albicans germtubes, but not blastospores, were able to bind to soluble human tenascin-C as revealed by an indirect immunofluorescence assay. However, materials present in cell wall extracts from both morphologies attached to tenascin-C immobilized in wells of a microtiter plate. The binding specificity was demonstrated by the inhibitory effect of antibodies against C. albicans cell wall components and an anti-tenascin antibody, but not anti-laminin antibody. Fibronectin, but not fibrinogen, inhibited binding, thus indicating a role of the fibronectin type III repeats in the interaction between the fungus and tenascin-C. Binding of C. albicans cell wall materials to tenascin was RGD- and divalent cation-independent. © 1999 Academic Press Key Words: Candida albicans; tenascin-C; adhesion.
Candida albicans is a dimorphic fungus that is both a commensal and opportunistic pathogen of man. Depending on the underlying host defect, the fungus is able to cause a variety of infections that range from mucosal to life-threatening disseminated candidiasis. Adhesion of the fungus to host cells and tissues is considered the initial step leading to establishment of infection (1–3). Adherence is mediated by complementary molecules on the surface of the fungus and the host. Host structures supporting attachment of C. albicans include the extracellular matrix (ECM) and different groups have described binding of C. albicans to
MATERIALS AND METHODS Organism and culture conditions. C. albicans strain 3153A was maintained on Sabouraud medium containing 2% (w/v) agar. Yeast cells (blastoconidia or blastospores) were grown in suspension culture in the medium of Lee et al. (26) at 22°C in an orbital shaker at 180 –200 rpm. Germ tubes (germinated blastoconidia) were induced from stationary phase yeast cells that were resuspended at 5 3 10 7 cells per ml in fresh prewarmed medium and incubated at 37°C for 4 h with shaking.
1 Present address: Departamento de Microbiologı´a, Facultad de Medicina y Odontologı´a, Universidad del Paı´s Vasco, Campus de Leioa s/n 48080 Leioa, Bizkaia, Spain. 2 Corresponding author. Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas 79430. Fax: 806-743-2334. E-mail: [email protected].
1522-4724/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Structural model of a human tenascin-C subunit depicting the different domains in the molecule. The constant fibronectin type III repeats (white squares) are numbered, while the additional alternatively spliced repeats intercalated between repeats 5 and 6 are shaded and letter coded. The position of the RGD motif, present in the third fibronectin-type III repeat, is also indicated.
Indirect immunofluorescence (IIF). Blastoconidia and germinated blastoconidia were harvested, washed in water and resuspended at approximately 10 6 organisms per ml in phosphate buffered saline (PBS). Organisms were incubated with 0.5 mg per ml human tenascin-C (Chemicon International Inc., Temecula, CA) at 37°C with gentle agitation for 2 hr. After four washes with PBS they were incubated 1 hr incubation at 37°C in a 1:50 dilution of rabbit anti-tenascin immunoglobulin (Chemicon) in PBS containing 1% bovine serum albumin (PBSB). The organisms were washed as before and incubated with fluorescein-conjugated goat anti-rabbit immunoglobulin (Boehringer Mannheim, Indianapolis, IN) diluted (1:10) in PBSB. Cells were examined with a Nikon Labophot microscope equipped for epifluorescence and digital images were taken with a cooled charge-coupled device camera. Images were processed for brightness and contrast (Adobe Photoshop), and printed. Negative controls included the omission of tenascin-C or anti-tenascin antibody.
recovered and washed four times with 50 mM phosphate buffer (pH 6) and once with PBS. Binding of extracted cell wall components to immobilized tenascin-C. Wells of a microtiter plate (Immulon 2, Dynex Technologies, Inc., Chantilly, VA) were coated with tenascin-C diluted in 100 mM borate buffer, pH 8.2. The plates were incubated overnight at 4°C, washed with PBS and treated with 5% (wt/v) nonfat dry milk in PBS for 2 h at 37°C. Biotinylated components were diluted in PBS 1 0.05% Tween 20 (PBST) containing 1% BSA, 1 mM Ca 21 and 1 mM Mg 21 (binding buffer) and added to the appropriate wells (6 mg/well) and incubated for 1 h at 37°C. Following extensive washing of the plate with PBST, peroxidaseconjugated ExtrAvidin (Sigma Chemical Co., St. Louis, MO) diluted 1:3,000 in PBST containing 1% BSA was added to the wells for a 1 h incubation at 37°C. The plates were washed again. Reactivity was determined by standard colorimetric methods. Background values from uncoated wells were subtracted from experimental values. To determine RGDS inhibition, 2 mg of cell wall extract in binding buffer containing 200 mg/ml of RGDS peptide was incubated for 1 h at 37°C and then transferred to wells of a microtiter plate coated with 2 mg tenascin-C. The assay was completed as described above. For inhibition by antibodies, pooled rabbit polyclonal antiserum to cell wall proteins (11), rabbit polyclonal anti-tenascin antiserum (Chemicon) or rabbit polyclonal anti-laminin antiserum (Sigma) were diluted 1:50, 1:20 and 1:20 respectively in binding buffer and added to the appropriate wells and incubated for 1 hr at 37°C. The cell wall extract was added and the assay continued as described above. For competition with fibronectin and fibrinogen, 50 mg of the respective component was added to the wells of a microtiter plate coated with 2 mg of tenascin-C and the assay completed as described before. All experiments were performed in
Preparation of cell wall extracts. Cell wall extracts were prepared from collected intact cells (labeled with biotin or unlabeled) by treatment with b-mercaptoethanol (bME) (5, 27). Organisms were resuspended in 1/10 the original culture volume in ammonium carbonate buffer containing 1% (v/v) of bME and incubated with shaking for 30 min at 37°C. The supernatant fluid was recovered, dialyzed against distilled water (four changes) for 48 h at 4°C, and lyophilyzed. Quantitation of cell wall extract was based on total sugar content that was determined colorimetrically with mannose as standard (28). Intact cells were labeled with biotin as previously described (27). Organisms were washed with 100 mM phosphate buffer (pH 8), and resuspended (0.1 original culture volume) in buffer containing 1 mg/ml NHSbiotin (Sigma Chemical Co., St. Louis, MO). After incubation for 1 h at 22°C with shaking, the cells were 59
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Material present in the bME extracts from both C. albicans morphological phases bound to tenascin in a dose-dependent and saturable manner (Fig. 3). The levels of binding were similar for materials extracted from germ-tubes and yeast cells. This indicated that yeast cells of C. albicans possess the receptor for tenascin-C, but apparently it is not expressed at the surface as demonstrated by lack of fluorescence in IIF experiments. Effect of RGD and divalent cations on C. albicans binding to tenascin-C. The integrin recognition motif Arg-Gly-Asp (RGD) is present in the third fibronectin type-III repeat of tenascin-C, where it can support adhesion to different cell types (18, 21, 22, 31). Since RGD-mediated binding of C. albicans to other ECM components has been described previously (9, 11, 13, 32, 33), this motif could mediate binding of C. albicans to tenascin-C also. However, the presence of an RGDcontaining peptide (RGDS) enhanced rather than inhibited the binding of C. albicans cell wall extracts to immobilized tenascin-C (Fig. 4). This observation clearly indicates that the interactions between C. albicans and tenascin-C are not mediated by the RGD motif. The enhanced binding observed in the presence of RGDS may be due to a bridge effect of the peptide between tenascin-C and its receptor on C. albicans. However, a similar effect has been described for fibronectin binding to C. albicans, that appeared to be non-specific since irrelevant peptides at similar high concentrations also enhanced binding (12). The microtiter plate adhesion assay was performed in the presence of increasing concentrations (0-10 mM) of either Mg 21 or Ca 21. The interaction between C. albicans and tenascin-C does not require presence of
FIG. 2. Binding of soluble tenascin-C to whole cells of C. albicans as detected by IIF. Only germ-tubes, but not mother blastospores from which the tubes emerged, showed positive reactivity. Binding of the ECM component to isolated germ-tubes is shown in A and B, where arrows point to the mother blastospores that did not show fluorescence. Bars are 5 mm for all panels.
duplicate or triplicate wells and repeated with similar results. To assess the effect of divalent cations on binding, wells of a microtiter plate were coated with 1 mg of tenascin-C per well. Biotinylated cell wall extract (6 mg) in PBST with 1% BSA containing various concentrations of Mg 21 or Ca 21 were added to the wells and the assay completed as described before. RESULTS Binding of soluble human tenascin-C to intact C. albicans cells. Binding of human tenascin-C to the surface of C. albicans organisms was demonstrated by IIF (Fig. 2). Binding was restricted to the filamentous forms of the fungus. Blastospores growing at 28°C and mother blastospores from which the germ-tubes emanated showed no detectable levels of fluorescence (Fig. 2, arrows). Thus, as reported for other adhesive activities in C. albicans (4, 10, 11, 29, 30), binding of tenascin-C to C. albicans seemed to be dependent on morphology, and binding was heterogeneously distributed along the surface of germ-tubes.
FIG. 3. Binding of C. albicans extracted cell wall materials to immobilized tenascin-C in a solid-phase assay. bME cell wall extracts from biotinylated yeast cells (squares) or biotinylated germ tubes (circles) were reacted with immobilized tenascin-C as described in Materials and Methods. The wells on the plate were coated with increasing amounts of the respective ECM component, and incubated with biotinylated material from the correspondent morphological form. Values shown are the mean and standard deviation of duplicate samples. Levels of non-specific binding (binding to plastic in a non-coated well) were subtracted from the experimental values.
Binding of C. albicans extracted cell wall components to immobilized tenascin-C. The interaction was further analyzed using a modified solid phase adhesion assay in which biotinylated cell wall extracts of C. albicans were added to wells of a microtiter plate previously coated with different amounts of tenascin-C. 60
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antibody reduced binding approximately 40% at a 1:20 dilution, but more concentrated preparations of the antibody did not exhibit any additional effect on binding (not shown). An anti-laminin antiserum, used as a control, did not affect binding of C. albicans extracted materials to immobilized tenascin-C. DISCUSSION Adhesion of microorganisms to host cells and tissues represents a critical step in the infectious process (34). C. albicans is no exception as its survival in the host as a commensal and its pathogenic potential depend greatly on its ability to adhere to and invade host structures (35). Adhesion of C. albicans to host cells and tissues is mediated by a number of cell surface adhesins that specifically recognize complementary molecules present on host cells and tissues (reviewed in 3). The extended repertoire of adhesins displayed by C. albicans may reflect the range of sites that it can invade in the host, and the contribution of the different receptor-like molecules to adhesion may be differ depending on the type of cell or tissue in consideration. C. albicans interactions with ECM components have received special attention since they are likely to play an important role in the development of life-threatening disseminated disease (36). Binding of C. albicans to ECM components laminin, fibronectin, fibrinogen, collagens, entactin, vitronectin, and thrombospondin has been reported (4 –11, 13, 37). The enormous complexity of the interactions between this pathogenic fungus and ECM arises from four observations: (i) the existence of promiscuous receptors with the ability to interact with different ECM components (11, 38, 39), (ii) the presence of multiple receptor-like molecules that bind to the same ECM component (4, 6, 10), (iii) the existence of structural domains and recognition motifs shared by multiple ECM components (Fig. 1), and (iv) the fact that in vivo binding to ECM is likely to involve multiple receptors binding to different ligands (3). The strong fluorescence exhibited by hyphal elements of C. albicans after incubation with soluble tenascin-C was indicative of their ability to bind to this ECM component. This capacity expands the number of ECM components that the fungus is able to interact with. Tenascin-C also supports binding of certain pathogenic bacteria such as Streptococcus pyogenes (40). The fluorescence was patchy (Fig. 2) similar to that observed for other receptor-like molecules on the fungal surface (10, 11, 30). All mother blastoconidia from which germ-tubes emerged, as well as nongerminating yeast cells were not labeled. Contrary to these results, extracted cell wall materials from both morphologies were able to bind to immobilized tenascin-C to a similar extent and binding was dosedependent and saturable (Fig. 3). Thus, although both germ-tubes and blastospores posses the receptor(s) for
FIG. 4. Effect of RGDS, ECM components, and antibodies on binding of C. albicans cell wall extracts to tenascin-C. Wells of a microtiter plate were coated with 2 mg of tenascin-C and incubated with biotinylated cell wall extracts in adhesion buffer in the presence or absence of RGDS, fibronectin, fibrinogen, a 1:50 dilution of antiC.albicans cell wall antibodies (pooled-Pab), a 1:20 dilution of antitenascin-C antiserum (Pab anti-tenascin), and a 1:20 dilution of anti-laminin antiserum (Pab anti-laminin) (see Materials and Methods for details). Binding in the absence of the inhibitor was considered 100% (0% blocking). Values shown are mean 6 standard deviation of the percent inhibition of binding in the presence of inhibitor of duplicate samples in the same experiment.
divalent cations, since levels of binding were similar in the absence and in the presence of cations (data not shown). Effect of fibronectin and fibrinogen on binding of C. albicans cell wall components to immobilized tenascin-C. The tenascin-C molecule contains both fibronectin- and fibrinogen-like domains. Since C. albicans is able to bind to both ECM components, the interaction between human tenascin-C and C. albicans may be mediated by these domains. The addition of fibronectin markedly reduced adhesion of extracted cell wall components to immobilized tenascin-C (62% inhibition), whereas addition of fibrinogen to the adhesion mixtures had only a minor effect on binding (14% reduction) (Fig. 4). These results suggested that the interaction between human tenascin-C and C. albicans is likely to be mediated by the fibronectin-like domains in the ECM component, but not the terminal fibrinogen-like globular domain. Effect of antibodies on the interaction between C. albicans cell wall extracts and tenascin-C. The specificity of binding was supported by the fact that a pooled polyclonal antisera raised against materials present in different C. albicans cell wall extracts (11) and an anti-tenascin antiserum but not an anti-laminin antiserum were able to partially inhibit binding of biotinylated C. albicans extracts to immobilized tenascin-C (Fig. 4). The anti-cell wall antibody was the most effective inhibitor (approximately 50% at a 1:50 dilution), and almost completely abolished binding at a higher concentrations (not shown). The anti-tenascin 61
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groups have reported RGD- and divalent cationdependent binding of C. albicans to fibronectin, mediated mainly by the fibronectin cell-binding domain and consistent with an integrin-like type of interaction (7, 9, 13, 32, 33, 49), whereas Ne´gre and colleagues identified additional domains of fibronectin mediating binding to C. albicans, including the collagen-, heparin-, and fibrin-binding domains (12). The same group also identified an hemoglobin-induced receptor in C. albicans recognizing the cell binding domain of fibronectin which is composed of type III repeats similar to those found in tenascin-C, in an RGD- and divalent cation independent manner (12, 14, 37, 39). Thus, C. albicans interaction with tenascin-C resembles that of the hemoglobin-induced fibronectin receptor in that binding is mediated by fibronectin type III repeats, is inhibited by fibronectin, and does not require RGD or divalent cations. In summary, we have studied the characteristics of the interaction between C. albicans and human tenascin-C, thus expanding the number of ECM components with which the fungus interacts. As suggested for binding to other ECM components, C. albicans interaction with tenascin-C may facilitate adhesion to different tissue sites. Moreover, the regulatory properties of tenascin-C on adhesion may also modulate binding of C. albicans to other ECM components. Since the structure of the other members of the tenascin family is similar to tenascin-C, it is likely that the fungus can also interact with tenascin-R, tenascin-X and tenascin-Y, which have different spatio-temporal tissue distribution. Further studies will focus on the biochemical characterization of the molecules in the C. albicans cell wall responsible for tenascin binding and their uniqueness or homology with other candidal receptors for ECM components.
tenascin, it may be in a cryptic state in yeast cells and expressed only at the surface of germ-tubes. Similar differences in cell wall location have been reported previously for several binding proteins such as the C3d receptor (41, 42). Also, it is possible that the interactions between C. albicans blastospores and tenascin-C are dependent on conformational changes in the ECM component upon immobilization on the solid surface (43). The specificity of the interaction between C. albicans and tenascin-C was further demonstrated by the ability of an anti-tenascin antibody and antibodies against fungal cell wall components to partially inhibit binding (Fig. 4). RGD-dependent binding of C. albicans to a number of ECM components is well characterized (9, 11, 13, 32, 33) and this motif is present in the third fibronectin type III repeat of tenascin-C where it mediates interaction with a number of cell types mainly via integrins (21, 22, 31). However, the interactions between C. albicans and tenascin-C did not occur through this recognition motif since an RGD-bearing peptide (RGDS) failed to inhibit binding (Fig. 4). Moreover, these interactions were independent of the presence of divalent cations. Together these observations differed from that expected for an integrin-like receptor and suggested the putative “integrin-analogue” of C. albicans (44 – 46) does not mediate binding of the fungus to tenascin-C. The tenascin-C molecule exhibits a multidomain structure that includes fibronectin type III repeats and a globular fibrinogen-like terminal domain. Both domains display adhesive properties mediating the interaction between tenascin-C and different types of cells (18, 22–25, 31, 40, 47, 48). Also, the ability of C. albicans to bind fibrinogen and fibronectin is well established (5–9, 12, 14, 32, 33, 37, 39, 49). Inhibition experiments revealed that fibronectin, but not fibrinogen, was an effective competitor of the binding of C. albicans cell wall materials to immobilized tenascin-C. Since tenascin-C is also able to interact with fibronectin (17), it is possible that the residual binding observed in these competition experiments results from binding of C. albicans materials to tenascin-bound fibronectin, or possibly to the EGF-like repeats in the tenascin molecule (although there are no known reports of candidal binding to EGF motifs). Together, these observations suggested that the C. albicans-tenascin interactions are mediated by the fibronectin type-III repeats in the tenascin molecule and that fibronectin and tenascin-C share, at least to some extent, the same receptor(s) in C. albicans. This is also the case in Streptococcus pyogenes where fibronectin type III repeats in both ECM components showed affinity for the bacterium, also in an RGD-independent fashion (40). Discrepancies in studies of the candidal fibronectin receptor may reflect the presence of multiple adhesins on the surface of C. albicans with the ability to bind to fibronectin (reviewed in 3). Different
ACKNOWLEDGMENTS This work was supported by Public Health Service grants from the National Institutes of Health R29 AI42401 and AI23416 to J.L.L.-R. and W.L.C., respectively. R.S.M. acknowledges the receipt of a postdoctoral fellowship from the Direccio´n de Polı´tica Cientı´fica of the Basque Government (Spain).
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