Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Abstract Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using ¹²⁵I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cell. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant ( K _d) of 5.2 × 10⁻⁸ M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one compotent of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.     

Dynamic changes of the cell wall surface of Candida albicans associated with germination and adherence

The distribution of mannoproteins at the cell wall surface of Candida albicans was analyzed during the process of germination in conditions favoring adherence of germ tubes to a plastic matrix. Three cytochemical methods allowing the detection of concanavalin A binding sites, anionic sites and the enzyme acid phosphatase, respectively were used. All three methods gave similar results, indicating a spatial and temporal reorganization of some cell wall mannoproteins: a strong labeling was observed on blastoconidia; in contrast, as soon as the emergence of germ tubes took place, these reactions decreased dramatically at the surface of mother cells, whereas the germ tube surface was strongly stained. Some new components with multiple biological activities were detected at the germ-tube surface. Indeed, among mannoproteins responsible for an enhanced adhesion to plastic surfaces, two components with molecular weights of 68 and 60 to 62 kDa were shown to interact with laminin, fibrinogen, and C3d. This study therefore indicates that germination, and then adherence of germ tubes, imply a degradation of surface mannoproteins, and a simultaneous presentation of new molecules which can interact with their nonbiological materials or host proteins.     

Characterization of binding of human fibrinogen to the surface of germ-tubes and mycelium of candida albicans

The binding of human fibrinogen to germ-tubes and mycelium of Candida albicans, forms usually found in infected tissues, was studied in vitro by an immunofluorescence assay. Binding was quantified by using 125I-labelled fibrinogen. The degree of binding differed according to the morphological form of the fungus. Binding relative to that of the yeast form was greater for mycelium (12-fold) than for germ-tube (7.7-fold). Pretreatment of yeasts with fragments D and E (terminal degradation products of fibrinogen) before fibrinogen binding showed that fragment D possessed a higher affinity for C. albicans than fragment E. Binding of fibrinogen was diminished when C. albicans was pretreated with 2-mercaptoethanol alone or in combination with pronase, or pretreated with alpha-mannosidase or trypsin. Binding was not decreased by pretreatment with pronase alone or chitinase. Inhibition experiments using C. albicans dialysed culture filtrate, C. albicans mannan, chitin, sugars or amino sugars were done by preabsorbing the fibrinogen with each of the above mentioned compounds. C. albicans dialysed culture filtrate inhibited the binding more strongly than C. albicans mannan. However, fibrinogen binding to C. albicans was not significantly reduced by mannose, several other sugars or chitin. These studies demonstrate the existence of a fibrinogen-binding factor (FBF) strongly associated with the surface of germ-tube and filamentous forms of C. albicans, and indicate a possible role for FBF in the pathogenicity of C. albicans.     

Interaction of fibrinogen and its derivatives with fibrin

The binding between complementary polymerization sites of fibrin monomers plays an essential role in the formation of the fibrin clot. One set of polymerization sites involved in the interaction of fibrin monomers is believed to pre-exist in fibrinogen, while the complementary set of binding sites is exposed after the cleavage of fibrinopeptides from fibrinogen. The polymerization sites present in fibrinogen and its derivatives mediate their binding to fibrin. Although the binding of fibrinogen and its derivatives to fibrin have been qualitatively studied, there has been no systematic, quantitative investigation of their interaction with forming or preformed clots. In the present study, the binding of fibrinogen and fragments DD, D1, and E1 was measured using a sonicated suspension of plasminogen- and thrombin-free human cross-linked fibrin as a model of a preformed clot. Dissociation constants of 0.056, 0.19, and 2.44 microM, and the number of binding sites corresponding to 0.10, 0.21, and 0.13/fibrin monomer unit of fibrin polymer were found for fibrinogen, fragment DD, and fragment D1, respectively. Fragment E1 did not bind to sonicated noncross-linked or cross-linked fibrin suspensions. However, it was bound to forming fibrin clots as well as to fibrin-Celite, suggesting that the binding sites on fibrin involved in the interaction with fragment E1 may have been altered upon sonication. Affinity chromatography of various fibrinogen derivatives on a fibrin-Celite column showed that only part of the bound fragment DD was displaced by arginine, whereas fragments D1 and E1 were completely eluted under the same conditions. The results indicate that interaction of fibrinogen with the preformed fibrin clots is characterized by affinity in the nanomolar range and that binding between fibrin monomers, in the process of clot formation, could be characterized by even a higher affinity.     

Characteristics of fibrinogen binding to the domain of CD11c, an alpha subunit of p150,95.

beta2 integrins on leukocytes play important roles on cell-cell or cell-matrix adhesion through their ability to bind multiple ligands. The alpha subunits of leukocyte CD11/CD18 integrins contain an approximately 200-amino-acid inserted domain (I-domain) which is implicated in ligand binding function. To understand the characteristics of ligand binding to the alpha subunit of beta2 integrin p150,95 (CD11c/CD18), a recombinant form of the I-domain of CD11c was generated and analyzed for the interaction with fibrinogen, one of the ligands of p150,95. It was found that the CD11c I-domain bound fibrinogen specifically. Fibrinogen binding to the CD11c I-domain was inhibited by a molar excess of fragment E, a central domain of fibrinogen, and not by that of fragment D, a distal domain of fibrinogen, suggesting that CD11c/CD18 recognizes a central domain of fibrinogen. Divalent cations such as Mg(2+) and Mn(2+) were required for fibrinogen binding to the CD11c I-domain. Also alanine substitutions on the putative metal binding sites of the CD11c I-domain such as Asp(242) and Tyr(209) reduced its ability to bind fibrinogen. These data reinforce the fact that the divalent cation is a prerequisite for ligand binding of the CD11c I-domain.     

Recognition of Candida albicans Als3 by the germ tube-specific monoclonal antibody 3D9.3

Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity between MAb 3D9.3 and an als3 Δ /als3 Δ mutant strain, and the fact that an immunoglobulin preparation enriched for Als3 specificity recognized the purified 3D9 antigen. PCR demonstrated that C. albicans strain 66396 has two different-sized ALS3 alleles that correspond to the two purified protein bands. Strain- and species-specificity of the 3D9 epitope were studied with various C. albicans strains and Candida species, such as closely related Candida dubliniensis . The 3D9 epitope was detected only in C. albicans , demonstrating the utility of MAb 3D9.3 for differentiation between C. albicans and C. dubliniensis . Adhesion assays demonstrated that MAb 3D9.3 blocks adhesion of C. albicans germ tubes to human buccal epithelial cells and vascular endothelial cells.     

Differential binding of mannose-specific lectins to the carbohydrate chains of fibrinogen domains D and E

The interaction of fibrinogen with the mannose-specific lectins concanavalin A (ConA), its acetyl derivative (Ac-ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen B beta and gamma chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac-ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1 = 2.4 microM and Kd2 = 16.6 microM; n1 = n2 = 2) while only two moles of LcH are bound per mole of fibrinogen (Kd = 2.6 microM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac-ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac-ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectin-fibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac-ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen. The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.     

Inhibition of Candida albicans attachment to extracellular matrix by antibodies which recognize hydrophobic cell wall proteins

Cell surface hydrophobicity influences the adhesive properties of the opportunistic fungal pathogen Candida albicans. Hydrophobic proteins are present in the C. albicans cell wall. These proteins were used to generate a polyclonal antiserum and monoclonal antibodies. We characterized three of these monoclonal antibodies (designated 6C5, 5F8 and 5D8) that recognize different hydrophobic cell wall proteins. Initial characterization of the three antigens, and assessment of their distribution among various Candida species was also carried out. Further, pretreatment of germ tube initials with the mAb inhibits binding of these cells to immobilized extracellular matrix. These results suggest that these hydrophobic proteins are involved in C. albicans adhesion events.     

Interaction of heavy and light chains of plasmin with fibrinogen E and D fragments

It was demonstrated that plasminogen and the plasmin heavy chain form a complex with an immobilized fibrinogen fragment E. The E-fragment interacts, in its turn, with the immobilized heavy chain; this interaction is provided for by the lysin binding sites of the plasminogen molecule. The plasmin light chain having no lysin binding sites is specifically absorbed on the immobilized fragment D, whereas the D-fragment--on the immobilized light chain. The elution is caused by arginine or benzamidine; 6-aminohexanoic acid does not affect this interaction. It is assumed that the interaction of plasminogen and plasmin with fibrin is provided for not only by the lysine binding but also by the benzamidine binding sites of the plasminogen molecule.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

Fibrinogen fragment D is necessary and sufficient to anchor a surface plasminogen-activating complex in Streptococcus pyogenes

In this study, the importance of different domains of the fibrinogen molecule in the binding and assembly of a surface plasminogen (plgn) activator has been analyzed. This was achieved using SELDI technology that enabled dissociation of bound fragments from intact bacteria and accurate distinction between fibrinogen fragments based on their molecular mass. These studies indicate that Streptococcus pyogenes binds directly to human fibrinogen fragment D but not fragment E. The predominant surface proteins binding to fragment D were associated with the mrp gene product. Surface-associated fibrinogen fragment D was capable of anchoring a functional surface plgn activator complex. Taken together, these data indicated that fragment D of fibrinogen is necessary and sufficient to anchor a plgn activator complex on the surface of Streptococcus pyogenes.     

Thrombospondin interaction with fibrinogen. Evidence for binding to the A alpha- and B beta-chains of fibrinogen

Platelet thrombospondin interacts with fibrinogen in a specific and saturable manner. Thrombospondin was found to specifically bind to the A alpha- and B beta-chains of fibrinogen; binding was independent of divalent ions. Binding could be blocked either by preincubation of thrombospondin with 9.4 microM fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. Thrombospondin bound only to the beta-chain component of the D and DD plasmin fragment of fibrinogen. Thrombospondin interaction with fibrinogen was not blocked by preincubation with synthetic peptides which have previously been identified as either the fibrinogen receptor (alpha 572-575, the synthetic tetrapeptide arginyl-glycyl-aspartyl-serine) or cell attachment (gamma 400-411) domains. Fibrinogen, therefore, possesses at least two unique and distinct sites, within the A alpha- and B beta-chains, for its interaction with thrombospondin.     

Fibronectin receptors of mononuclear phagocytes: Binding characteristics and biochemical isolation

Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, we have investigated the hypothesis that proteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. We report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. The Ki for inhibition of attachment (0.5 microM) was consistent with the Kd for binding of the 3H-labeled 80-kDa fragment (0.34 microM) to U937 cells in suspension. There were 4-5 x 10(5) 80-kDa binding sites per cell. The relatively high affinity of the 80-kDa fragment for the monocyte surface permitted the isolation and characterization of fibronectin-binding proteins from U937 cells and peripheral blood monocytes by affinity chromatography. When octylglucoside lysates of lactoperoxidase iodinated cells were applied to 80-kDa-Sepharose columns, a polypeptide complex of 152/125 kDa was eluted with the synthetic peptide GRGDSPC, but not with GRGESP. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.     

Morphological studies of N-acetylglucosamine induced germ tube formation by Candida albicans

In N-acetylglucosamine induced germ tube formation by Candida albicans, multiple (up to five) protuberances appeared within 90 min at 37 degrees C on each yeast cell. The protuberances were extensions of the cytosol and contained vesiclelike structures. Usually only one protuberance subsequently developed into a germ tube. The germ tubes emanated from all aspects of the cell surface but seldom from the budding (long axis) poles. Pseudohyphae, which originated from the budding pole, exhibited a marked constriction at the site of emergence and were 0.6-2.5 microns in diameter compared with a diameter of 0.6-0.8 micron for germ tubes. The presence of septa confirmed that germ tubes are precursors of septate mycelia. Ultrathin-section transmission electron microscopy of aldehyde plus osmium fixed cells revealed electron-lucent walls with a thin electron-dense outer layer. A fibrillar border was also routinely associated with germ tubes. Poststaining with potassium permanganate revealed, in addition, a previously invisible fuzzy layer on the outer region of the cell wall which extended over bud scars and germ tubes and which coalesced at sites of contact between cells.     

Extracellular fibrinogen-binding protein, Efb, from Staphylococcus aureus blocks platelet aggregation due to its binding to the alpha-chain.

Extracellular fibrinogen-binding protein (Efb) secreted by Staphylococcus aureus has previously been shown to contribute to pathogenesis in a rat wound infection model. Also antibodies against Efb exhibited a protective effect in a mouse mastitis model. The interaction between Efb and fibrinogen is divalent, with one binding site within the N-terminal repeat region in Efb and one at the C terminus. In this study we show that the distal D domain of fibrinogen contains at least one of the binding domains recognized by Efb. Efb stimulates fibrinogen binding to ADP-activated platelets. Furthermore, Efb inhibits ADP-induced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner. This implies that Efb modifies platelet function by amplifying a non-functional interaction between fibrinogen and platelets. Efb recognizes the A alpha-chain of the D fragment of fibrinogen. The RGD sequence on the A alpha-chain is located close to the region recognized by Efb and contains a putative binding site for the platelet integrin GPIIb/IIIa receptor complex involved in platelet aggregation.     

Binding of lys-plasminogen to E-fragment of fibrinogen

The interaction of Lys-plasminogen and its fragments with fibrinogen fragment E was studied by equilibrium affinity binding. A quantitative analysis of binding parameters revealed two types of binding sites responsible for Lys-plasminogen interaction with the immobilized fragment E, i.e., with a high (Kd = 1.5 x 10(-6) M) and low (Kd = 82 x 10(-6) M) affinity ones. Among plasminogen fragments, only miniplasminogen and KI-3 bound immobilized fragment E and were eluted by epsilon-aminocaproic acid. Hence, two lysine binding sites may be involved in the binding of Lys-plasminogen to fragment E; they are localized in the KI-3 and K5 kringle structures.     

Binding of Glu-plasminogen by fibrinogen and byproducts of its proteolysis

The ability of the native form of plasminogen (Glu-plasminogen) to form complexes with fibrinogen and its fragments immobilized on CNBr-agarose was studied. It was found that unlike Lys-plasminogen, the native form of the proenzyme does not bind to fibrinogen agarose. Limited proteolysis of fibrinogen by plasmin involving alpha C-domains results in the appearance of Glu-plasminogen binding sites at fibrinogen surface. The X2 fragment of fibrinogen binds to about 0.5 moles of Glu-plasminogen at an equimolar ratio of the interacting proteins. Under these conditions, the amount of bound Glu-plasminogen does not increase as a result of subsequent hydrolysis of fibrinogen down to end products, fragments E and D. It was found that Glu-plasminogen interacts with both E- and D-fragments of fibrinogen. Similar to Lys-plasminogen, Glu-plasminogen exhibits a high affinity for the E-fragment. The maximal quantity of the bound protein under the given experimental conditions is 2 moles per mole of the immobilized E-fragment. The interaction of Glu-plasminogen with the E-fragment is mediated by the lysine-binding sites of the proenzyme with a high and low affinity [Kd = 1.8.10(-6) and 7.5.10(-5) M, respectively]. Glu-plasminogen, unlike Lys-plasminogen, shows a low affinity for the D-fragment (Kd = 2.10(-5) M). Glu-plasminogen cannot be adsorbed by arginine-binding sites at the DH fragment-agarose.     

Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.     

An investigation of the role of true hypha production in the pathogenesis of experimental oral candidosis

From 427 cases of human oral candidosis 2135 yeast clones were screened for the presence of germ tube-negative C. albicans and variants that formed only pseudohyphae in serum; one strain of each was found. The pathogenic potential of the serum-pseudohyphal and germ tube-negative C. albicans variants was investigated in the oral cavity of the rat; both variants failed to induce palatal candidosis, in contrast to a germ tube-positive C. albicans control strain. The pathogenic potential of C. albicans strains appears to be dependent on the formation of true germ tubes.