Binding Specificity of Mouse Serum Amyloid P-Component for Fibronectin

Binding between purified mouse serum amyloid P-component (SAP) and plasma fibronectin (Fn) occurred when either one of the proteins was immobilized by specific antibody and the second protein was offered in a soluble form. Binding of Fn to immobilized SAP was cooperative and saturable at a molar ratio of SAP/Fn = 7.1. The molar ratio at saturation was 3.7 for SAP/Fn when SAP was allowed to bind to immobilized Fn. The binding required 2 to 3mM amounts of Ca⁺⁺. The binding of SAP to Fn was selectively inhibited by a monoclonal antibody specific for the mid-molecule region of Fn, by soluble gelatin, and by heparin in the presence of 3mM Ca⁺⁺. We conclude that the SAP binding site was localized at the mid-molecule region of Fn that includes the adjacent gelatin-binding domain and the heparin-I binding domain.     

Collagen receptor on T lymphocytes and the control of lymphocyte motility

Human lymphocytes, freshly isolated from blood, were allowed to settle on surfaces coated by collagen type 1, fibronectin, laminin, IgG or albumin at different concentrations. In separate cultures the lymphocytes were also exposed to these proteins in soluble form. The lymphocytes, predominantly T cells, attached to two-dimensional collagen substrata both in the presence and absence of serum but did not adhere or adhered poorly to substrata coated with fibronectin, laminin, IgG and albumin. In contrast, T blasts induced in a mixed lymphocyte culture adhered to fibronectin-coated substratum. During contact with substratum-bound collagen for a 24-h period, 47 +/- 15% of the freshly purified lymphocytes from separate individuals developed motile behavior whereas 16 +/- 4% of the cells became motile on fibronectin. Gelatin (denatured collagen) also mediated attachment of lymphocytes to surfaces but only at comparatively high concentrations (40 mg/ml). Collagen and gelatin in solution also caused agglutination and motility of the vast majority of freshly isolated T lymphocytes whereas fibronectin and other proteins, when presented in soluble form, did not. Cell agglutination was maximal at moderate (10 or 20 mg/ml) and cell motility at low gelatin concentrations (1 to 10 mg/ml). High gelatin concentrations (20 and 40 mg/ml) did not induce motile behavior. Cytochalasin B augmented the proportion of adherent cells on gelatin-coated substrata. In the presence of cytochalasin B gelatin mediated substrate-adhesion at concentrations below those which normally induced adhesion indicating that motile behavior counteracted persistent lymphocyte adhesion to the substratum. Noteworthy, gelatin/collagen is unique among ligands (e.g. plasma fibronectin and other serum proteins) in its capacity to induce motility in the vast majority of resting lymphocytes freshly isolated from blood within a relatively short period. Taken together these results indicate that circulating lymphocytes have a collagen/gelatin-binding plasma membrane component. Cross-linking of this component is a likely explanation for the selective inducing effect of gelatin and collagen on lymphocyte motility. The present results showed that the lymphocyte plasma membrane contains collagen-binding components with a relative molecular mass of 130 and 55 kDa. The 55-kDa component also reacted with an anti-fibronectin antibody. Thus, interactions with the extracellular matrix may control lymphocyte locomotor capacity.     

Identification of a fibronectin binding protein from Streptococcus mutans

The interaction of viridans streptococci with components of the extracellular matrix (ECM) plays an important role in the pathogenesis of infective endocarditis. We have identified a surface protein of Streptococcus mutans which binds the ECM constituent fibronectin (Fn). Initially, we found that S. mutans could adsorb soluble Fn in plasma, but with lower efficiency than Streptococcus pyogenes. In addition, S. mutans could bind immobilized Fn in a dose-dependent manner when tested using an enzyme-linked immunosorbent assay. Crude extracts of cell wall-associated proteins or extracellular proteins from S. mutans MT8148 specifically bound Fn through a protein with the molecular mass of ca. 130 kDa, as detected by far-Western immunoblotting. The candidate Fn binding protein (FBP-130) was purified to near homogeneity by using Fn coupled Sepharose 4B affinity column chromatography. A rabbit polyclonal antibody against FBP-130 reacted specifically with a protein of molecular mass of ca. 130 kDa in both cell wall and extracellular fractions, and the abundance of FBP was higher in the former than in the latter fractions. The purified FBP bound specifically to immobilized Fn, whereas the binding of soluble Fn to coated FBP could only be detected in the presence of high concentrations of Fn. The purified FBP, as well as anti-FBP immunoglobulin G, inhibited the adherence of S. mutans to immobilized Fn and endothelial cells (ECV304) in a dose-dependent manner. These results demonstrated that FBP-130 mediated the adherence of S. mutans specifically to Fn and endothelial cells in vitro. The characteristics of S. mutans and FBP-130 in binding Fn confirmed that viridans streptococci adopt different strategies in their interaction with ECM.     

Identification with monoclonal antibodies of different regions of human plasma fibronectin, including that which interacts with human monocyte fibronectin receptors.

The determinant specificities of five monoclonal anti-fibronectin antibodies, designated BC7, CE9, BD4, AB3 and CPG1, were defined and mapped within intact human plasma fibronectin by immunoblot analyses with defined fragments of fibronectin. The latter were derived by tryptic, chymotryptic or cathepsin D digestion of the intact molecule and fractionated by DE-cellulose chromatography and gelatin and/or heparin affinity chromatography. Monoclonal BC7 recognizes intrachain disulphide-formed determinants within the 27,000 MW N-terminal domain; monoclonal CE9 recognizes determinants within an 18,000 MW fragment immediately adjacent to the carboxyl end of the gelatin-binding domain; monoclonal BD4 recognizes determinants within the cell-adhesive domain and within 150,000 of the N-terminus; monoclonal AB3 recognizes intrachain disulphide-formed determinants within 35,000 of the COOH-terminus of the intact molecule and detectable only on the alpha-chain polypeptide subunit; and monoclonal CPG1 recognizes determinants present on both chains of the intact molecule and immediately adjacent to the interchain disulphide bonds at the COOH-terminus. None of the epitopes recognized by these monoclonal antibodies is present at alternative regions of the intact molecule. Fab fragments of each of these monoclonal antibodies were incubated with gelatin-coated sheep erythrocytes which had been reacted with a fixed amount of intact plasma fibronectin. When these target particles were incubated with monolayers of human monocytes and the resultant rosettes were quantitated, the Fab fragments of BD4 markedly inhibited the proportion of monocytes binding these fibronectin-bearing targets, whereas none of the other Fab fragments had an inhibitory effect. Thus, monocyte fibronectin receptors which mediate adherence of fibronectin bridges to a target via gelatin recognize regions within the cell-adhesive domains of intact fibronectin but not regions at the amino or carboxy terminals.     

Binding of plasma fibronectin to Candida albicans occurs through the cell binding domain

Candida albicans yeast cells bind soluble human plasma fibronectin (Fn) through a glycoprotein receptor (adhesin) located on the cell surface. This work demonstrates that a 120 kDa proteolytic fragment of Fn encompassing the cell binding domain binds more avidly to the yeast cell adhesin than does the parent Fn molecule. The presence of binding of Fn fragments containing heparin- and gelatin-binding domains of Fn could not be detected.The binding of the 120 kDa fragment is inhibited by a monoclonal antibody to the cell binding domain containing the amino acid sequence, Arginine-Glycine-Aspartic acid (RGD) as well as by an RGD-containing ∼23-mer Fn peptide, but not with heparin or GRGDSPL. The fact that the cell binding domain of soluble Fn binds more avidly than does the parent molecule may explain the difference in the interaction of soluble Fn and immobilized Fn with Candida. It is possible that, upon immobilization, Fn may expose domains of the molecule previously unexposed when the molecule is in the soluble state.     

Interaction of human plasma fibronectin with cariogenic and non-cariogenic oral streptococci

The interaction of purified human plasma fibronectin (Fn) with bacteria was studied with a variety of oral streptococci. Each of the strains of Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis tested was aggregated by Fn to various degrees, depending on the concentration of Fn added to the test mixtures. Binding assays performed with radiolabeled Fn and various strains of streptococci demonstrated various capabilities to bind Fn, and the amount of Fn bound by each strain was paralleled by its Fn-induced aggregation, with S. mutans 6715 giving the highest values in both assays. Because of the avid binding of Fn by certain strains of potentially cariogenic streptococci, we investigated the possibility that Fn may be present in human saliva and may be adsorbed from saliva onto artificial tooth pellicles. Immunoreactive Fn was detected in paraffin-stimulated whole saliva by enzyme-linked immunosorbent assays of saliva adsorbed onto gelatin-coated cuvettes and by immunoelectroblots (Western blots) of salivary components separated on sodium dodecyl sulfate-polyacrylamide slab gels. Furthermore, immunoreactive Fn was found to be present in artificial tooth pellicles formed by incubating hydroxyapatite beads with whole human saliva. These results demonstrate that certain strains of oral streptococci bind to and are aggregated by Fn. The presence of Fn in artificial tooth pellicles suggests that this macromolecule may play a role in the attachment of potentially cariogenic and other oral streptococci to dental tissues.     

Lipopolysaccharide enhances monocyte adherence to matrix-bound fibronectin

Fibronectin (Fn), an extracellular matrix glycoprotein with binding sites for collagen, fibrin, heparin, and cell surfaces, is a nonimmune opsonin which up-regulates phagocytic function and facilitates adherence of human monocytes. We have developed a simple assay to study adherence of peripheral blood monocytes to Fn on a gelatin matrix. While cell adherence was enhanced by the presence of Fn in a dose-dependent manner, it was inhibited by peptides containing the Arg-Gly-Asp (RGD) cell attachment sequence or by coating the matrix with antibodies directed against Fn. Preincubation of monocytes for 30 min with Escherichia coli lipopolysaccharide (LPS) at doses of 1-50 micrograms/ml increased adherence to Fn-gelatin but not to gelatin alone, while longer preincubation (24 hr) resulted in similar changes at lower doses (0.01-1.0 micrograms/ml). Enhanced Fn adherence may be essential for monocyte localization to sites of inflammation.     

The Haemophilus influenzae Hap autotransporter binds to fibronectin,laminin, and collagen IV

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Hap(s), from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Hap(s). Purified Hap(s) binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Hap(s) to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract.     

Specific attachment of Staphylococcus aureus to immobilized fibronectin.

Staphylococcus aureus cells have been shown to possess surface-associated proteins with affinity for soluble fibronectin. We have investigated the ability of these surface proteins to mediate attachment to immobilized fibronectin and collagen. Attachment was quantified by determination of bacterial ATP in a bioluminescence assay. The ability to attach to fibronectin- or collagen-coated plastic surfaces was investigated for four S. aureus strains: Cowan 1, Newman, SA113(83A), and Wood 46. Cells from the different strains varied in their attachment properties, but all cells except those of strain Wood 46 attached readily to substrates coated with fibronectin. Only cells from strain Cowan 1 attached reproducibly to collagen-coated substrates in the absence of fibronectin. The attachment of cells from strain SA113(83A) to fibronectin-coated surfaces was shown to be dependent on time, fibronectin concentration, and bacterial growth phase. Soluble fibronectin or NH2-terminal fibronectin fragment (Mr, 29,000) disturbed the attachment to surfaces coated with fibronectin bound to denatured collagen type I. The attachment process to such substrates was also effectively inhibited by preincubating the substrate with fibronectin-binding proteins isolated from S. aureus Newman and SA113 (83A) and purified with affinity chromatography.     

Rapid identification of fibronectin, vitronectin, laminin, and collagen cell surface binding proteins on coagulase-negative staphylococci by particle agglutination assays.

Seventeen strains of ten different species of coagulase-negative staphylococci were shown to interact with collagen, laminin, fibronectin, and vitronectin immobilized on latex beads. Different species of coagulase-negative staphylococci have different capacities to agglutinate proteins. Cells of 18 strains of Staphylococcus haemolyticus reacted more strongly than did cells of 18 Staphylococcus epidermidis strains with proteincoated latex beads, although no significant difference in cell surface hydrophobicity or charge could be shown. The cell surface receptors of S. haemolyticus were more heat and protease resistant than were Staphylococcus aureus receptors. Strains of Staphylococcus saprophyticus isolated from urinary tract infections showed a high capacity to adhere to laminin. The ability to agglutinate fibronectin and collagen was common among coagulase-negative staphylococci isolated from other infections; 55% (31 of 56) and 63% (35 of 56) agglutinated fibronectin and/or collagen. S. haemolyticus and S. epidermidis bound to both N-terminal (29-kDa) and C-terminal (120-kDa) fragments of fibronectin.     

Fibronectin and the adhesive properties of rat lymphocytes obtained from different peripheral lymphoid tissues

A comparative investigation has been carried out on the effect of plasma fibronectin (Fn) on the adhesive properties of normal rat lymphocytes obtained from different lymphoid tissues: blood, spleen, mesenteric and tonsillar lymph nodes. Fn was immobilized on the basis of its ability to bind to gelatin. We established that concentrations of 40-50 micrograms/ml are sufficient for a saturation effect on Fn coating. For spleen cells an adhesion of 55.7 +/- 9.3%, for mesenteric lymph nodes 34.5 +/- 8.7% and for tonsillar cells 33.8 +/- 3.2% was observed. Blood lymphocytes showed the lowest adhesion, 21.3 +/- 4.2%. Compared to the other lymphoid tissues, the spleen cells exhibited a "basal" adherence to surfaces coated with gelatin only: 19.2 +/- 4.1%. T lymphocytes participate to a greater extent in the process, since their number was significantly reduced in cell suspensions after adhesion to both gelatin and gelatin-Fn coated surfaces. The addition of soluble Fn leads to a competitive inhibition of the lymphocyte adhesion to gelatin-Fn coated surfaces. The data demonstrated the important role of Fn for the adhesive interactions of lymphocytes during their functional distribution in the tissues.     

Interaction of plasma fibronectin with gelatin and complement C1q

A variety of techniques have been used to examine the interaction of human plasma fibronectin (Fn) with complement C1q in comparison to that with gelatin in phosphate buffered saline at pH 7.4. The precipitation of 3H-Fn by polyethylene glycol (PEG) was shifted to much lower concentrations of the polymer by addition of gelatin, and to a lesser extent, by C1q. Precipitation of 3H-Fn in the presence of C1q was close to that of C1q alone under identical conditions suggesting an affinity of Fn for solid phase C1q; a similar interaction was seen with heat-insolubilized C1q. Fibronectin bound tightly to gelatin-Sepharose and C1q-Sepharose and this binding could be inhibited by gelatin but not by C1q. The presence of gelatin retarded the anodal migration of Fn during immunoelectrophoresis under physiological conditions whereas C1q had an effect only at low ionic strength. Exclusion chromatography of Fn, alone and preincubated with gelatin or C1q, was also consistent with the formation of strong complexes with gelatin but not with C1q, whereas similar mixtures of Fn and gelatin exhibited a fast-sedimenting boundary and marked depletion of the 12S Fn peak. Titration of fluorescein-labeled alpha 2 chains of type I collagen with Fn produced an increase in fluorescence polarization which could be reversed by addition of unlabeled alpha 2 chains or gelatin but not by C1q or the pepsin-derived collagen-like domain of C1q. These observations indicate that the fluid-phase interaction of Fn with C1q is much weaker than that with gelatin but that Fn does have appreciable affinity for solid-phase C1q. Such interaction could signify a role for Fn in the clearance of immune complexes from circulation.     

Involvement of fungal cell wall components in adhesion of Sporothrix schenckii to human fibronectin

Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable. S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca(2+), inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.     

Fibronectin fragments released from phorbol ester-stimulated pulmonary artery endothelial cell monolayers promote neutrophil chemotaxis.

We have recently shown that monolayer cultures of calf pulmonary artery endothelial (CPAE) cells pretreated with phorbol myristate acetate (PMA) generate a conditioned medium that is chemotactic for human polymorphonuclear leucocytes (PMNL). Fibronectin (Fn) is a multidomain protein found in the plasma and subendothelial extracellular matrix that induces attachment and migration of a variety of cell types. The present study was designed to evaluate the role of Fn or fragments of Fn present in conditioned medium from phorbol ester-stimulated endothelial cells as potential chemotactic factors for human PMNL. A large number of Fn fragments were revealed by Western immunoblotting of serum-free conditioned medium 4 hr after treatment of CPAE monolayers with PMA. Gelatin-Sepharose affinity chromatography of 4-hr conditioned medium demonstrated chemotactic activity for PMNL in both gelatin-binding and non-gelatin-binding fractions. The addition of bovine Fn antiserum to the conditioned medium inhibited PMNL chemotaxis in a dose-dependent manner while having no effect on PMNL chemotaxis generated by zymosan-activated serum. One site on the Fn molecule known to interact with phagocytic cells is the cell-binding domain containing the Arg-Gly-Asp (RGD) sequence. Pretreatment of PMNL with a RGD-containing peptide (1 mM GRGDSPK) for 10 min completely inhibited the expression of chemotactic activity present in conditioned medium and in the gelatin-binding and non-gelatin-binding fractions. PMNL chemotaxis was not stimulated by either intact Fn or the RGD-containing septapeptide tested over a wide concentration range. However, incubation of PMNL with a purified 120,000-MW fragment of Fn containing the cell-binding domain stimulated chemotaxis in a dose-dependent manner. In contrast, a purified 45,000 MW fragment of Fn containing the gelatin-binding domain was not chemotactic for PMNL. When a monoclonal antibody directed against the cell-binding domain of Fn was incubated with conditioned medium, a significant reduction in PMNL chemotaxis was observed. These results demonstrate that phorbol ester-stimulated pulmonary artery endothelial cells release Fn fragments and suggest an important role for Fn fragments containing the cell-binding domain in stimulating the migration of PMNL.     

Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. We studied the quantitative adsorption of fibronectin onto polymethylmethacrylate (PMMA) cover slips by using a 125I-labeled preparation of the purified plasma glycoprotein. Fibronectin in buffer solutions showed a high affinity to PMMA coverslips. Adherence of S. aureus Wood 46 was studied on PMMA pre-exposed to fibronectin, using an assay specifically adapted to the cover slip model. Whereas S. aureus adherence in an albumin-containing buffer was less than or equal to 10(3) CFU on control uncoated cover slips, adherence in the same medium increased up to maximum of 7.7 X 10(4) CFU on cover slips preincubated in a solution of fibronectin (125-micrograms/ml). At intermediate fibronectin concentrations, bacterial adherence was a linear function of both the quantity in solution and of the quantity adsorbed on the PMMA cover slips. The presence of human serum proteins, as represented by a fibronectin-depleted pool, essentially prevented adsorption of radiolabeled fibronectin on PMMA and subsequent bacterial adherence on the cover slips. Precoating of PMMA with denatured collagen resulted in increased fibronectin adsorption on PMMA, even in the presence of serum proteins, and S. aureus adherence was optimal on such surfaces. Collagen may therefore play a role as a cofactor contributing to S. aureus adherence onto fibronectin-coated substrata or foreign bodies.     

Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.     

Enhanced levels of attachment of fibronectin-primed Treponema pallidum to extracellular matrix.

Freshly extracted Treponema pallidum organisms treated with exogenous human fibronectin (Fn) (Fn-primed treponemes) showed a 6- to 15-fold increased level of attachment to Fn-coated cover slips and to extracellular matrix (ECM) when compared with unprimed treponemes. Treponemes primed with collagen or laminin showed no similar enhanced binding to immobilized Fn or ECM. Preexposure of immobilized Fn and ECM to anti-Fn serum but not to anti-collagen or anti-laminin serum prevented treponemal adherence. Also, the presence of proteoglycanlike molecules such as dextran sulfate or heparan sulfate inhibited Fn-primed treponemal attachment to Fn or ECM. In contrast Fn-primed treponemes did not exhibit elevated levels of attachment to eucaryotic cell monolayers. To understand the increased tropism of Fn-primed T. pallidum organisms for Fn and ECM-like surfaces, we radiolabeled freshly extracted treponemes with [35S]methionine and examined them for the presence of surface immunoreactive Fn. Magnetic protAspheres and glass beads coated with monospecific anti-Fn serum bound only 20 to 30% of radiolabeled treponemes. Nonadherent treponemes failed to bind to gelatin-agarose, further confirming the absence of surface Fn or Fn-like material. Fn-free organisms, however, did attach to Fn-coated cover slips and to cell monolayers like treponemes of the original population. Incubation of Fn-free treponemes with human Fn resulted in almost total binding of organisms to anti-Fn antibody on glass beads and also produced increased attachment to Fn-coated cover slips and ECM. These results suggest that enhanced interactions between T. pallidum and the host are dependent on the presence of Fn on syphilis spirochetes and the specific location and orientation of Fn in vivo.     

Binding of Bartonella henselae to extracellular molecules: identification of potential adhesins

Bartonella henselae, the etiologic agent of cat scratch disease, bacillary angiomatosis and other clinical syndromes initiates infection through a trauma or wound to the skin suggesting involvement of extracellular matrix molecules. We have demonstrated in this study that B. henselae bound strongly fibronectin, collagen IX and X, but comparatively less laminin and collagen IV. B. henselae bound primarily the N- and C-terminal heparin (Hep-1 and Hep-2, respectively) and the gelatin-binding domains of fibronectin (Fn) but not the cell-binding domain. Binding to the Hep-binding domain was significantly inhibited by Hep suggesting common binding sites on the Fn molecule. Furthermore, glycosaminoglycans-mediated binding of B. henselae to soluble Fn showed that Hep but not dextran sulfate inhibited the bacterium binding to Fn. Unlike Fn, B. henselae bound strongly vitronectin only in the presence of Hep or dextran sulfate. Also, the binding of B. henselae to host cells could be inhibited by anti-B. henselae surface-reactive antibodies, the exogenous Fn or the anti-Fn polyclonal antibodies. Ligand blots, batch affinity purification and MALDI-TOF peptide fingerprinting identified B. henselae Pap31, Omp43 and Omp89 as the three major putative Fn-binding proteins (FnBPs) in B. henselae outer membrane proteins. We hypothesized that B. henselae wound associated infections involved interactions with extracellular matrix molecules. Taken together, the above data suggest that interactions between B. henselae and ECM molecules such as Fn may play an important role in the bacterium adherence to and invasion of host cells.     

In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.     

High-affinity binding of fibronectin to cultured Kupffer cells

Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative "fibronectin receptors" per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.