Adherence of Streptococcus pneumoniae to Immobilized Fibronectin

INFECTION AND IMMUNITY, Nov. 1995, p. 4317–4322 0019-9567/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 63, No. 11

Adherence of Streptococcus pneumoniae to Immobilized Fibronectin MICHIEL

FLIER,1 NOK CHHUN,1 THERESA M. WIZEMANN,1 JENNY MIN,1 JAMES B. MCCARTHY,2 AND ELAINE I. TUOMANEN1*

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Laboratory of Molecular Infectious Diseases, The Rockefeller University, New York, New York 10021,1 and Department of Laboratory Medicine and Pathology, Biomedical Engineering Center, University of Minnesota Medical School, Minneapolis, Minnesota 554552 Received 1 June 1995/Returned for modification 6 August 1995/Accepted 22 August 1995

Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade injured epithelia. Streptococcus pneumoniae was found to adhere to immobilized fibronectin more avidly than other streptococci and staphylococci do. Binding was dose, time, and temperature dependent. Trypsin treatment of the bacteria resulted in decreased binding, suggesting that the bacterial adhesive component was a protein. Fragments of fibronectin generated by proteolysis or by expression of recombinant gene segments were compared for the ability to bind pneumococci and to compete against bacterial binding to immobilized fibronectin. Fragments from the carboxy-terminal heparin binding domain were consistently active, suggesting that this region contains the pneumococcal binding site, a region distinct from that supporting the attachment of most other bacteria. teins identified and sequenced for S. aureus, Streptococcus pyogenes, and Streptococcus dysgalactiae each demonstrate a similar amino acid sequence motif, EDTxxxxxxxGGxxxxxEF (14), suggesting common features required for binding to the Nterminal region of fibronectin (12, 14, 32). In addition to protein-mediated adherence to fibronectin, the lipoteichoic acid of S. pyogenes has been suggested to mediate binding by bridging the bacteria to fibronectin, particularly in the context of bacterial adherence to oral epithelia (5). Since soluble fibronectin is ubiquitous in body fluids, it stands to reason that bacteria might need to avoid saturation of fibronectin-binding proteins by plasma fibronectin in order to become attached to immobilized cells or matrix. Several microorganisms have been reported to interact differently with soluble versus solid-phase fibronectin, presumably reflecting the considerable difference in presentation of epitopes in the two forms (28). Group B streptococci, Streptococcus sanguis, and Yersinia spp. bind fibronectin adherent to solid phase but not soluble fibronectin (11, 20, 25). S. aureus binds to material in both phases, but it has been suggested that the mechanisms of binding are different (10). Studies on the ability of S. pneumoniae to bind to fibronectin have been conducted uniformly in solution and have yielded conflicting results. Several studies have reported no binding (9, 13, 24). In contrast, Courtney and colleagues detected binding that was quantitatively and qualitatively similar to that of S. aureus (5, 8), and Andersson and colleagues noted that the attachment of pneumococci to epithelial cells increased after pretreatment of the eukaryotic cells with fibronectin (1, 2). Insight into these discrepancies is provided by the observations described in this report. We show that S. pneumoniae adheres avidly to immobilized but not soluble fibronectin. We characterize the variables which affect this property and present evidence that the binding site in the fibronectin molecule is within the C-terminal heparin binding domain, a rare site for microbial targeting.

Streptococcus pneumoniae is a major, gram-positive pathogen which causes invasive infections such as sepsis, meningitis, and, less frequently, endocarditis (26). The pneumococcus colonizes the nasopharyngeal epithelium (3) and then is presumed to penetrate the epithelium of the lung or nasopharynx in order to reach the vascular compartment. Such translocation would involve, of necessity, an encounter with basement membrane and extracellular matrix components, such as fibronectin. Colonization and invasion in the presence of mucosal injury which exposes basement membrane components would also be expected to be enhanced by an ability to recognize fibronectin. In support of this scenario, pneumococci have been shown to bind poorly to ciliated cells of the airways but to bind avidly to basement membrane components which become exposed following injury to respiratory epithelium induced by pneumolysin or viral infection (15, 17). Fibronectin is a mammalian glycoprotein present as a soluble dimer (molecular mass, 550 kDa) in body fluids such as plasma (200 to 700 mg/ml), cerebrospinal fluid, and amniotic fluid and as a less soluble multimer in the extracellular matrix and basement membrane (18). Fibronectins from different species have a high degree of similarity at both the amino acid and nucleotide sequence levels. Fibronectin serves diverse biological functions, including roles in metastasis, wound healing, embryogenesis, blood clotting, phagocytosis of cell debris, and possibly opsonization of microorganisms. Fibronectin has specific binding sites for a number of proteins, including collagen and integrins, and has two binding sites for heparin (see Fig. 1). Since the finding that Staphylococcus aureus binds to fibronectin, many microorganisms, including oral streptococci and some gram-negative bacteria, have been shown to bind to the molecule (12, 14, 32). This property has been associated with a propensity to establish colonization of endovascular lesions, an important step in the pathogenesis of infective endocarditis (8, 19). These diverse pathogens most commonly target the type 1 repeats of the N-terminal heparin binding domain of fibronectin. The cognate fibronectin-binding pro-

MATERIALS AND METHODS Bacterial strains and growth conditions. The serotypes of S. pneumoniae described below (with strains given in parentheses) were tested. Types 4 (p31), 7F (p27), 9V (p10), 14 (p30), 18C (p57), 19F (p115), and 23F (p26) were clinical isolates obtained from J. Weiser and R. Austrian, University of Pennsylvania, Philadelphia, Pa. (31). Type 2 (D39) and the isogenic, unencapsulated derivatives

* Corresponding author. Mailing address: The Rockefeller University, Box 104, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8276. Fax: (212) 327-7428. 4317

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FIG. 1. Schematic representation of the domains of the fibronectin molecule. Fragments tested for the ability to bind pneumococci are indicated by their molecular masses and by their known binding specificities. Alternative fragment sizes generated by different enzymatic cleavage techniques are shown in parentheses. Fn51 is a recombinant protein representing the 33-kDa heparin binding domain. Bacterial binding regions are indicated in italics. This study supports pneumococcal binding to the C-terminal heparin binding domain.

R6x and R6xSpxB2 and Escherichia coli DH5a were obtained from R. Masure, The Rockefeller University, New York, N.Y. (23). S. pyogenes D471 (type 6) was obtained from V. Fischetti, The Rockefeller University. S. aureus PES was a clinical isolate provided by A. Tomasz, The Rockefeller University. Bacteria were grown on tryptic soy agar containing 5% sheep blood for 16 to 18 h at 378C in 5% CO2 or in a candle extinction jar. Alternatively, bacteria were grown in semisynthetic medium to control for growth phase and possible adsorption of components of blood to the bacterial surfaces. Under no conditions were differences in adherence to fibronectin detected for bacteria grown on plates versus those grown in broth. For some experiments, bacteria were exposed to trypsin (1 mg/ml at pH 7.0; Sigma, St. Louis, Mo.) and incubated at 378C for 60 min (1). The trypsin was inactivated by trypsin inhibitor (10 mg/ml; Sigma) at 378C for 60 min. For some experiments, bacteria were killed by heating for 10 min at 608C. Cell wall preparation. Pneumococcal cell wall was prepared as described previously (27). Briefly, logarithmically growing pneumococci were heat killed. Crude cell wall was extracted in 5% sodium dodecyl sulfate (SDS) at 1008C for 15 min. The pellet was washed to remove detergent and then was vortexed with glass beads to mechanically break any remaining intact cells. The cell wall was pelleted and then treated with DNase, RNase, and then trypsin to remove associated proteins. Cell wall was reprecipitated in SDS and then washed and finally dried with a speed vacuum and was stored at 2208C. Fibronectin and derived proteolytic fragments. Fibronectin from human plasma was purchased from Sigma and from Gibco BRL (Grand Island, N.Y.). The fragments, designated by molecular mass, are depicted schematically in Fig. 1. Proteolytic fragments purchased from Gibco BRL (indicated in parentheses in Fig. 1) included a tryptic 30-kDa collagen binding fragment, an a-chymotryptic 120-kDa cell binding fragment, and an a-chymotryptic 40-kDa heparin binding fragment. Proteolytic fragments prepared by following the method of Vercelotti et al. (29) (indicated in the boxes in Fig. 1) included a 27-kDa amino-terminal fragment, a 46-kDa collagen binding fragment, a 75-kDa cell binding fragment, a 33-kDa heparin binding fragment associated with a second 66-kDa fragment (33/66-kDa fragment), and a 31-kDa carboxy-terminal fragment. A recombinant fragment from the 33-kDa domain (Fig. 1) was prepared as described elsewhere (11a). Assay for binding to immobilized fibronectin and immobilized proteolytic fragments. Fibronectin or proteolytic fragments were noncovalently immobilized through passive adsorption to 60-well Terasaki trays (wettable polystyrene [plasma treated]; Robbins Scientific, Sunnyvale, Calif.). Briefly, fibronectin was reconstituted (50 mg/ml) in phosphate-buffered saline (DPBS) or 0.5 M NaCl–0.05 M Tris, pH 7.5, and incubated 4 to 24 h with the Terasaki trays at room temperature. The polystyrene surface was subsequently blocked with 5% bovine serum albumin (Sigma) for at least 3 h at 378C. Prior to use, the plates were washed five times with DPBS and excess liquid was removed from the wells. To quantify the amount of fibronectin adsorbed to each well by this procedure, 14 C-methylated fibronectin (Sigma) was used to coat the wells. Adsorbed fibronectin was eluted from the surface with trypsin (1 mg/ml) followed by 1 M NaOH (7), and the radioactivity was quantified. The amount of adsorbed fibronectin for plates coated with a 50-mg/ml solution was calculated to be 2.7 mg/cm2. This implies that the fibronectin was packed as a multilayer, since the saturation limit for monolayers is ,0.7 mg/cm2. Adsorption to a hydrophilic surface and dense packing of the molecules is preferable since it results in small conformational changes in the fibronectin (7, 28). In wells precoated with fibronectin fragments, the binding of fibronectin was reduced to ;1 mg/ml, indicating comparable binding of the fragments to the immobilized surface. Bacteria were labeled with fluorescein isothiocyanate as described previously (6) and were resuspended in DPBS supplemented with 0.05% glucose and Ca21 and Mg21. Bacterial suspensions were brought to an A620 of 0.04, previously established to equal 107 CFU of S. pneumoniae per ml. Bacteria (10 ml) were added to each well and incubated statically for 1 h at 378C. Unbound bacteria were eliminated by washing five times with DPBS. For cell wall competition experiments, the fibronectin-coated wells were preincubated with 5 ml of cell wall (50 mg/ml) for 30 min at 378C and then the bacterial suspension (5 ml of 2 3 107

INFECT. IMMUN. bacteria per ml) was added to each well. For heparin competition experiments, fibronectin-coated wells were preincubated (15 min, 378C) with 1, 10, 100, or 1,000 U of heparin (Sigma) per ml for 10 min at 378C. Bacteria (5 ml of 2 3 107 bacteria per ml) were added for 30 min at 378C, and then the wells were washed. Bound bacteria were fixed to the surface by incubation with 2.5% glutaraldehyde solution for 3 min. Bacteria were counted visually with an inverted microscope (Nikon) equipped with an IF DM 510 filter for viewing fluorescence. Binding was expressed as the number of attached bacteria per 0.25 mm2 of surface area. Values were corrected for nonspecific binding by subtracting adherence to fibronectin-uncoated, albumin-blocked wells and are presented as the means 6 standard deviations of at least three experiments with three to six wells per plate per experiment.

RESULTS Comparison of degrees of bacterial adherence to immobilized fibronectin. S. pyogenes, S. aureus, and E. coli have been shown to bind to fibronectin (8, 14, 32). The adhesion of S. pneumoniae R6x to immobilized fibronectin was significantly greater than that of any of these pathogens. The number of adherent bacteria per 0.25 mm2 of surface area for S. pneumoniae (range, 3,452 to 4,248) was ;2 to 3 times higher than that for S. pyogenes (range, 1,232 to 2,084), ;2 to 4 times higher than that for S. aureus (range, 716 to 2,636), and ;500 times higher than that for E. coli. Kinetic parameters. Binding was a function of the concentration of both the bacteria and the fibronectin. With a constant amount of fibronectin (50 mg/ml) to coat the wells, the effect of variation in bacterial density on adherence was determined (Fig. 2A). A dose dependence was observed for densities between 106 and 108 bacteria per ml. At bacterium densities of .108/ml, the numbers of bacteria per 0.25 mm2 of surface area were too high to count. Binding was also dependent on the fibronectin concentration. Binding was detectable at $0.4 mg/ml and increased with higher fibronectin concentrations to a plateau at ;6 mg/ml (Fig. 2B). Variation of the incubation time from 5 min to 3 h indicated that binding increased in a linear fashion until ;2 h, when an equilibrium was reached (Fig. 3). The incubation temperature influenced the equilibrium. With the number of bacteria bound per 0.25 mm2 of surface area after 2 h of incubation at 378C defined as 100% (3,017 6 700), binding was 91% 6 23% when the assay was performed at 308C and 51% 6 17% when the assay was performed at 48C (n 5 3). Heat killing the bacteria (10 min, 608C) did not decrease adherence significantly (85% 6 14% of the values for live bacteria; n 5 13). Bacterial components effecting adhesion. To assess the effect of the polysaccharide capsule on binding, we compared the fibronectin binding capacities of a series of clinical isolates of various serotypes which were chosen for their high prevalence in causing disease. The isolates of types 7F, 14, 18C, and 19F showed binding similar to that of the unencapsulated strain R6x, while types 9V and 23F were moderately impaired and types 2 and 4 bound very poorly (Fig. 4A). Strain D39 (type 2), which bound very poorly, is isogenic with the avidly adhering, unencapsulated R6x, indicating that in some strains the capsule might affect the presentation of adherence determinants. Switching of colonial morphology from transparent to opaque phenotypes has been shown to decrease pneumococcal virulence in a rat model of nasopharyngeal colonization and, concomitantly, to compromise the ability of pneumococci to adhere to eukaryotic cells in vitro (26, 31). However, no consistent difference in adherence to fibronectin between isogenic pairs of opaque and transparent pneumococci was noted (Fig. 4B). This was observed regardless of whether the parental strain demonstrated high or low levels of adherence within a designated serotype, as shown for type 9V in Fig. 4. This suggested that binding to fibronectin was not coregulated with

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FIG. 2. Effect of concentration of bacteria or fibronectin on adherence. (A) Wells were coated with fibronectin (50 mg/ml), blocked with albumin buffer, and incubated with various concentrations of bacteria at 378C for 2 h. The number of adherent fluorescein isothiocyanate-labeled bacteria is expressed as a percentage of the maximum observed at a bacterial concentration of 108/ml on the same plate. The 100% values ranged from 3,044 to 15,332 adherent bacteria per 0.25 mm2 of surface area. Points represent the mean percentages 6 standard deviations of all individual wells (n 5 6 to 18) for two to five separate plates. (B) Plates were coated with various concentrations of fibronectin, blocked with albumin buffer, and incubated with 107 bacteria at 378C for 2 h. Values are expressed as percentages of the value for 50 mg/ml on the same plate. The 100% value is 1,735 6 1,181 adherent bacteria per 0.25 mm2 of surface area. Points represent the means 6 standard deviations for three to five plates.

the ability to bind to other substrates. Consistent with this hypothesis, insertional inactivation of spxB, which results in a global decrease in adherence of pneumococci, did not affect the ability of pneumococci to adhere to fibronectin (control, 2,813 bacteria per 0.25 mm2; spxB2, 2,100 bacteria per 0.25 mm2; standard deviation, 400). Pretreatment of S. pneumoniae with trypsin to remove surface proteins resulted in loss of binding capacity. Trypsin treatment caused a decrease in adhesion of 93% 6 4% compared with that of the control without trypsin treatment (n 5 3). Protein-free pneumococcal cell wall (50 mg/ml) did not compete with the bacteria for binding to the immobilized fibronectin (104% 6 21% compared with control preincubated with DPBS; n 5 3). These results suggested that a protein may be the ligand on the pneumococcal surface mediating the binding to fibronectin. Localization of pneumococcal binding site within fibronectin. To test the role of different domains of the fibronectin molecule on the adhesion of S. pneumoniae, the ability of the bacterium to bind to wells coated with a series of proteolytic fragments of fibronectin was tested. Two of the fragments supported adhesion: the 27-kDa N-terminal fragment and the 33/66-kDa heparin binding fragment (Fig. 5A). The 31-kDa C-terminal fragment, the 30- and 46-kDa collagen binding fragments, and the 75- and 120-kDa cell binding fragments bound only low numbers of bacteria. The 40-kDa heparin binding fragment had a binding capacity similar to that of the structurally related 33/66-kDa fragment. To further distinguish the binding activities of the heparin binding domain and the amino-terminal domain, the abilities of fragments to competitively inhibit pneumococcal binding to immobilized fibronectin were tested (Fig. 5B). Plasma fibronectin (3 mM) did not inhibit binding of bacteria to the immobilized fibronectin. The 27- and 33/66-kDa fragments also failed to inhibit binding. However, the 40-kDa heparin binding fragment inhibited binding with a 50% inhibitory concentration of ;0.6 mM. The participation of the heparin binding domain in pneumococcal attachment was consistent with the ability of heparin to competitively inhibit bacterial attachment (Fig. 6). It is recognized that this effect of heparin could arise by several mechanisms not explored herein.

Further analysis of the activity of the C-terminal heparin binding domain was undertaken by using a recombinant fragment as a substrate in the direct binding assay (as described in the legend to Fig. 5). When compared with intact fibronectin (1,385 6 450 bacteria per 0.25 mm2; n 5 6), recombinant fragment Fn51 (Fig. 1) supported 65% 6 27% pneumococcal attachment, a value equivalent to that of the proteolytically derived, structurally analogous 33/66-kDa fragment (69% 6 42%). DISCUSSION Fibronectin is found within the basement membranes underlying epithelia, as well as at cell junctions. During tissue injury, fibronectin and other matrix proteins become prominently exposed and pneumococci have been shown to localize to these sites of denuded basement membrane under damaged cells in vivo (15, 17). Fibronectin also plays a role in endovascular bacterial adhesion during the formation of vegetations in

FIG. 3. Effect of time on pneumococcal adherence. The binding assay was performed as described in the legend to Fig. 2 but at a bacterial density of 107/ml. The incubation time was varied from 5 to 300 min. Points represent means 6 standard deviations for three to five wells; data from three separate experiments are shown.

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FIG. 4. Adherence of encapsulated pneumococci to immobilized fibronectin. The binding assay was performed as described in the legend to Fig. 2. Values are from a representative experiment and are expressed as percentages of the mean value for the unencapsulated strain R6x on the same plate. The 100% value is 1,266 6 140 adherent bacteria per 0.25 mm2 of surface area. (A) Encapsulated strains of the designated serotypes. (B) Opaque (shaded bars) and transparent (white bars) subpopulations of encapsulated strains of the designated serotypes.

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bacterial endocarditis (16, 19). These reports indicate that pneumococci are likely to encounter fibronectin at many sites during the progression of disease. This study indicates that pneumococci bind avidly to immobilized fibronectin. The results also suggest how the apparently conflicting data found in the literature on this activity for pneumococcus may be reconciled. Firstly, studies performed a decade ago would likely have suffered from contamination of fibronectin preparations by several other matrix components, such as tenascin, which alter the bioactivities of fibronectin in many assay systems (18). Secondly, the conformation of immobilized fibronectin may be more favorable for binding than that of soluble fibronectin. This is supported by the inability of soluble fibronectin to block pneumococcal adherence to immobilized fibronectin in this study. Finally, variability in fibronectin binding by clinical isolates could reflect true differences in adhesive capacity between serotypes, as found in the present study. The presence of capsules that influence the binding of both S. pyogenes (22) and S. aureus (29) to fibronectin has been described. On the basis of our results, the capsular type may affect adherence in a minority of cases, but major differences in adherence capabilities between strains arise from as yet unknown, noncapsular determinants. The ligand on the pneumococcus which supports binding to fibronectin could be either a protein, such as for staphylococci and streptococci, or a cell wall component, such as for group A streptococci (5, 12). The ability of heat-killed pneumococci to adhere as well as living bacteria would favor a heat-stable, cell wall-mediated mechanism. However, the failure of purified cell wall preparations to inhibit pneumococcal attachment to fibronectin argues against adhesion mediated by cell wall proper. The dramatic reduction in binding of trypsin-treated bacteria favors a protein-mediated adherence mechanism. The mechanism appears to be independent of the regulon associated with colonial morphology variation between opaque and transparent. Given the difference in the binding domain targeted by pneumococci in comparison with other known bacterial fibronectin binding proteins, sequence homology to known fibronectin-binding proteins would not be a predicted feature of the pneumococcal ligand. To identify possible surface proteins mediating this adhesion, efforts to screen a library of

FIG. 5. Localization of the fibronectin fragment supporting adherence. (A) Direct binding assay. Plates were coated with 50 mg of each fragment (indicated by size [in kilodaltons]) per ml, and the binding assay was performed as described in the legend to Fig. 2. Values are from a representative experiment and are expressed as percentages of the mean value of adherence to immobilized intact fibronectin (220-kDa fragment). The 100% value is 2,234 6 330 adherent bacteria per 0.25 mm2 of surface area. (B) Competition assay. Pneumococci were preincubated with a fibronectin fragment at 378C for 15 min and then tested for attachment to immobilized intact fibronectin as described in the legend to Fig. 2. All fragments were tested at 1.5 mM, as indicated by the group of symbols at the right; the 40- and 33/66-kDa fragments were tested at a greater range of doses. Symbols for fragments (designated by their sizes [in kilodaltons]): ■, 40; }, 33/66; E, 220; å, 27; h, 46; 1, 31; 3, 75.

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melanoma cells in eukaryotic systems (18). The details of the protein-protein interaction between pneumococci, one of few bacteria described to target this domain, and fibronectin will be important to understand from the point of view of prokaryotic as well as eukaryotic cell trafficking.

FIG. 6. Inhibition of pneumococcal attachment to fibronectin by heparin. The binding assay was performed as described in the legend to Fig. 2, with the addition of preincubating fibronectin-coated wells with heparin prior to exposure to the bacteria. Values represent the means 6 standard deviations for three experiments with six wells per experiment.

mutants of pneumococci with specific defects in exported proteins for clones deficient in binding to fibronectin are currently under way. Most bacterial binding to fibronectin involves recognition of N-terminal domains of fibronectin: either the heparin binding domain (12, 14, 32) or, less frequently, the collagen binding domain (21, 25). However, the ability of pneumococci to agglutinate S. aureus coated with fibronectin monomers suggested that the binding sites for the two bacteria were distinct (22). The results of both our direct binding studies and inhibition assays with fibronectin fragments and heparin suggest that pneumococci bind within the C-terminal heparin binding site. Both the N-terminal and the C-terminal heparin binding fragments directly supported pneumococcal adherence when generated as proteolytic fragments of native fibronectin. However, only the C-terminal heparin binding domain blocked pneumococcal binding to immobilized fibronectin in a competition assay, suggesting that specific binding may be targeted to the C-terminal heparin binding domain while interactions with the N-terminal domain may arise by weaker, or less specific, charged interactions. This same difference between the two assays has been observed for S. aureus, for which only the N-terminal domain retains activity in competition assays (4). This suggests that pneumococci preferentially bind to the Cterminal heparin binding domain of fibronectin but does not eliminate consideration of the N terminus as a relevant binding site. The E and F proteins of the P-fimbrial filament and a 55-kDa protein of enterotoxigenic E. coli have been shown to bind to this region of fibronectin (30, 32). Two different preparations of the C-terminal heparin binding domain (40 and 33/66 kDa) behaved differently in the bacterial binding assays. Both supported adhesion when immobilized on plastic, but only the 40-kDa fragment was active in the competition assay. This may relate to masking of the 33-kDa fragment by the accompanying 66-kDa fragment in the inactive preparation but not in the active 40-kDa preparation. Alternatively, the 40-kDa fragment may be conformationally distinct or contain important residues not present in the 33kDa fragment. As these proteolytic products are poorly defined, a recombinant product was used to analyze the interaction in more detail. The ability of a recombinant analog of the heparin binding domain shared by the 33- and the 40-kDa fragments to support pneumococcal attachment further supports the targeting of this region of fibronectin by pneumococci. It also indicates that the interaction is not dependent on glycosylation of fibronectin. The C-terminal heparin binding domain of fibronectin has been implicated in the trafficking of

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