Oct 23, 1991 - well-known S. aureus 8325-4, was obtained from Tim J. * Corresponding author. ...... Boden, M. K., and J.-I. Flock. 1989. Fibrinogen-binding ...
Vol. 60, No. 3
INFECrION AND IMMUNITY, Mar. 1992, p. 899-906 0019-9567/92/030899-08$02.00/0 Copyright © 1992, American Society for Microbiology
Binding of Heparan Sulfate to Staphylococcus aureus OLIN D. LIANG,1 FELIPE ASCENCIO,1 LARS-AKE FRANSSON,2 AND TORKEL WADSTROM"* Department of Medical Microbiology, University of Lund, S-223 62 Lund,1 and Department of Medical and Physiological Chemistry, University of Lund, 5-221 00 Lund,2 Sweden Received 23 October 1991/Accepted 24 December 1991
Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of
'251-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 1251-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80°C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 1251-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10-5 M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37°C for 2 h. Proteins of this extract that competitively inhibited binding of 1251-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 1251-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-ThrGly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr. Foster, Department of Microbiology, Moyne Institute, Trinity College, Dublin, Ireland. Strains of 10 other staphylococcal species were the same as for previous studies on fibronectin binding (29) and vitronectin and collagen binding (22). Culture conditions. Unless otherwise stated, bacteria were grown in Todd-Hewitt (T-H) broth at 37°C for 18 h on a gyratory shaker with vigorous agitation. Bacterial cells were harvested at late stationary phase by centrifugation (5,000 x g for 30 min at 4°C), washed with 0.1 M sodium phosphate buffer (pH 7.0) twice, then suspended to a final density of 1010 cells per ml, and used for binding assay promptly. All media used in the study were purchased from Difco (Detroit, Mich.). Chemicals. Highly purified preparations of various glycosaminoglycans (Table 1) were bovine lung heparan sulfate, (heparan sulfate 3), bovine cartilage chondroitin sulfate, porcine skin dermatan sulfate, and porcine intestinal heparin obtained as described elsewhere (10, 11, 35). They were all prepared from the tissue by exhaustive proteolytic digestion to yield corresponding glycosaminoglycans with reducingterminal serine residues. Pepsin (from pig), pronase E (from Streptomyces griseus), proteinase K (from Tritirachium album), trypsin (from beef), chymotrypsin (from beef), neuraminidase (from Clostridium perfringens), dextran sulfates (Mr, 5,000 and 500,000), DEAE-dextran (Mr, 500,000), hyaluronic acid (from beef), polygalacturonic acid, alginic acid, pectin, lipoteichoic acid (from S. aureus), RNA (from bakers' yeast), and colominic acid were from Sigma Chemical Co. (St. Louis, Mo.). Bovine serum albumin (BSA) was purchased from KEBO (Spanga, Sweden). All other chemicals were of analytical grade. Preparation of '251-heparan sulfate. Heparan sulfates were prepared according to the procedures described previously (11, 35). Heparan sulfate 3 was derivatized at the reducingterminal serine (Ser) residue with p-hydroxyphenyl (HOPh) propionate and radio-iodinated (10). The purity of the radiolabelled heparan sulfate {i.e., [1 I]HOPh-(CH2)2-CO-HN-
Staphylococcus aureus and other staphylococcal species capable of causing diseases in humans and animals interact with various connective tissue proteins, such as fibronectin (25, 29), collagens (27, 30), laminin (20), and vitronectin (22). Although the consequences of these binding phenomena for host-parasite relationships are still to be explored, they could promote staphylococcal adherence to host tissues. As Streptococcus pyogenes and other streptococci bind heparan sulfate (2, 7), we wanted to determine whether heparan sulfate and other components of connective tissues and endothelial cell surfaces bind to staphylococci, which has been suggested in previous studies (la, 3, 18, 31). We now report on the interaction between S. aureus and heparan sulfate. The latter constitutes a heterogeneous family of glycosaminoglycans that are normally linked to protein, forming proteoglycan (9, 12). As such, they are components of connective tissues and basement membranes and they are also located on the eukaryotic cell surfaces. Heparan sulfate can interact with other tissue proteins, like collagen (16) and fibronectin (9, 14), and also plays an important role in cell adhesion (5, 9, 12, 24). Our results suggest that S. aureus has cell surface adhesins for heparan sulfate, in addition to the known fibronectin- and collagenbinding proteins of the bacterium, all of which could be used to colonize host tissues. MATERUILS AND METHODS Bacterial strains. Common laboratory S. aureus strains (Wood 46, Cowan 1, Newman, 8325-4, and V8, etc.) and about 40 other S. aureus strains were from our own collection (29). Originally strain V8, a well-known protease producer, was from Staffan Arvidsson, Karolinska Institute, Stockholm, Sweden. Strain DU1090, a derivative of the well-known S. aureus 8325-4, was obtained from Tim J. *
Corresponding author. 899
900
INFECT. IMMUN.
LIANG ET AL.
TABLE 1. Structure of glycosaminoglycansa Glycosaminoglycan
Chondroitin sulfate Dermatan sulfate
Heparan sulfate
Glycan core structure
-4-D-GlcAP1-3GalNAc P13-4-D-GlcA-4-L-IdoAa1-3GalNAc otl-4-D-GlcA-
-4-D-GlcAP1-4GlcNAc cxl-4-D-GlcA-
Heparinb
-4-L-IdoAa1-4GlcNS03 xl-4-L-IdoA-
1
2
3
4
Substituents/variants
i"'
4-/6-0-sulfate at
F
GalNAc As above, plus 20-sulfate at IdoA N-Ac or N-sulfate, 0-sulfate at
GlcNS03, some L-IdoA Mostly 6-0-sulfate at GlcN and 2-0sulfate at IdoA
a Abbreviations: GlcA, D-glucuronic acid; GalNAc, N-acetyl-D-galactosamine; IdoA, L-iduronic acid; GlcNAc, N-acetyl-D-glucosamine; NSO3, N-sulfate group. b Heparin in general contains more iduronate 2-sulfate and N-sulfate glucosamine residues in the polymer than the closely related heparan sulfate.
7060SO
-
4030-
20-
Ser-heparan sulfate} was checked by degradation with heparan sulfate lyase (26) followed by gradient polyacrylamide gel electrophoresis (10). The radiolabel appeared in the positions of the expected carbohydrate-serine components corresponding to segments extending from the reducing end (iodine labelled) to the point of enzymatic cleavage
(Fig. 1).
Binding assay. Fifty microliters of 125I-labelled heparan sulfate (approximately 25,000 cpm; specificity, 0.6 x 106 cpm/,g) solution in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.05% sodium azide and 0.1% BSA were mixed with 100 ,ul of S. aureus cell suspension (109 cells) in a polystyrene centrifuge tube and kept at room temperature for 1 h. After addition of 2 ml of ice-cold phosphate buffer containing 0.1% Tween 20, the mixtures were centrifuged (5,000 x g for 10 min) and the radioactivity of the pellets was measured in a gamma counter (1272 CliniGamma; WALLAC, Abo, Finland). Other binding assays were carried out in the presence of divalent cations (Ca2+, Mg2+, Mn2+, Fe2+, and Cu2+; final concentration of 1 mM) and chelating agents (EDTA or dipyridyl; final concentration of 1 mM) and in buffers with various pHs (0.1 M different buffers with appropriate capacity ranges were used) and ionic strengths (various molarities of NaCl in 0.1 M sodium phosphate buffer, pH 6.0). The bound 1251-heparan sulfate was expressed as a percentage of the total radiolabelled heparan sulfate added to 109 bacterial cells. For each experiment extra negative control tubes with all ingredients except bacteria were included and the radioactivity was determined. This value was then subtracted from the value of other tubes in the experiment before the percentage of binding was calculated. Duplicate determinations were made and averaged in all cases. Enzymatic digestion and heat treatment of bacteria. S. aureus suspensions (100 ,u containing 109 cells) were treated with pepsin, pronase E, proteinase K, trypsin, chymotrypsin, or neuraminidase. The conditions for each enzyme were those described in the Worthington Enzyme Manual (Worthington Biochemical Corp., Freehold, N.J.), and the ratio of enzyme to substrate was 100 ,ug/100 RI of bacterial cell suspension. Cells were carefully washed twice with 4 ml of phosphate buffer to remove enzyme residue, resuspended in the same buffer at the original cell concentration, and used in the binding assays as described above. In heat treatment experiments, fresh cell suspensions (100 RI containing 109
10-
FIG. 1. Heparan sulfate lyase digestion of '25I-heparan sulfate. Lane 1, untreated heparan sulfate; lanes 2 to 4, heparan sulfate treated with heparan sulfate lyase for 30 min, 1 h, and 2 h, respectively. Molecular masses are indicated in kilodaltons. Procedures were described previously by Fransson et al. (10) and Schmidtchen et al. (26).
cells) were kept at various temperatures (40, 60, 80, and 100°C) for 10 min and then used in the binding assay. '251-heparan sulfate binding inhibition assay. S. aureus suspensions (100 ,ul containing 109 cells) were incubated at 20°C for 1 h with 100 ,ug of unlabelled homologous glycosaminoglycans (hyaluronic acid, heparan sulfate, chondroitin sulfate, dermatan sulfate, and heparin), anionic polysaccharides (alginic acid, polygalacturonic acid, pectin, lipoteichoic acid, RNA, and colominic acid), glycoproteins (fetuin, hog gastric mucin, orosomucoid, and transferrin), various monosaccharides (fucose, galactose, mannose, and N-acetyl-D-glucosamine), or other polysaccharides (highand low-molecular-weight dextran sulfate, high-molecularweight dextran, and DEAE-dextran). The treated cells were washed with phosphate buffer, resuspended in 100 RI of the buffer, and used in the binding assay as described above. Isolation of heparan sulfate-binding proteins from S. aureus. All isolation procedures were carried out at 4°C. S. aureus was grown and harvested as described above, washed with 0.1 M phosphate buffer (pH 7.0) three times,
VOL. 60, 1992
40
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FIG. 2. Heparan sulfate-binding to 11 staphylococcal species (grown in T-H broth). Each solid circle indicates one staphylococcal strain. Four common laboratory S. aureus strains are indicated.
and suspended in 1 M LiCl at a final concentration of 1 g (wet weight) of cells per 5 ml. The suspension was incubated with gentle stirring at 37°C for 2 h, the cells were removed by centrifugation (8,000 x g, 30 min), and the supematant fluids were dialyzed against 0.1 M sodium phosphate buffer (pH 6.0) containing 0.1 mM phenylmethylsulfonyl fluoride. The crude extract (300 ml) was applied to a heparin-Sepharose (Pharmacia LKB) column (2 by 3.5 cm) (precycled for a few hours with 0.01 M NaOH, which does not hydrolyze heparin from the matrix under these circumstances) equilibrated with the same phosphate buffer, and then the column was washed with about 10 bed volumes of buffer (until a steady baseline was obtained). The column was then eluted with a linear 0 to 1 M NaCl gradient followed by 2 M NaCl (both in 0.02 M Tris-HCl, pH 8) and with 0.01 M NaOH, and the effluent was collected in 1-ml fractions at a flow rate of approximately 30 ml/h. The fractions in each 280 nm-absorbing peak were pooled, dialyzed against 0.01 M ammonium bicarbonate (pH 8) containing 0.1 mM phenylmethylsulfonyl fluoride, and assayed for the ability to competitively inhibit binding of 1251-heparan sulfate as described above. Immunization. Antisera against the isolated heparan sulfate-binding proteins of S. aureus V8 (i.e., the peak fraction
obtained by heparin-Sepharose chromatography and found
to inhibit binding of '1I-heparan sulfate) were raised in BALB/c mice. The mice were injected intraperitoneally on day 0 with a water-in-oil emulsion containing 25 ,ug of protein in complete Freund adjuvant (Difco) and on day 28 with protein in incomplete adjuvant. The mice were bled on day 38, and their sera were stored at -20°C. SDS-PAGE, immunoblotting, and autoradiography. So-
dium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% separating gel, 4.0% stacking gel) was performed according to the method of Laemmli (17), and the gels were stained with Coomassie brilliant blue R-250. Either a Mini-PROTEAN II cell or a PROTEAN II xi vertical cell (Bio-Rad) was used for the electrophoresis experiments. In Western blotting (immunoblotting) experiments, the separated proteins were electrophoretically transferred to nitrocellulose (Ancos, Vig, Denmark) in a Trans-blot cell (Jancos Biotechnical Instruments ApS, Olstycke, Denmark). Additional binding sites were blocked by incubating the membranes with 5% skim milk in 0.1 M phosphate-buffered saline (PBS), and the membranes were washed with PBS-Tween (0.1% Tween 20 in PBS) at room temperature. The membranes were probed with either immunized BALB/c mouse
902
INFECT. IMMUN.
LIANG ET AL.
serum (diluted 1:500 in PBS-Tween) raised against the isolated heparan sulfate-binding proteins or with 125I-heparan sulfate (2,000 cpm/10 ,ul in 0.1 M sodium phosphate buffer, pH 6) for 2 h at room temperature. After washing them with PBS-Tween, the membranes probed with mouse antiserum were incubated with a 1:1,000 dilution of peroxidase-conjugated rabbit immunoglobulins against mouse immunoglobulins (DAKOPATTS, Glostrup, Denmark) for 2 h at room temperature and developed with 0.02% carbazole and 0.3% HO2 in 0.05 M acetate buffer (pH 5). The blots probed with 12 I-heparan sulfate were dried and examined by autoradiography (-70°C, about 48 h) with X-Omat AR film (Eastman Kodak). Amino-terminal amino acid sequence. Samples of purified heparan sulfate-binding proteins were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, Mass.) after SDS-PAGE. Membranes were stained with 0.1% Coomassie blue R-250 in 50% methanol and destained with 10% acetic acid in 50% methanol. Protein bands were cut from the dried polyvinylidene difluoride membranes, and their amino-terminal amino acid sequences were determined by the BioMolecular Resource Facility (University of Lund, Lund, Sweden).
RESULTS Binding of 1251I-heparan sulfate to S. aureus grown in several different culture media was optimal with cells grown in T-H broth. In general, S. aureus showed better binding than many of the strains of coagulase-negative staphylococci examined (Fig. 2). S. aureus prototype strain V8 and S. aureus DU1090 (a derivative of the commonly used laboratory strain NCTC 8325-4) bound considerable amounts of heparan sulfate. However, another S. aureus prototype strain (Cowan 1) bound only moderate amounts of heparan sulfate. Strain V8 was selected for further characterization of heparan sulfate binding (Fig. 3 and 4). Binding was saturable within the concentration range used (Fig. 3); Scatchard plot analysis of the binding data fit a straight line (Fig. 3, inset) and suggested the presence of one class of heparan sulfate-binding sites on the bacterial cell surface. From the slope of the line, a dissociation constant of 3 x 10-5 M was calculated for the S. aureus-heparan sulfate interaction. Binding was maximal within 30 min (Fig. 4A), and cell-bound 1251-heparan sulfate was displaced by an excess of unlabelled heparan sulfate or heparin (Fig. 4B). Binding was most pronounced in the acidic range (optimal at pH 6; Fig. 4C) and was reduced by high ionic strength (data not shown). Inhibition studies performed by adding various unlabelled substances to the incubation mixtures showed that unlabelled heparan sulfate and heparin inhibited binding of 1251I-labelled heparan sulfate to S. aureus V8 (Table 2). Other glycosaminoglycans of comparable size, such as chondroitin sulfate and dermatan sulfate, also inhibited binding. Some of the anionic components (RNA, lipoteichoic acid, and alginic acid but not colominic acid) partially inhibited binding, and high- and low-molecular-weight dextran sulfate (but not dextran of the same molecular weight), high-molecularweight DEAE-dextran, and hog gastric mucin inhibited binding. On the other hand, highly glycosylated glycoproteins such as fetuin and orosomucoid did not affect binding (Table 2). Fucose, galactose, mannose, and N-acetyl-Dglucosamine, as well as divalent cations, chelating agents, and so42-, did not affect binding (data not shown). Heating S. aureus V8 cells at 80°C for 10 min or exposing
the bacterium to pronase E, proteinase K, pepsin, or chymotrypsin drastically reduced its ability to bind 125I-heparan sulfate (Table 3), thus suggesting that the binding component(s) is proteinaceous. Therefore, we began studies to isolate and identify the heparan sulfate-binding surface protein(s) from S. aureus V8. A cell surface extract was obtained by stirring the bacteria with 1 M LiCl at 37°C for 2 h, which eliminated their ability to bind 1251-heparan sulfate (data not shown). The extract was applied to a heparinSepharose column which was then washed with a NaCl gradient and with 0.01 M NaOH, and the effluent was assayed for the ability to competitively inhibit 1251-heparan sulfate binding to S. aureus V8. Fractions that inhibited binding of 1251-heparan sulfate to S. aureus (more than 90% inhibitory activity) were obtained by elution with 0.01 M NaOH (Fig. 5). The material in the peak of inhibitory activity was subjected to SDS-PAGE, and three bands were revealed by Coomassie blue R-250 staining (Fig. 6A, lane 1). Immunoblotting showed that two of the three polypeptides reacted with immunoglobulins from mice immunized against the fractions comprising the peak of inhibitory activity, while the smallest protein, presumably with lower immunogenicity, did not interact with the immunoglobulins (Fig. 6A, lane 2). These three polypeptides did not react with normal serum from a BALB/c mouse, which excludes the possibility of protein A contamination, since this is a well-known abundant surface protein of S. aureus. Also, probing with 1251_ heparan sulfate autoradiography revealed that these two proteins (approximately 66 and 60 kDa) were responsible for the binding (Fig. 6A, lane 3). Furthermore, the crude LiCl extract and isolated putative heparan sulfate-binding proteins were simultaneously probed with 125I-heparan sulfate, showing identical results (Fig. 6B). This confirmed that the TABLE 2. Influence of potential inhibitors on binding of 1251_ heparan sulfate to S. aureus V8 Inhibitor
PBS (control)
Glycosaminoglycans Heparin Heparan sulfatea Dermatan sulfate Chondroitin sulfate Hyaluronic acid Anionic polysaccharides Dextran sulfate (Mr, 500,000) Dextran sulfate (Mr, 5,000) RNA
Lipoteichoic acid Alginic acid' Polygalacturonic acid Pectinc Colominic acidd Neutral and cationic polysaccharides Dextran (Mr. 500,000) DEAE-dextran (Mr, 500,000) Glycoproteins Hog gastric mucin Fetuin Orosomucoid Transferrin
% Binding
% Inhibition
32.0
0.0
0.3 2.2 5.4 12.5 26.6
99.0 93.2 83.0 61.0 17.0
0.3 0.5 14.2 17.0 17.8 19.5 26.6 34.3
99.0 98.0 55.6 46.9 44.4 39.1 17.0 0.0
33.0 5.6
0.0 82.5
8.7 26.3 30.0 37.9
72.8 18.0 6.3 0.0
a Heparan sulfate 3 (SO4/hexosamine ratio, 1.00; L-iduronic acid/glycuronic acid ratio, 35; and L-iduronic acid-SO4/glycuronic acid ratio, 20). h i.e., polyuronic acid. A partially methoxylated polygalacturonic acid. d i.e., poly-2,8-N-acetyl-neuraminic acid.
VOL. 60, 1992
BINDING OF HEPARAN SULFATE TO S. AUREUS
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Heparan sulphateadded ( 1OOjjg) FIG. 3. Saturability of binding of 1251-heparan sulfate to S. aureus V8. Various amounts of 1251-heparan sulfate were mixed with 109 bacteria, and binding assays were performed as described in Materials and Methods. (Inset) Scatchard plot analysis of the data.
two cell surface proteins of S. aureus V8 did bind 125I1 heparan sulfate, although they were minor components in the extract (cf. lanes 2 and 3 in Fig. 6B). In addition, Western blots of concentrated T-H broth failed to bind the mouse immunoglobulins (diluted 1:500 in PBS-Tween) that interacted with the 60- and 66-kDa proteins, which rules out the possibility that these two proteins were derived from the T-H broth; note that Streptococcus mutans was found to bind T-H broth antigens (28). The amino-terminal amino acid residues of the heparan sulfate-binding proteins isolated from S. aureus V8 were as follows: 66-kDa protein, AspTrp-Thr-Gly-Trp-Leu-Ala-Ala-Ala; and 60-kDa protein, Met-Leu-Val-Thr. TABLE 3. Effect of heating and enzymatic treatments on the ability of S. aureus V8 to bind 1251-heparan sulfate Relative binding (% of control) 100.0 ...................... 20°C (control .................. 400C ........................................ 72.5 600C ......... 54.1 ............................... 800C ........................................ 15.6 1000C ........................................ 7.2 Treatment
Chymotrypsin ........................................
Proteinase K ........................................ Pronase E .............. ..........................
Pepsin ........................................ Trypsin ........................................
Neuraminidase ........................................
7.0 8.8 16.3 30.3 90.8 100.0
DISCUSSION
S. aureus has recently been reported to bind to undamaged vascular endothelial cells possessing heparan sulfate on their surface and to bind to heparinized intravascular catheters (6, 8, 13, 18, 21, 23, 32). In the latter case, polymerassociated foreign body infections, especially those caused by staphylococci, have become a problem of increasing importance in modern medicine (33, 34). Thus, it seemed appropriate to screen S. aureus and strains of various coagulase-negative staphylococci for their ability to bind 1251I-labelled heparan sulfate, by an assay similar to that previously used to define fibronectin- and collagen-binding surface proteins of staphylococci (25, 27, 29). In the present investigation we examined the ability of strains belonging to 11 species of the genus Staphylococcus to bind 1251-heparan sulfate. Different strains bound variable amounts of heparan sulfate, whereas more-virulent S. aureus strains appeared dominant. Nevertheless, it is improper to differentiate staphylococci into virulent binders and avirulent nonbinders in term of heparan sulfate binding, since some coagulasenegative staphylococci strains were isolated from patients with staphylococcal infections. A bacterial cell may simultaneously express adhesins for several matrix components under various circumstances; none of these adhesins should be solely responsible for colonizing host tissues (30). However, we cannot exclude the possibility that the expression of heparan sulfate-binding surface components is suppressed in strains of various staphylococcal species grown in vitro or can be suppressed under certain growth conditions, since it is well known that growth conditions influence plasma or
904
INFECT. IMMUN.
LIANG ET AL.
40C
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Displacer (jig) FIG. 4. Characterization of '25I-heparan sulfate-binding to S. aureus V8. (A) Kinetics of binding. Staphylococcal cells were incubated with 1251-heparan sulfate for the periods of time indicated. (B) Reversibility of binding. Cell-bound 251I-heparan sulfate was displaced by increasing amounts of unlabelled heparan sulfate (circles) or heparin (triangles). (C) Effect of pH on binding. Bacterial preparations and binding assays are described in Materials andMethods.
Incubation time (min)
C
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also used in the studies on S. aureus-human endothelial cell interactions (la, 18). Binding of 125I-heparan sulfate to S. aureus V8 was time dependent, saturable, reversible, and sensitive to heat and protease treatment. Also, unlabelled heparan sulfate, heparin, chondroitin sulfate, and dermatan sulfate inhibited binding of 1251-heparan sulfate to S. aureus V8. However, the inhibitory activity may be very much dependent on the density of anionic charges along the polymers, as several other nonsulfated polyanionic molecules also inhibited binding. Furthermore, the observation that the interaction between S. aureus V8 cell surface components and heparan sulfate was affected by high ionic strength and alkaline pH suggests that binding was probably mediated mainly by ionic interactions between free amino groups of S. aureus cell
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connective tissue protein binding and other cell surface properties of staphylococci (34). Yet, how S. aureus surface protein production is selectively expressed or enhanced is still an obscure matter. Interestingly, T-H broth, which was optimal for 125I-heparan sulfate binding in this study, was 0.7
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65 60 25 55 15 20 Fraction number FIG. 5. Isolation of heparan sulfate-binding proteins from S. aureus V8 by affinity chromatography on heparin-Sepharose. The fractions which competitively inhibited binding of '251-heparan sulfate were obtained by elution with 0.01 M NaOH (cross-hatched area). Experimental conditions are described in Materials and Methods. 0
10
BINDING OF HEPARAN SULFATE TO S. AUREUS
VOL. 60, 1992
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bind fibrinogen (4), fibronectin (25), laminin (20), collagen (27, 30), and thrombospondin (15), which bind their respective ligands with dissociation constants of 10-9 M (10-7 M for collagen). The observation that the dissociation constant for the binding of heparan sulfate to S. aureus V8 is relatively high suggests that if the heparan sulfate-binding proteins are important in the pathogenesis of staphylococcal infections, they promote binding of the bacteria to host cells during early stages of the disease process. Thus, because of their relatively low affinity for heparan sulfate, the bacteria could readily detach and subsequently bind to connective tissue proteins by other adhesins. As an implication in a previous study by Herrmann et al. (15), heparin (closely related to heparan sulfate) could compete with the thrombospondin-binding site on staphylococci to promote the bacterial adherence to surfaces. Herpes simplex virus types 1 and 2 have been reported to initiate infection by first binding to cell surface heparan sulfate glycosaminoglycans and subsequently by interacting with other receptors at the host cell surface (19, 36). Also, dermatan sulfate, chondroitin sulfate, and other polyionic carbohydrate polymers (natural as well as synthetic) affect binding of heparan sulfate to staphylococci. Therefore, the heparan sulfate-binding proteins of S. aureus may be general glycosaminoglycan-binding proteins. Further studies on characterization of these proteins are now in progress.
B
FIG. 6. SDS-PAGE, immunoblotting, and autoradiography. (A) Electrophoresis experiments were performed with a Mini-PROTEAN II cell. Analysis of the peak fraction obtained by heparinSepharose chromatography and found to inhibit heparan sulfate binding to S. aureus V8. Lane 1, SDS-PAGE of the peak fraction (20 ,ug of protein); lane 2, immunoblot of the peak fraction (30 ,ug of protein) probed with mouse antiserum raised against the peak fraction; lane 3, autoradiograph of the peak fraction (30 p.g of protein) probed with 1251-heparan sulfate. (B) Electrophoresis experiments were performed with a PROTEAN II xi vertical cell. Lane 1, high-molecular-weight markers (Bio-Rad); lane 2, crude LiCl extract of S. aureus V8 (50 pLg of protein); lanes 3 and 4, autoradiographs of crude LiCl extract (50 ,ug of protein) and isolated putative heparan sulfate-binding proteins (30 jig of protein) probed with 1"I-heparan sulfate. Molecular masses are indicated in kilodaltons at left side of each panel. The arrow indicates S. aureus protein A (SpA) revealed from crude extract by normal mouse serum. Experimental conditions are described in Materials and Methods.
surface proteins and the sulfate and carboxyl groups of heparan sulfate, as has been reported for Streptococcus pyogenes and Streptococcus mutans (2, 7) and for herpes simplex virus (36). The observation that the ability of S. aureus to bind heparan sulfate was destroyed by heat and protease treatment suggests that the binding components are proteinaceous. In fact, two heparan sulfate binding-proteins having molecular masses of 66 and 60 kDa were obtained by affinity chromatography on heparin-Sepharose of a cell surface extract of S. aureus V8. The sizes of the heparan sulfatebinding proteins of S. aureus V8 are different from those of Streptococcus pyogenes (2) and Helicobacter pylori (1). The binding of heparan sulfate to S. aureus V8 does not appear to be mediated by any of the staphylococcal surface proteins so far described, since the Mrs of the heparan sulfate-binding proteins differ from those of other described staphylococcal adhesins. The apparent affinity of the heparan sulfate-binding proteins for their ligand is considerably lower than those of the staphylococcal adhesins which
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ACKNOWLEDGMENTS
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