T H E tJOIIRNAL OF BlOI.OGICAL CHEMISTRY Vol. 266, No. 34, Issue of December 5, pp. 23399-23406,1991 Printed In l i S. A.

Binding of Elastin to Staphylococcus aureus* (Received for publication, February 21, 1991, and in revised form, August 5 , 1991)

Pyong Woo Park$§, David D. Roberts(, Leonard E. Grossoll , William C. Parks$, Joel Rosenbloom**, William R. Abrams**, and Robert P. Mecham$ $$§§ From the $Department of Cell Biology and Physiology, Washington Uniuersity School of Medicine, St . Louis, Missouri 631 10, the Departments of $$Medicine and )(Pathology, Jewish Hospital at Washington University Medical Center, St. Louis, Missouri 631 10, the BLaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and the **School of Dental Medicine, University of Pennsyluania, Philadelphia, Pennsyluania 1 9104

Many pathogenic bacteria specifically bind to com- ponents of the extracellular matrix. In this study, we report the specific association of Staphylococcus au- reus with elastin, a major structural component of elastic tissue. Competition assays in which the binding of radiolabeled tropoelastin was inhibited by excess unlabeled elastin peptides, but not by other proteins, established the specificity of the interaction. Kinetic studies showed that tropoelastin binding to the bacteria was rapid and saturable. Scatchard analysis of the equilibrium binding data indicated the presence of a single class of high affinity binding sites (&- 4-7 nM) with approximately 1000 sites per organism. Protease susceptibility suggested that the elastin binding moiety on S. aureus was a protein, which was confirmed by the isolation of a 25-kDa elastin-binding protein from S. aureus extracts through affinity chromatography. Using a truncated form of tropoelastin, the bacterial binding domain on elastin was mapped to a 30-kDa fragment at the amino end of the molecule. Although the precise amino acid sequence recognized by the staphylococcal elastin receptor has not been character- ized, it is clearly different from the region of tropoe- lastin that specifies binding to mammalian elastin receptors.

Interactions between ECM’ molecules and cell surface receptors play important roles in numerous physiological and pathological events. In higher animals, cell-matrix interac- tions are important in hemostasis, cellular differentiation, tumor cell invasion, and a variety of other functions (1, 2). In addition to animal cells, various pathogenic microorganisms are known to possess specific receptors for ECM molecules. For example, Trypanosoma cruzi (3), Treponemapallidum (4),

* This work was supported by National Institutes of Health Grants HL26499 and HL41926 (to R. P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

Q This work represents partial fulfillment of the requirements for a Ph.D. degree in the Department of Cell Biology and Physiology, Washington University.

$ 3 TO whom correspondence and reprint requests should be ad- dressed: Pulmonary Research, Jewish Hospital at Washington Uni- versity Medical Center, 216 South Kingshighway, St. Louis, MO 63110.

I The abbreviations used are: ECM, extracellular matrix; nTE, native tropoelastin; rTE, recombinant full-length tropoelastin; trTE, truncated recombinant tropoelastin; CFU, colony forming unit; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; HPLC, high performance liquid chro- matography; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

Pneumocystis carinii (5), and streptococci (6) specifically bind fibronectin, and Staphylococcus aureus interacts with fibro- nectin (6, 7), vitronectin (8), collagen (9), and laminin (10). The physiologic role of these microbial interactions is specu- lative at present. Several observations, however, suggest that the interaction between ECM molecules and pathogenic mi- croorganisms is of major importance in the initiation and progression of infection.

Elastic fibers are components of the mammalian ECM and are present in abundance in tissues that require elasticity. Mature elastin is a polymer of tropoelastin molecules (the monomeric form of elastin secreted from the cell) covalently cross-linked one to another via modified lysine side chains (11,12). Since S. aureus infects and colonizes elastin-contain- ing organs, such as lung, skin, and aorta (13-16), and because invasive S. aureus has a high probability of interacting with the elastin matrix of blood vessels, we investigated whether this bacteria might interact with purified elastin. Using tro- poelastin and solubilized forms of mature elastin, we demon- strated specific binding of both forms of the molecule to S. aureus and have shown that binding occurs within a region of elastin distinct from the binding site for the mammalian receptor.

MATERIALS AND METHODS

Materials-Kits for cell-free translation were purchased from Pro- mega (Madison, WI). Engelbroth-Holm-Swarm mouse tumor laminin was purchased from Collaborative Research, Inc. (Bedford, MA). Iodogen was from Pierce Chemical Co., and N a Y was obtained from ICN (Costa Mesa, CA). PD-10 columns (Sephadex G-25M) were purchased from Pharmacia (Uppsala, Sweden). IgGsorb was pur- chased from the Enzyme Center (Malden, MA). Tryptic soy broth (TSB) and TSB-agar were purchased from Remel (Lenexa, KS) . Horseradish peroxidase-conjugated goat anti-rabbit IgG, horseradish peroxidase color development reagent, Affi-Gel 10, and Poly-Prep columns were from Bio-Rad. Papain and lysostaphin were from Sigma. ProBlot paper was from AB1 (Foster City, CA).

Purification and Iodination of Elastin Ligands-Because mature elastin is insoluble, it could not be used directly in ligand binding studies. For this reason, we utilized two soluble forms of the protein: tropoelastin, the naturally soluble uncross-linked precursor molecule, and n-elastin, an oxalic acid-solubilized form of insoluble elastin (17) that consists of a polydisperse mixture of peptides with an average molecular mass between 60 and 85 kDa. Although tropoelastin is the precursor molecule for insoluble elastin, the two forms of the protein exhibit important differences. The most important being the presence of lysyl-derived cross-links in a-elastin which are absent in tropo- elastin. For this reason, we felt it was important to test the biological activity of both preparations in the binding assays.

Full-length native bovine tropoelastin (nTE) was isolated from fetal bovine ligamentum nuchae as described (18). To ensure purity, the lyophilized protein was dissolved in water containing 0.05% trifluoroacetic acid and chromatographed on a Hamilton PRP-3 column using a water-propanol gradient a t a flow rate of 0.5 ml/min. Column fractions containing tropoelastin (eluting between 60 and

23399

23400 Binding of Elastin to 5'. aureus 70% propanol) were identified by dot blot assay using an anti- tropoelastin polyclonal antibody, pooled, and dried in a Speed-Vac concentrator. The dried sample was dissolved in ice-cold water, separated into small aliquots, and stored at -70 "C.

Recombinant bovine tropoelastin, lacking the first 170 amino acids from the amino terminus, was from a cDNA clone in a bacterial expression system. The specific protocols for the generation and purification of truncated recombinant tropoelastin (trTE) have been described (19). Full-length human recombinant tropoelastin (rTE) was also prepared from a bacterial expression system (20), and a- elastin peptides were prepared from bovine ligamentum nuchae in- soluble elastin as detailed in Mecham and Lange (21). Purity of the native and recombinant proteins was assessed by SDS-PAGE, im- munoblotting, amino acid analysis, and sequencing of 20 amino acids at the NH2 terminus.

rTE and trTE were iodinated by incubating 5-10 pg of each protein with 1 mCi of Na'"1 for 10 min at room temperature in an Iodogen- coated Microfuge tube. Unincorporated radioactivity was separated from the radiolabeled protein by PD-10 gel chromatography. For both proteins, greater than 85% of the recovered material was precipitated with 10% trichloroacetic acid. The specific activity for rTE was approximately 10 pCi/mg and 30 pCi/mg for trTE.

RNA I.wlation and Cell-free Translation-Total RNA was isolated from fetal bovine nuchal ligament by isopycnic centrifugation through CsCl (22). RNA was translated in uitro using (~"H]Ieucine and rabbit reticulocyte lysate, and tropoelastin proteins were immunoprecipi- tated and resolved as described (22).

Bacteria-S. aureus strains 12598 (Cowan), 8095, 10832 (Woods 46), and coagulase-negative staphylococci Staphylococcus epidermidis (strain 155), Staphylococcus saprophyticus (153051, and Staphylococ- cus xylosus (29971) were purchased from the American Type Culture Collection. Biochemical classification strategies for the coagulase- negative staphylococci have been described elsewhere (23). Following the supplier's instructions, the bacteria were hydrated with TSB (pH 7.2) and propagated overnight in 20 ml of TSB at 37 "C with shaking. A 10-pl aliquot of the resulting culture was plated onto a sheep blood agar plate. After overnight incubation at 37 "C, a single colony was inoculated in 20 ml of TSB and grown for 24 h a t 37 "C with agitation. The resulting culture was stored in 1-ml aliquots which were frozen at -70 "C in sterile TSB with 10% glycerol. For all experiments, one aliquot was gradually thawed on ice and inoculated into fresh TSB. The cells were grown for 24 h a t 37 "C with agitation to obtain late stationary growth phase bacteria. The bacterial concentration in colony forming units per milliliter was determined from the turbidity at 620 nm and by comparing the values with standard curves derived from diluted samples of known numbers of bacteria. The bacteria were washed twice with fresh TSB before use in the assays.

IgGsorb (formalin-fixed S. aureus of the Cowan strain) was hy- drated with deionized water to a 10% (w/v) suspension and stored a t 4 "C. Aliquots of fixed S. aureus were equilibrated with TSB prior to each experiment.

Papain and Lysostaphin Digestion of S. aureus-Papain digestion of live S. aureus was performed by incubating 1.15 X 10" CFUs for 2.5 h a t 37 "C in 400 11 of papain digestion buffer (20 mM cysteine- HCI, 1 mM EDTA, 20 mM NaH2P04 (pH 7.0)) containing 50 or 200 pg of papain (10-20 units/mg). The reaction was terminated by the addition of iodoacetamide to a final concentration of 100 mM and incubating for 30 min at 37 "C. For lysostaphin treatment, 1.15 X 10"' CFUs were incubated with 0.5 mg/ml lysostaphin (480 units/mg) in 400 p1 of digestion buffer (1 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmaleimide, 20 pg/ml DNase I in TBS (20 mM Tris, 150 mM NaCI, (pH 7.5)) for 1 h at 37 "C. Another 400 p l of the digestion buffer was added, and the reaction was continued for 2 h. At the end of all enzyme treatments, the mixtures were centrifuged, the cell pellets were washed twice with TSB, and the cell suspension adjusted to the desired concentration with fresh TSB. As positive controls, S. aureus was incubated in digestion mixtures without enzymes. Other cultures were heat-killed by incubating at 88 "C for 30 min in TSB.

Binding Assay-Unless otherwise indicated, 1.5 X 10' CFUs of S. nureus of the Cowan strain (12598) were used for all binding assays. The total volume of the reaction was 300 pl, and TSB was used as the assay buffer. Cells were added to a 1.5-ml Microfuge tube con- taining the respective ligands and agents as specified in the different experiments and incubated for 1, 6, 10, 30, or 60 min on an orbital shaker at room temperature. Binding studies were performed at room temperature rather than 37 "C to minimize problems associated with coacervation of tropoelastin (24) and proteolytic digestion of ligand by endogenous bacterial enzymes. The assay was terminated by

centrifugation for 8 min in a Beckman Microfuge and the superna- tants either discarded or immunoprecipitated. The pellets were re- suspended in 0.5 ml of TSB which was then transferred to new Microfuge tubes. Since tropoelastin readily adsorbs to plastic surfaces (18), transferring the pellet to fresh tubes was necessary to avoid counting radiolabeled tropoelastin that had nonspecifically adsorbed to the original tube. Following transfer of the pellet, the original tubes were washed with 0.5 ml of TSB which was added to the bacterial suspension in the new tubes. The samples were centrifuged for 8 min, the supernatants discarded, and the pellets washed once with 1 ml of TSB. Additional washes did not further decrease the amount of radioactivity associated with the cell pellet. Radioactivity associated with the pellet was measured with an LKB 1280 Ultro- gamma counter. In some experiments, the pellet was incubated with SDS-PAGE sample buffer, and the products were analyzed by SDS- PAGE followed by autoradiography or immunoblotting.

Cell Surface Labeling and Preparation of Surface-labeled Extract- 1.7 X 10"' CFUs of S. aureus of the Cowan strain were pelleted by centrifugation and washed three times with 1 ml of ice-cold TBS. The bacteria were resuspended in 600 p l of cold TBS and transferred to a 12 X 75-mm glass tube coated with 150 pg of Iodogen. 600 pCi of Na'*'I was added and the tube incubated for 10 min on ice. The reaction was continued a t room temperature for 5 min. The reaction mixture was then transferred to a Microfuge tube and centrifuged for 1 min in a bench-top Microfuge a t 10,000 rpm. The supernatant was discarded, and the radiolabeled bacterial pellet washed four times with 1 ml of cold TBS to separate free from incorporated iodine. Surface-labeled S. aureus were then digested with lysostaphin in the presence of protease inhibitors and DNase 1 as described above. At the end of the digestion, the labeled bacterial extract was collected by microcentrifugation for 20 min. Specific activity of the extract was approximately 600 pCi/l.7 X 10"' bacteria.

Electrophoresis, Autoradiography, and Immunoblotting-Samples in SDS sample buffer were analyzed by SDS-PAGE using 0.75-mm thick 7.5-12% gradient or 12% straight acrylamide gels (22). Gels with radioactive samples were fixed with 5% MeOH, 7% acetic acid for 60 min, dried, and exposed to Kodak XAR-2 film a t -70 'C. Nonradioactive samples were transferred from the SDS-PAGE gel to nitrocellulose a t 200 mA for 90 min a t 4 "C. The nitrocellulose with transferred proteins was incubated for 2 h at room temperature with 5% (w/v) nonfat dry milk in TBS to block unbound sites and then washed twice with TBS containing 0.1% (v/v) Tween-20 and 0.5% (w/v) non-fat dry milk (TTM) for 15 min a t room temperature. Tropoelastin antiserum a t a dilution of 1:300 in TTM was added and the blot incubated for 1 h. After washing twice with TTM, 1500 dilution of horseradish peroxidase-conjugated goat anti-rabbit im- munoglobulin in TTM was added and the blot incubated for 1 h. Color development reagent (4-chloro-naphthol) was then added after washing the blot twice with TBS for 15 min. The color reaction was stopped by rinsing the membrane with distilled water, and the blot was air-dried for photography.

Purification of the S. aureus Elastin-binding Protein-@-Elastin (160 mg) or BSA (80 mg) in 8 ml of 100 mM NaHCOn (pH 8.0) were coupled to 6 ml of Affi-Gel-10 resin according to the manufacturer's instructions. Coupling efficiency, as measured by reading absorbance at 280 nm, was approximately 50 and 65% for a-elastin and BSA, respectively. 1.5 ml of Affi-Gel resin with bound protein were trans- ferred to polypropylene columns and washed thoroughly with binding buffer (20 mM Tris, 2 mM CaC12, 2 mM MgCI?, 2 mM N-ethylmaleim- ide, 2 mM benzamidine HCI, 5 mM glucose, 500 mM NaCI, pH 7.4). Surface-labeled extract diluted to 5 ml with binding buffer was added to the columns in the presence or absence of 2 mg/ml @-elastin or BSA. The BSA column served as a control for nonspecific absorption. After a 30-min incubation a t room temperature, the resins were washed with binding buffer until the radioactivity of the eluates reached background levels (approximately 50 ml). Bound proteins were eluted from the columns with 1% (w/v) SDS buffer. 300-pl aliquots were collected and analyzed by SDS-PAGE.

T o determine the elution position for the staphylococcal elastin- binding proteins on reverse-phase HPLC, surface-labeled lysostaphin extracts were first pre-absorbed with BSA-Affi-Gel and then frac- tionated by a-elastin affinity chromatography. Bound proteins were eluted with CAPS buffer (30 mM CAPS, 2 mM N-ethylmaleimide, 2 mM benzamidine-HC1 (pH 11)). The eluted fraction was lyophilized, resuspended in water, 0.05% trifluoroacetic acid to 1/4 the original volume and fractionated by reverse-phase HPLC (Beckman C18 column, 0-80% water-acetonitrile gradient containing 0.05% trifluo- roacetic acid over 30 min, flow rate of 0.5 ml/min). One-minute

Binding of Elastin to S. aureus 23401

lractions were collected and counted for radioactivity in a y counter. The peak fraction (eluting a t 50% acetonitrile, fraction 25) was dried in a Speed-Vac concentrator and analyzed by SDS-PAGE. For se- quence analysis, extract from a large scale preparation of unlabeled S. aureus (8 X 10" CFUs) was fractionated by HPLC and the fraction eluting at the position of the radioactive peak collected, dried, sub- jected to SDS-PAGE, and electrophoretically transferred to Pro-Blot using the manufacturer's suggested protocol. Two bands a t approxi- mately 25 and 40 kDa, visualized by staining the blot with Coomassie Brilliant Blue R-250, were excised and directly sequenced using an Applied Biosystems 473 protein sequencer.

RESULTS

Binding of Tropoelastin to S. aureus-This study originated from the chance observation that fixed S. aureus precipitates cell-free translated tropoelastin in the absence of elastin- specific antibody (22). To further characterize this interac- tion, we compared the relative efficiency of tropoelastin pre- cipitation by fixed S. aureus (IgGsorb) alone or S. aureus plus specific antibodies. As shown in Fig. 1, fixed S. aureus precip- itated cell-free translated tropoelastin as efficiently as did the combination of S. aureus and tropoelastin-specific mono- clonal antibody. Nonimmune mouse ascitic fluid, goat anti- mouse immunoglobulin, or elastin antibody alone failed to precipitate tropoelastin to the same degree as did fixed S. aureus. A similar result was obtained with native tropoelastin (nTE) purified from bovine ligamentum nuchae. As can be seen in the immunoblot in Fig. 2, not only did native tropo- elastin bind to the bacteria, but the amount bound increased as a function of S. aureus concentration. The multiple bands evident in Figs. 1 and 2 are isoforms of tropoelastin that arise through alternative splicing. I t is important to note that S. aureus interacts with all of the major isoforms present in the reaction mixture.

To eliminate the possibility that the S. aureus-tropoelastin interaction is an artifact of bacterial fixation, we examined the binding of iodinated human recombinant tropoelastin (rTE) to live S. aureus. The recombinant form of the molecule, which is a single isoform that includes all of the exons and is functionally similar to native tropoelastin (20), was used because native tropoelastin is difficult to obtain in the quan-

B A - 4 + + - - - - + - N M A - - - + + - - -

St0ph-A + - + t - - - - G A M - - - - - - + +

FIG. 1. Formalin-fixed S. aureus binds to tropoelastin in cell-free translation reactions. Total RNA (5 pg), isolated from a 270 day fetal calf nuchal ligament, was translated in uitro using rabbit reticulocyte lysate and ["H]leucine. The reaction mixtures were in- cubated for 18 h a t 4 "C with anti-bovine elastin monoclonal antibody ( H A - 4 ) and antigen-antibody complexes were precipitated with 30 ml of a 10% suspension of formalin-fixed S. aureus or goat anti-mouse IgG ( G A M ) for 1 h a t room temperature in immunoprecipitation buffer (50 mM NaHyPO,, 150 mM NaC1, 1 mM benzamidine HCI, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100 (pH 7)). Control reactions were incubated with nonimmune mouse ascitic fluid ( N M A ) . Precipitated products were resolved by 7.5-1276 SDS-PAGE and gels were processed for fluorography.

- 110

e 3 3

10 30 50 S. aureus suspension @I)

FIG. 2. Binding of native tropoelastin to formalin-fixed S. aureus. Native tropoelastin (1 pg) was incubated with 10 P I , 30 pl, or 50 pl of a 10% suspension of formalin-fixed S. aureus for 1 h a t room temperature in TSB. Reactions were stopped by centrifugation in a Beckman Microfuge and the pellets washed with fresh TSR. The S. aureus-tropoelastin complex was dissociated by boiling in SDS- PAGE sample buffer and analyzed by SDS-PAGE and immunoblot- ting.

->-. , .~

- 200

- 92.5

Live Fixed FIG. 3. Both live and f ixed S. aureus bind full-length recom-

binant tropoelastin (rTE). Five ng of '"I-rTE was incubated with 1.5 X 10" CFUs of live S. aurew or 30 p1 of a 10% suspension of formalin-fixed S. aurew for 1 h at room temperature in TSB. A t the conclusion of the assay, the bacteria were pelleted by centrifugation, washed with fresh TSB, and heated to 100 "C in SDS-PAGE sample buffer. An aliquot of the solubilized sample was analyzed by SDS- PAGE and autoradiography.

tities required for multiple binding assays. Live or fixed bacteria were incubated with the radiolabeled ligand for 1 h at room temperature and the amount of bound protein was determined by SDS-PAGE and autoradiography. As shown in Fig. 3, "'I-rTE bound to both live and fixed forms of the bacteria.

Specificity and Kinetics of Radiolabeled rTE Binding to S. aureus-The specificity of tropoelastin binding was examined by including increasing amounts of unlabeled elastin-derived peptides (&-elastin) or BSA in the incubation buffer. The addition of unlabeled m-elastin peptides, but not BSA, inhib- ited the binding of "'I-rTE to live S. aureus (Fig. 4). Inhibition of binding by a-elastin was dose-dependent, and no specific binding of the ligand was observed in the presence of 5 pg or more of unlabeled elastin peptides, whereas up to 20 pg of cold BSA had no inhibitory effect.

Measurements of the time course of "'I-rTE binding to live

23402 Binding of Ela

140 1

& 0 Y

20 - 1 0 1 . 1 . 1 ' 1 . 1

3 5 10 1 5 2 0 25

Protein added (pg) FIG. 4. Competition of '''I-rTE binding to S. aureus. Binding

of 10 ng of 12511-rTE to live S. aureus was measured in the presence of 0.5, 1, 5, and 20 pg of unlabeled a-elastin peptides or 5 and 20 pg of BSA. Incubations were at room temperature for 60 min. The assay was terminated by centrifugation for 8 min in a Beckman Microfuge and the supernatants discarded. The pellets were resuspended in 0.5 ml of TSB which was then transferred to new Microfuge tubes. The original tubes were washed with 0.5 ml of TSB which was added to the bacterial suspension in the new tubes. The samples were centri- fuged for 8 min, the supernatants discarded, and the pellets washed again with 1 ml of TSB. Radioactivity associated with the pellet was measured with a y counter. The data points represent mean f S.D. of triplicate measurements. The background value is the radioactivity recovered from reaction tubes containing no bacteria. 0, BSA; 0, a- elastin; - - - - , background.

4 1

0 4 . I . , . I . , . , . , . (

0 10 20 30 4 0 5 0 60 70

Time (min) FIG. 5. Time-dependent binding of radiolabeled rTE to S.

uureus. Bacteria were incubated a t room temperature with 20 ng of '"I-rTE in the presence (nonspecific binding, W) and absence (total binding, 0) of 20 fig of unlabeled elastin peptides. At the indicated times, the bacteria were washed and processed as described in Fig. 4. The amount of tropoelastin bound was calculated as amount bound = (counts/min bound/total cpm added) X (amount added in nano- grams). Specific binding (0) represents the difference between the means of duplicate determinations of total and nonspecific binding.

bacteria at room temperature showed an initial rapid phase of binding over 10 min that reached maximal levels by 30 min (Fig. 5). Maximal specific binding of the ligand was still maintained at 60 min. To ensure optimal association of the

s t in to S. aureus

ligand with the bacteria, incubation was carried out for 60 min in all other experiments.

Equilibrium Binding of lZ5I-rTE to S. aureus-Fig. 6 shows specific binding at equilibrium for several concentrations (30- 400 ng/ml) of radiolabeled rTE to live S. aureus. Nonspecific binding was measured in the presence of a 500-fold excess of elastin peptides, and specific binding was determined as the difference between total and nonspecific binding. Scatchard analysis (25) gave a linear plot consistent with the presence of a single type of high affinity binding sites (Fig. 6, inset). In the plot shown, the dissociation constant ( KD) was calculated to be 4.3 nM, with 1023 binding sites per CFU. In a second experiment, the K D and number of sites/CFU were, respec- tively, 6.7 nM and 1113 sites/CFU (data not shown).

S. aureus Binding Requires the Amino One-third of Tropo- elastin-The interaction of S. aureus with native tropoelastin demonstrates that the signal peptide is not necessary for bacterial binding. To better define the domain on tropoelastin that mediates interactions with bacteria, the ability of live S. aureus to bind to a truncated form of tropoelastin (trTE) was examined. The truncated molecule lacks approximately 30 kDa of the NH2 terminus corresponding to exons 1-13 of the tropoelastin gene. trTE does, however, contain sequences that define the binding domain of the mammalian receptor (exon 24) and is active in cell adhesion assays for mammalian cells (19). Interestingly, trTE did not bind to live S. aureus (Fig. 7).

The inability of trTE to bind to S. aureus suggests that the staphylococcal binding region on tropoelastin is located within the amino one-third of the molecule and is separate and distinct from the mammalian receptor binding region. To further compare the binding properties of staphylococcal and mammalian elastin receptors, we did competition experiments with reagents known to inhibit mammalian cell binding to tropoelastin. A major elastin receptor on many cells is a 67- kDa protein that recognizes both elastin and laminin (26). Receptor affinity for these ligands is regulated by a carbohy- drate-binding site such that little or no protein binding occurs in the presence of 0-galactoside sugars (27). To determine

M E m E cp

.I

.I qyJ 0.20 .

0.12

0.10 0.08

50 150 250 350 450 Bound (pM)

0 30 60 90 120

rTE added (ng) FIG. 6. Specific binding of 1251-rTE to S. aureus at equilib-

rium. Cells were incubated with increasing concentrations of rTE in the presence and absence of 500-fold excess unlabeled elastin peptides for 60 min at room temperature and processed as described in Fig. 4. The data points represent specific binding obtained by determining the difference between the means of duplicate total and nonspecific binding values. Binding results were analyzed by Scat- chard analysis (inset) . The dissociation constant and number of binding sites were calculated using a value of 70 kDa for the molecular mass of ' T r T E .

Binding of Elastin to S. aureus 23403

1 '5 ] rTE

Bacteria + + + ng ligand 6 6 6 6 20 20

FIG. 7. S. aureus does not recognize the truncated form of tropoelastin. Radiolabeled rTE and t rTE were incubated with or without 1.5 X 10% live S. aureus as indicated. The binding assay was as described in Fig. 4. Data are presented as means of duplicate determinations.

200

100

0

binding of lZ5I-rTE to the bacteria whereas laminin, anti-67- kDa receptor antibody, and lactose had no effect. The slight enhancement of binding evident in Figure 8 was due to the inhibition of '2sII-rTE adsorption to the wall of the assay tube (data not shown).

General Characteristics of the Elastin-binding Component of S. aurew"Bacteria1 interactions with ECM molecules are mediated by binding molecules that require specific conditions for efficient binding. To better understand the mechanisms involved in the S. aureus-tropoelastin interaction at the mo- lecular level, we performed binding studies in the presence of agents that would provide a general characterization of the elastin-binding moiety. As seen in Table I, papain- and lyso- staphin-treated bacteria lost about 65 and 90%, respectively, of the binding capacity relative to the control, whereas heat- killing the bacteria did not decrease the binding ability. Inclu- sion of 0.05% (w/v) SDS in the binding buffer had a dramatic inhibitory effect on Iz5I-rTE binding to S. uurew, indicating that the interaction is sensitive to this detergent. In contrast, a moderate enhancement of binding was observed with thio- reductants. In the enzyme treatment experiments, unbound "51-rTE in the supernatants was immunoprecipitated to en- sure that the negative results were not due to degradation of the radiolabeled probe by residual protease activity in the bacterial digestion mixture. In both cases, intact unbound tropoelastin in the supernatants was detected at the end of the assay period (not shown).

Binding of '251-rTE to Different Strains of Staphylococci- To determine whether tropoelastin binding is a common property of S. aureus, binding of radiolabeled rTE to three " - different strains of S . aureus- and three coagulase-negative staphylococci species was examined. Under similar condi- tions, all three S. aureus strains specifically bound lZ5I-rTE, although the capacity to bind the ligand differed (Fig. 9). Relative to strain Cowan, strain 8095 bound about 1.6 times more Iz5I-rTE, whereas the Woods strain (10832) bound 60% less radiolabeled tropoelastin. Among the other staphylococci species tested, only S. epidermidis specifically interacted with '"I-rTE, although to a lesser degree relative to S. aureus. S. saprophyticus and S. xylosus showed no binding.

TABLE I Effects of various treatments on binding of "'I-rTE to S. aureus 1.15 X 10" CFUs of live S. aureus were treated with papain,

lysostaphin, or heat and prepared for the binding assay as described under "Materials and Methods." 5 ng of ?-rTE and 1.5 X 10" treated bacteria were incubated for 1 h a t room temperature and processed as described. For the other treatments, 5 ng of '*'I-rTE was incubated

control alpha LN BCZ lactose with 1.5 X lo8 CFUs of S. aureus in the presence of the agents tested. Values are for duplicate determinations and are exDressed as mean

FIG. 8. Antagonists of the mammalian cell-tropoelastin in- teraction do not affect s. aureus binding to '*'I-rTE. 5 ng of "'1-rTE was incubated with 1.5 X 10" CFUs in the presence of 10 pg of welastin, 10 pg of laminin, 10 pg of anti-67 kDa receptor antibody ( B C Z ) , or 30 p~ lactose and processed as described in Fig. 4. Results of duplicate determinations are presented as mean percentage of the binding level measured in the absence of antagonists.

whether the tropoelastin binding site on S. aureus has prop- erties similar to the mammalian receptor, live bacteria were incubated with "'I-rTE in the presence of 33 lg/ml a-elastin or 33 pg/ml laminin (both competitive inhibitors of elastin binding to mammalian cells), 33 pg/ml mouse monoclonal antibody (BCZ) to the 67-kDa mammalian receptor or 30 mM lactose, a disaccharide that inhibits tropoelastin and laminin binding. The antagonists at these concentrations inhibit mammalian cell-tropoelastin binding under similar condi- tions. As shown (Fig. 8), a-elastin peptides inhibited the

percentage of the-positive control level (no treatment). Nonspecific binding was measured in the presence of 67 pg/ml of n-elastin peptides, and this value was subtracted from each determination.

Treatment Concentration p ~ ~ ~ ~ ~ ~ f

No treatment 100 Heat-killed (30 min at 88 "C) 116.7 Papain 125 pg/ml 31.5

500 pg/rnl 33.8

SDS Lysostaphin 500 pg/ml 9.2

/3-Mercaptoethanol 10 mM 140.9 100 mM 142.5

Dithiothreitol 10 mM 142.3 100 mM 142.4

0.05% (w/v) 0.0" 1.0% (w/v) 0.0

Binding values in SDS-containingreactions were lower than those for nonspecific binding, resulting in negative relative percent binding. The values, however, are presented as 0.0%.

23404 Binding of Elastin to S. aureus

S. aureus (8095)

S. aureus (Cowan)

S. aureus (Woods)

S. epiderrnidis

S. saprophyticus 1 I s. xylosus 1 -~-

0 100 200

Relative specific binding (Cowan=100%) FIG. 9. Binding of staphylococci strains to ““I-rTE. 1.5 X

10” CFUs of various staphylococci strains were incubated with 10 ng of radiolabeled rTE in the presence or absence of 20 pg of tr-elastin peptides. The binding assay was performed as described. The data in this figure represent relative specific binding of duplicate determi- nations except for S. aweus (12598) and 5’. saprophyticus, where specific binding was determined from quadruplicate measurements. S . aureus of the Cowan strain was designated as the reference bac- teria. Negative specific binding values are shown as O R .

Isolation of the S. aureus Elastin-binding Protein-The inhibitory effects of papain and lysostaphin pretreatments on the bacteria suggested that the S. aureus cell surface molecule mediating binding of tropoelastin is a protein and is released from the surface when the organism is lysed with the pepti- doglycase lysostaphin. To identify the putative cell surface elastin-binding protein, affinity chromatography with elastin peptides as the active ligand was performed. Lysostaphin extracts prepared from surface-labeled S. aureus were incu- bated with a-elastin peptides coupled to Affi-Gel 10 for 30 min at room temperature. Taking advantage of the inhibitory effect of 1% SDS on rTE binding to intact bacteria, surface- labeled molecules bound to the affinity resin were eluted with 1% SDS buffer. Subsequent SDS-PAGE and autoradiography revealed three proteins, eluting after 1.5 ml of SDS buffer, with a molecular mass of approximately 25, 55, and 29 kDa, in the order of band intensity (Fig. 10, lane A ) . The 29- and 55-kDa proteins, however, also bound to the BSA column (Fig. 10, lane B ) .

To further examine binding specificity of the three proteins, surface-labeled lysostaphin extracts were incubated with cy-elastin-Affi-Gel 10 in the presence of 2 mg/ml a-elastin or BSA. The affinity resin was washed, and bound materials were eluted with 1% SDS buffer as described previously. As shown in Fig. 10, inclusion of BSA (lane C) or n-elastin (lane D) in the incubation mixture did not inhibit binding of the 29- and 55-kDa proteins to the affinity resin, whereas in the presence of n-elastin, binding of the 25-kDa protein was effectively inhibited (Fig. 10, lane D). Thus, only the 25-kDa protein specifically binds elastin.

With reverse-phase HPLC, it was possible to obtain the radiolabeled 25-kDa protein isolated through affinity chro- matography in a single fraction (Fig. 11, lane A ) . To obtain sufficient protein for sequence analysis, a lysostaphin extract of unlabeled bacteria was fractionated by HPLC and the fraction eluting at the position of the radiolabeled 25-kDa protein (fraction 25, eluting a t 50% acetonitrile) analyzed by SDS-PAGE. Two proteins were clearly visible by silver stain: the 25-kDa protein and a protein migrating at approximately

69 -

46 -

30 -

14.3 -

A B C D

FIG. 10. Isolation of the S. uureus elastin-binding protein. Surface-labeled lysostaphin extracts of S. aureus were incubated with elastin or RSA affinity resins in the presence or absence of 2 mg/ml tr-elastin or BSA for 30 min at room temperature. The affinity resins were washed thoroughly with binding buffer, and bound proteins that were eluted with 1% SDS elution bulfer were analyzed by SDS-PAGE on a 12% gel followed by autoradiography. Lane A, proteins bound to tu-elastin-Affi-Gel. Lane R, proteins bound to BSA-Affi-Gel. Lane C, proteins bound to tr-elastin-Affi-Gel in the presence of 2 mg/ml BSA or 2 mg/ml welastin (lane 11).

7001

40

25

A B C

0 10 20 30 40 50

Retention Time (minl

FIG. 11. Fractionation of S. uureus elastin-binding proteins with reverse-phase HPLC. Radiolabeled S. aurcus elastin-hinding protein isolated by affinity chromatography was subjected to reverse- phase HPLC, and the peak radioactive fraction (graph) was analyzed by SDS-PAGE on a 12% gel followed by autoradiography (lane A ). Unlabeled lysostaphin extract was then fractionated using the same gradient, and the peak eluting at the position of the radiolabeled elastin-binding protein was collected and analyzed by SDS-PAGE and silver stain (lane R ) . Elastin binding activity of the isolated proteins was assessed by elastin affinity chromatography of the iodinated column fraction. Both the 25- and 40-kDa proteins bound to the elastin column ( h e C ) .

40 kDa (Fig. 11, lane B ) . To examine the elastin-binding activity of each protein, the HPLC fraction was iodinated and fractionated by elastin affinity chromatography. As shown in Fig. 11 (lane C), both the 25- and 40-kDa proteins bound to the affinity resin. For sequence analysis, these proteins were transferred to Pro-Blot membrane and subjected to 14-18 cycles of amino-terminal sequence analysis. Interestingly,

Binding of Elastin to S. aureus 23405

both proteins had the same amino acid sequence over this region (Table 11). A search of GenBank showed that the sequence was unique.

DISCUSSION

Adherence of bacteria to components of the ECM is thought t o play a critical role in the infectious process. Several bac- teria, including strains and species of Streptococcus and Staphylococcus, have been shown to have specific receptors that mediate attachment to matrix components such as fibro- nectin, laminin, and collagen (10, 28, 29). Thus, by binding to ECM components, bacteria may enhance their colonization of sites where the epithelial layer may be compromised or attach to artificial prosthetic devices with adsorbed ECM molecules. The invasiveness of these bacteria is typified by S. aureus, which when introduced into wounds or breaches in the mucous membranes, moves quickly into the blood stream, possibly resulting in endocarditis and widespread abscesses (14). In entering and leaving the circulatory system, S. aureus most likely encounters elastin, a major ECM component of the vessel wall. Indeed, S. aureus is thought to have a potent elastase (30, 31) that may facilitate its movement through the elastin-rich layer. For these reasons, and to investigate the evolution of the elastin receptor by comparing binding and molecular properties of the bacterial and mammalian systems, we chose to investigate the presence of a specific interaction between S. aureus and elastin.

In ligand binding studies, S. aureus interacts with cell-free translated, native and full-length recombinant forms of tro- poelastin. Evidence that this binding is specific is the inhibi- tion of tropoelastin binding by @-elastin peptides but not by other proteins. The ability of a-elastin peptides to inhibit tropoelastin binding suggests that the bacteria interacts with mature insoluble elastin, which is the physiologically relevant form of the protein that would be encountered by the bacteria during infection and invasion. These data, together with the ability of S. aureus to bind the various forms of tropoelastin, suggest the following conclusions about recognition of elastin by the bacteria: 1) interaction with nTE indicates that the lysine-derived crosslinks found in mature elastin are not critical for binding; 2) binding to rTE eliminates hydroxypro- line as an important determinant since rTE contains none; and 3) the inability of S. aureus to bind to trTE strongly suggests that the binding domain for S. aureus is located within the NH2-terminal 30-kDa fragment of tropoelastin. We are currently in the process of generating smaller frag- ments from the NHP-terminal 30-kDa region to better char- acterize the staphylococcal binding domain.

Tropoelastin binding was not a universal function shared by all staphylococci, but rather a unique property of some

TABLE I1 Amino-terminal sequence of the 25,000- and 40,000-dalton elastin-

binding proteins isolated from S. aureus Lysostaphin extracts of unlabeled S. aureus were fractionated by

reverse-phase HPLC. The fraction eluting a t 50% acetonitrile was collected, dried, subjected to SDS-PAGE and electrophoretically transferred to Pro-Blot using the manufacturer's suggested protocol. The 25- and 40-kDa proteins, visualized by staining the Pro-Blot membrane with Coomassie Brilliant Blue R-250, were excised and directly sequenced using an Applied Biosystems 473 protein sequen- cer. The 25- and 40-kDa proteins were subjected to 14 and 18 cycles of sequence analysis, respectively. X represents the uncertain amino acids of the sequence.

5 10 15 25-kDa protein A N N F K D D F E K N R Q Q 40-kDaprotein A N N F K D D F E K N R Q X S D X N

species of this organism. All three strains of S. aureus tested in this study specifically bound Y - r T E , although to different degrees. Strain 8095 showed the strongest binding, followed by strains Cowan and Woods 46. A similar heterogeneous binding pattern has been reported for the interaction between type I collagen and various strains of S. aureus (9) and is thought to result from variations in the expression of the collagen binding molecule among the various S. aureus strains. We do not yet know if alterations in elastin-binding protein expression is an appropriate explanation for the ob- served difference in '"I-rTE binding. Specific binding of 9 - rTE by the protein A-negative strain Woods 46 indicates that protein A is not mediating the binding to tropoelastin. In addition, expression of coagulase, which is the specific fibrin- ogen binding protein on s. aureus (32), is not a prerequisite for tropoelastin binding to staphylococci, since coagulase- negative s. epidermidis also interacts with 'T- rTE.

A candidate protein for mediating elastin binding to S. aureus is the 25-kDa protein identified in this study. Func- tional properties of the 25-kDa protein agree favorably with the results of our binding studies with live S. aureus. Similar to binding at the cellular level, elastin binding by the 25-kDa protein is inhibited by a-elastin but not by BSA. The presence of a single elastin binding molecule on the bacterial surface is also consistent with results of Scatchard analysis, demon- strating the presence of a single class of high affinity binding sites. We did not directly examine the elastin binding ability of other S. aureus surface molecules, such as lipoteichoic acid that have been implicated in bacterial adhesion (33), since our data implicate the 25-kDa protein as the primary surface molecule mediating tropoelastin binding.

The relationship between the 25- and 40-kDa elastin-bind- ing proteins shown in Fig. 11 is unknown. The molecular mass of the higher band is inconsistent with it being a dimer of the 25-kDa protein. The similar elastin binding properties, identical amino acid sequence at the amino end of both proteins, and the observation that the 40-kDa protein does not radiolabel when intact bacteria are surface iodinated suggest that the 40-kDa protein may be an intracellular precursor of the 25-kDa molecule. Further experimentation is necessary to establish this relationship.

The results of this study suggest that the staphylococcal elastin-binding protein has structural and functional proper- ties distinct from the 67-kDa mammalian elastin receptor. The difference in size of the two receptors suggest structural uniqueness, whereas functional differences are suggested by distinct binding domains on tropoelastin for the two receptors. The staphylococcal binding site is located in the 30-kDa amino-terminal fragment of tropoelastin while the mamma- lian receptor recognizes a Val-Gly-Val-Ala-Pro-Gly sequence about one-third the distance in from the carboxyl terminus. Similarly, agents that perturb elastin interaction with the mammalian receptor do not influence tropoelastin binding to S. aureus. These patterns are similar to fibronectin receptors and fibronectin binding to S. aureus. Previous investigations have shown that the staphylococcal fibronectin receptor has a different molecular mass from the mammalian fibronectin receptors (34) and that the primary binding site on fibronectin for S. aureus is localized to the NH2-terminal domain of the protein, whereas the mammalian fibronectin receptor binds near the carboxyl end (35, 36).

At present, the functional significance of elastin binding to S. aureus and the biosynthetic pathways of the 25-kDa elastin receptor are unknown. We do not know whether the elastin- binding protein contributes to the pathogenicity of the bac- teria or if it plays a role in S. aureus metabolic activities. It

23406 Binding of Elastin to S. aureus

should also be noted that only laboratory passaged strains were used in this study and that the observed results may or may not reflect in vivo binding. Further characterization of the elastin-binding protein and the development of better reagents to study its expression and activity will help answer these questions.

REFERENCES

1. Liotta, L. A., Rao, C. N., and Wewer, U. M. (1986) Annu. Ret..

2. Ruoslahti, E. (1988) Annu. Reu. Biochem. 57, 375-413 :I. Ouaissi, M. A., Afchain, D., Capron, A,, and Grimaud, J. A. (1984)

4. Thomas, D. D., Baseman, J . B., and Alderete, J. F. (1985) J . Exp.

5. Pottratz, S . T., and Martin 11, W. J . (1990) J . Clin. Inuest. 85,

6. Kuusela, P., Vartio, T., Vuento, M., and Myrhe, E. B. (1984)

7. Kuusela, P. (1978) Nature 2 7 6 , 718-720 8. Chhatwal, G. S., Preissner, K. T., Muller-Berghaus, G., and

9. Holderbaum, D., Hall, G. S., and Ehrhart, L. A. (1986) Infect.

10. Lopes, J. D., DosReis, M., and Brentani, R. R. (1985) Science

11. Rosenbloom, J. (1982) Connect. Tiss. Res. 10, 73-91 12. Sandberg, L. B., Gray, W. R., and Franzblau, C. (1977) Elastin

and Elastic Tissues, Plenum Press, New York 13. Oz, M. C., Brener, B. J., Buda, J. A., Todd, G., Brenner, R. W.,

Goldenkranz, R. J., McNicholas, K. W., Lemole, G. M., and Lozner, J. S. (1989) J. Vasc. Surg. 10, 439-449

14. Sheagren, J. N. (1984) N. Engl. J. Med. 310, 1437-1442 15. Wheat, L. J., Kohler, R. B., and White, A. (1983) in Staphylococci

and Staphylococcal Infections (Easmon, C. S. F., and Adlam, C., eds) pp. 121-148, Academic Press, New York

16. Noble, W. C., and White, M. I. (1983) in Staphylococci and staphylococcal infections (Easmon, C. S. F., and Adlan, C., eds)

Biochem. 55, 1037-1057

Nature 308, 380-382

Med. 161,513-525

351-356

Infect. Immun. 45,433-436

Blobel, H. (1987) Infect. Immun. 5 5 , 1878-1883

Immun. 54,359-364

2 2 9 , 275-277

pp. 165-192, Academic Press, New York 17. Partridge, S. M. (1962) Adv. Protein Chem. 17,227-302 18. Prosser, I. W., Whitehouse, L. A., Parks, W. C., Stahle-Backdahl,

M., Hinek, A,, Park, P. W., and Mecham, R. P. (1991) Connect. Tiss. Res. 25, 265-279

19. Grosso, L. E., Parks, W. C., Wu, L., and Mecham, R. P. (1991) Biochem. J . 273, 517-522

20. Indik, Z., Abrams, W. R., Kucich, U., Gibson, C. W., Mecham, R. P., and Rosenbloom, J. (1990) Arch. Biochem. Biophys. 280, 80-86

21. Mecham, R. P., and Lange, G. (1982) Methods Enzymol. 82,744- 759

22. Parks, W. C., Secrist, H., Wu, L. C., and Mecham, R. P. (1988) J. Biol. Chem. 263,4416-4423

23. Joklik, W. K., Willett, H. P., Amos, D. B., and Wilfert, C. M. (1988) in Zinsser Microbiology, pp. 343-356, Appleton & Lange, Norwalk, CT

24. Wrenn, D. S., Hinek, A., and Mecham, R. P. (1988) J . Bid. Chem.

25. Scatchard, G. (1949) Ann. N . Y. Acad. Sci. 51,660-672 26. Mecham, R. P., Hinek, A., Griffin, G. L., Senior, R. M., and

27. Mecham, R. P., Hinek, A., Entwistle, R., Wrenn, D. S., Griffin,

28. Switalski, L. M., Speziale, P., Hook, M., Wadstrom, R., and

29. Switalski, L. M., Murchison, H., Timpl, R., Curtiss, R., 111, and

30. Potempa, J., Dubin, A., Korzus, G., and Travis, J. (1988) J . Biol.

31. Murphy, R. A. (1974) J . Dent. Res. 53,832-834 32. Bodkn, M. K., and Flock, J . (1989) Infect. Immun. 57,2358-2363 33. Haagen, I. A., Heezius, H. C., Verkooyen, R. P., Verhoef, J., and

34. Froman, G., Switalski, L. M., Speziale, P., and Hook, M. (1987)

35. Proctor, R. A,, Mosher, D. F., and Olbrantz, P. J . (1982) J. Bid.

36. Sottile, J., Schwarzbauer, J., Selegue, J., and Mosher, D. F. (1991)

263,2280-2284

Liotta, L. (1989) J. Biol. Chem. 264, 16652-16657

G. L., and Senior, R. M. (1989) Biochemistry 28,3716-3722

Timpl, R. (1984) J. Biol. Chem. 259,3734-3738

Hook, M. (1987) J. Bacteriol. 1 6 9 , 1095-1101

Chem. 263,2664-2667

Verbrugh, H. A. (1990) J. Infect. Dis. 161, 266-273

J. Biol. Chem. 262, 6564-6571

Chem. 257,14788-14794

J . Biol. Chem. 266,12840-12843