Zbl. Bakt. 277, 54-64 (1992) © Gustav Fischer Verlag, StuttgartlNew York

Vitronectin-Binding Surface Proteins of Staphylococcus aureus MARIANNE PAULSSON, OLIN D. LIANG, FELIPE ASCENCIO, and TORKEL WADSTRGM ,. Department of Medical Microbiology, University of Lund, Lund, Sweden

With 4 Figures· Received September 3, 1991 . Revision received November 5, 1991 . Accepted November 28, 1991

Summary S. aureus strain ISP546 was selected (of 55 strains tested) to define optimal conditions for expression of vitronectin binding. High binding was expressed when the strain was grown on blood agar and in Todd-Hewitt broth. Binding was optimal in the 6.0 to 7.2 pH range and was unaffected by divalent cations and ionic strength. Binding was partially inhibited by D-mannose, heparin, types I and IV collagen, fibronectin, fibrinogen and vitronectin, but was not affected by other carbohydrates or glycoproteins tested. Cell surface binding components were extracted with the aid of 1 M LiCI (pH 5.0) from strain ISP 546 grown in Todd Hewitt broth. Vitronectin binding proteins were purified by affinity chromatography on heparin-Sepharose. Fractions inhibiting binding of 125I-labelled vitronectin to strain ISP 546 were eluted by 0.01 M NaOH, dialysed, concentrated and subjected to SDS-PAGE. Silver staining revealed one major band (70kDa) and two minor bands (34 and 36 kDa).

Zusammenfassung Aus 55 gepruften Stammen wurde S. aureus ISP 546 ausgewahlt, urn die optimalen Bedingungen fur die Expression einer Vitronectin-Binding zu definieren. Bei Anzucht des Stammes auf Blutagar und in Todd-Hewitt-Bouillon kam es zur Expression einer starken Bindung. Im pH-Bereich zwischen 6,0 und 7,2 war die Bindung optimal; sie wurde durch divalente Kationen und die Ionenstarke nicht beeinflufst. Die Bindung wurde durch DMannose, Heparin, Collagen (I und IV), Fibronectin und Vitronectin, aber nicht durch andere geprufte Kohlenhydrate und Glycoproteine gehemmt. Die Komponenten der Bindung an die Zelloberflache wurden mittels 1 M LiCI (pH 5,0) aus dem in Todd-HewittBouillon angezuchteten Stamm ISP 546 extrahiert. Die Vitronectin-bindenden Proteine wurden durch Affinitiitschromatographie auf Heparin-Sepharose gereinigt, Die Bindung von 125I-markierten Vitronectin an den Stamm ISP546 hemmenden Fraktionen wurden mit 0.01 M NaOH eluiert, dialysiert, konzentriert und mittels SDS-PAGE abgetrennt. Silberanfarbung lielS eine Hauptbande von 70 kDa und zwei Nebenbanden von 34 bzw. 36 kDa erkennen. " Corresponding author

Vitronectin-Binding Surface Proteins of Staphylococcus aureus

55

Introduction Vitronectin (Vn), or serum spreading factor (synonym: S-protein), is a serum protein involved in the regulation of blood coagulation and certain immunoreactions such as binding to C5-C8 complement complexes (21, 26) and plasminogen(22). Vitronectin is also a constituent of dermis (11) and vascular walls associated with elastin (6, 7) and different types of collagen (10). Thus, tissue vitronectin is a possible receptor, in addition to fibronectin, various collagens and laminin, for colonization of staphylococci and other pathogens in wound tissues (1, 29). Vitronectin has recently been reported to exist in human plasma in two structurally and functionally distinct forms (12). Vitronectin binds to heparin at a heparin-binding domain located near its carboxyl terminus. It also binds to eucaryotic cells via a tripeptide Arg-Gly-Asp sequence located near its amino terminus (24). Interestingly, there is normally a subpopulation of vitronectin molecules in human plasma which have heparin-binding activity (12, 25). Binding of vitronectin to staphylococci was first described by Fuquay et al. (9) and streptococcal and E. coli Vn-binding was subsequently investigated (4). Vitronectinmediated adherence of streptococci to epithelial cells has been shown (8,27). We have previously demonstrated cooperative binding between vitronectin and collagen on the staphylococcal cell surface (19). The goals of the present investigation were (1) to determine the growth conditions required for optimal expression of vitronectin binding components by S. aureus, (2) to define receptor-ligand interactions between bacterial cells and vitronectin and (3) to evaluate a purification procedure for the vitronectin-binding components of S. aureus. Materials and Methods Bacterial strains and culture conditions. S. aureus strain ISP546 is a derivative of laboratory strain 8325-4 and was obtained by insertion of transposon Tn551 in the agr (accessory gene regulator) gene (2, 23). S. haemolyticus strain E2498/86 was isolated from an osteomyelitis patient. These strains have previously been used for vitronectin binding studies (19). Blood agar (BA) base was purchased from Lab M, Salford, U.K. and supplemented with 4% horse blood. Horse-sheep blood agar (HSBA) consisted of an underlayer of blood agar base No.2, and an overlayer of 10 ml of blood agar base containing 2% horse blood and 2% sheep blood. Haematinized blood agar (HBA) contained 7% horseblood. Trypticase Soy Broth (TSB) and Todd-Hewitt broth were purchased from Difeo Laboratories, Detroit, MI, USA. Cells were harvested during the stationary phase (i.e. at 24 h) and washed twice with 0.07M PBS, pH 7.2. The bacterial cells counts was calculated from a standard curve (absorbance at 540 nm) and adjusted to 5 x 109 organisms per m!. Chemicals. Human vitronectin (Vn) was affinity-purified after urea activation of human plasma on heparin-Sepharose according to Yatogho et a!. (30). Human fibronectin (Fn) was purified by affinity chromatography on gelatin-Sepharose (28). Heparin-Sepharose 6B and protein A were purchased from Pharmacia, Uppsala, Sweden. A collagen (Cn) preparation (Vitrogen'P 100, containing 95% Type-I and 5% of Type-III Cn, lot no 87H183) was purchased from Collagen Corporation, Palo Alto, Ca. Collagen Type IV was purchased from Collaborative Research, Inc, Massachusetts, USA. D-glucose and LiCI were purchased from BDH, Poole, U.K. N-acetylgalactosamine, D-mannose, fucose, porcine mucin, fetuin, guanidine-HCl, sucrose, copper sulphate, magnesium chloride, chymotrypsin, pepsin, pronase E, proteinase K and trypsin were purchased from Sigma Chemicals, St.Louis, MO, USA. Bovine serum albumin fraction V was obtained from Boehringer Mannheim GmbH, Germany and Iodobeads'" were from Piece Chemical Company, Rockford, IL, USA.

56

M . Paulsson, O . D. Liang , F. Ascencio, and T. Wadstrom

Orosomucoid, human fibrinogen and transferrin were purchased from Kabi, Stockholm, Sweden and human IgG from Serva , Heidelberg, GmbH, Germany. D-galactose, urea glycine, manganese chloride and calcium chloride were from Merck, Darmstadt, FRG, and heparin from Loven s Lakernedel, Malmo, Sweden. All chemicals used were of analytical grade. l25I-labelied protein bindin g assay. The method has been described in detail (19). Briefly, 100 ~l of bacterial cell suspension (5 X 10 8 cells) in 0.0 7 M sodium ph osphate-buffered sa line (PBS), pH 7.2, were incubated for one hour at 20 °C with 50 ~l of 12sI-labelled vit ro nectin (3 x 10 4 cpm ) in PBS with 1% BSA. After washing with PBS-Tween (0.1%), the radi oactivity remaining in th e pellet wa s measured. For each experiment, additional negative cont rol test tubes with all ingredie nts except bacteria were included and radioactivity wa s determined. Th is value was then substracted from the values mea sured in other tubes in the experiment before per centage of bind ing was calculated. Each exp eriment was performed in duplicate. Binding values below 5 % of added radioactivity were considered to represent nonspecific binding. Proteolytic digestion. Bacterial cells (100 ul) were suspended in appropriate buffers and incubated with 100 ug of the proteases for 1 h at The cells were then washed with 2 ml of PBS, and the supernatans obtained by centrifugation of the suspensions for 15 min at 1760 X g were discarded. The binding assay was performed with the cells. The buffers for the vario us proteases were 0.01 M ph osphate-buffered saline, 0.5 mM CaCb, (p H 7.2 ) for chymo trypsin, pronase E and pr ot einas e K, and 0.07 M PBS, (p H 7.2) for trypsin. The bacter ial cells were tested for Vn bind ing after being heated at 80 °C for 20 min . Ex traction of uitronectin-binding proteins. S. aureus, ISP 54 6, was cultured on blood agar for 24 h at The cells (approxima tely 1 g wet weight) were washed twi ce in cold PBS and the cells were suspended a nd ex trac ted with 1.5 ml of ext raction solutions. The extra ction meth od s were as follows; 1.0 M LiCl, pH 5. 0 at 45 °C for 2 h, 5 .0 M guanidineH Cl at 20 0 e for 15 min , 0.2 M glycine pH 2.2 at 20 0 e for 15 min , 20 % sucrose in 5 0 mM T ris pH 10 .0 at 20 0 e for 1 h, 3 .0 M and 6.0 M urea at 20°C for 30 min, 10 mM EDTA at 20 °C for 1 h. Th e extracted cell suspensio ns were centrifuged (9750 x g) for 60 min until the ex tracts were clear, an d dialyzed (LiCl , guanidine-H Cl, sucros e an d EDTA extracts aga inst PBS, and glycine and urea extracts against 0.02 M ammoni um bicarbon ate ). The ex tra cts were stor ed at -20 °C unti l use. Fermentor cultures and pu rification of uitronectin binding protein. S. aureus ISP 54 6 wa s cultured at in 9 liters of Todd Hewitt broth in a fermentor (Biotec FL 110, LKBPharm ac ia, Upp sala, Sweden ) and the cells were harvested when the culture had reached the late sta tio na ry phase (a fter 24 h of g ro w th), wash ed twi ce in PBS, a nd kept at -70 o e until use. O ne gra m (wet weight) of cells, was suspended in 2.5 ml of 1 M LiCl (p H 5. 0). The resuspe nded cells were incub ated at 45 °C for 2 h, centrifuged (5900 X g) for 30 min . The supe rn ata nt fluid was dialyzed agai nst 0.0 2 M ammonium bicarbon at e; 1 mM EDTA, pH 7.5 at 4 °C overnight, centrifuged at 39000 x g for 60 min and sup plement ed with 1 mM PMSF and the supernatants wer e kept at - 20°C, prior to chromat ography on a HeparinSepharose 6B column (6.5 X 2.5 em) equilibrated with PBS containi ng 0.1 M EDTA. 90 ml of ex trac t was applied to the column, whi ch was washed with a sodium chloride gradient (0 to 1 M ) followed by 2 M N aCl and , finally , 0.01 M NaOH. The fractions were dialysed aga inst PBS, pH 7.2 and ru n on SDS· PAGE (12 % ) according to Laemmli (14 ) and silver stained (silver-stain ing kit from Biorad, Richm ond , CA l. Inhibition assays. Th e a bility of d ifferent glyco pro teins and polysacch aride s to inh ibit Vn bind ing we re tested . Glycop rot eins (100 ~I of 0.1%) or polysaccharides (100 ~I of 0 .1 M ) we re preincubated with 100 111 of bacter ial cell suspensions for 1 h at 22 °C, 50 ~I o f labelled protein was added, and the bind ing assays were performed. The abili ty of th e cell ex tracts and of protein A to inh ibit Vn bind ing was also tested; cell extracts (100 ul) or prote in A (5 0 ug) were pr eincubated with 5 0 ~I of labell ed protein at 22 °C for 1 h before 100 J.lI of bac teria l cell suspensions were added and binding assa ys per formed .

3rc.

3rc.

3rc

Vitronectin-Binding Surface Proteins of Staphylococcus aureus

57

Competitive inhibition was studied by adding increasing amounts of Vn and heparin at the same time as the labelled Vn to the staphylococcal cell suspensions and the binding was tested. The effect of pH and divalent cations on uitronection binding. Buffers with different pH; 0.2 M glycine-HCl (pH 2.0), PBS (pH 4.0, 6.0, 7.2), and 0.1 M Tris-NaOH (pH 9.0 and pH 11.0) were used as incubation buffers in Vn binding tests. In some experiments, divalent cations (CUS04, MgCb, MnS04, CeCl-), 1 mM final concentration or EDTA (1 mM) were added to the incubation buffer.

Results

Vitronectin binding to staphylococcal cells was markedly influenced by culture medium and growth conditions (Table 1). Media containing blood enhanced the expression of vitronectin binding surface structure to strains of S. aureus and to coagulTable 1. Influence of culture media on vitronectin-binding by S. aureus ISP 546

a

b C

Culture media

% Vn-binding

Sheep blood agar Horse-sheep blood agar Blood agar-base" Todd Hewitt broth Denatured sheep blood agar" Trypticase soy broth TSBC + 5% human serum

52 52 20 59 11 10 9

Blood agar base for sheep blood agar. Denatured sheep blood agar (80°C, 14 min). Trypticase soy broth.

Table 2. Influence of different glycoproteins or carbohydrates on vitronectin binding to S.aureus ISP 546 Compound

% Vn binding

Control 0.1% BSA 0.02% type I collagen 0.02% type IV collagen 0.02% human fibronectin 0.1 % human IgG 0.1 % fibrinogen 0.1 % transferrin 0.1 M N-acetylgalactoseamine 0.1 M D-galactose 0.1 M D-glucose 0.1 % fetuin 0.1 M fucose 0.1 M D-mannose 0.1 % orosomucoid

48 40 10 15 23 53 35 41 43 46 45 43 45 26 46

% inhibition of Vn binding

17 57 68 52 -10 27 15 10 4

6 10 6 46 4

58

M. Paulsson, O. D. Liang, F. Ascencio, and T. Wadstrom

ase-negative staphylococci (although not as pronounced as for S. aureus , M. Paulsson , unpublished data). To exclude the pos sibility that this effect was cau sed by the agar, agar-base grown cells were tested for Vn binding and compared to the cells grown on blood agar. Cells grown on blood agar base bound 20% of the vitronectin added, while cells grown on blood agar bound 52% (Ta ble 1). The effect of various glycoproteins and carbohydrates on vitronectin binding to S. aureus ISP 546 is shown in Table 2. Unlabelled vitronectin totally inhibited vitronectin binding (Fig. la), whereas some glycoproteins (human fibron ectin , type I and IV collagen) and carbohydra te (mannose) had an inhibitory effect not as pronounced as that of vitronectin (T able 2). The almost total inhib ition of vitro nectin binding by heparin to S. aureus, ISP 54 6 is shown in Fig. 1b. Vitronectin purified from urea-activat ed plasm a binds to staphylococci differentl y as compared to vitronectin purified by the method described by Dahlback (6). The cooperative bind ing of collagen and vitro nectin on staphylococcal cell surfaces (19) which was observed using vitronectin purified according to the latter method, could not be demonstrat ed using the urea-activated vitronectin,

OJ

c '6 c

:0 I

5

c Q)

~

~ 10 50

100 150 vitronectin (~g/m l)

200

250

~ 50

'6 c

:0 I

40~

5 30 ~ 20 ~

~

10 8

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