Induction of a Putative Laminin-Binding Protein of Streptococcus

Vol. 60, No. 2

INFECTION AND IMMUNITY, Feb. 1992, p. 360-365

0019-9567/92/020360-06$02.00/0 Copyright C 1992, American Society for Microbiology

Induction of a Putative Laminin-Binding Protein of Streptococcus gordonii in Human Infective Endocarditis PASCAL SOMMER,'* CLAUDINE GLEYZAL,1 SYLVIANE GUERRET,1 JEROME ETIENNE,2 AND JEAN-ALEXIS GRIMAUD1 Department of Pathology, Centre National de la Recherche Scientifique URA 1459, Institut Pasteur of Lyon, Avenue Tony Garnier, 69365 Lyon Cedex 7,1 and Department of Bacteriology, Hopital Louis Pradel, Faculty of Medicine Lyon-Nord, 69394 Lyon Cedex 03,2 France Received 6 August 1991/Accepted 9 November 1991

There is evidence to suggest that the virulence of Streptococcus strains in infective endocarditis might be due to the expression of binding sites for the extracellular matrix proteins of damaged valves. In this communication, we draw attention to one laminin-binding protein from a strain of Streptococcus gordonii isolated from a patient with human endocarditis. This 145-kDa protein was found on the cell wall of the bacterium. The level of expression of this binding protein might be regulated by the presence of extracellular matrix proteins: the protein was lacking after in vitro selection of laminin, collagen I, and fibronectin nonbinding variants, and it was recovered after growth of the variants when laminin or collagen I was added to the growth medium. It was also missing after 10 subcultures in minimal medium, indicating some positive control. Furthermore, the 145-kDa protein was recognized as a major antigen by sera from patients treated for streptococcal infective endocarditis, while sera from patients with valvulopathies gave only slight recognition, suggesting an increase of the expression of this protein during infective endocarditis. It was also shown that the 145-kDa protein carried a collagen I-like determinant detected with anti-human collagen I antibodies.

used serum from an endocarditis patient, from whom the strain was isolated, as an immunological indicator of the in vivo level of protein expression of the bacteria.

Streptococcus gordonii (formerly Streptococcus sanguis I) is a general inhabitant of the buccal cavity, where it is commonly associated with dental caries (11). It is also one of the strains most often associated with endocarditis in humans (1). The virulence determinants in caries and endocarditis have not been well established, but they are most probably different and multifactorial. However, the role of receptors in the first step of infection is likely to be significant. Streptococcal receptors for saliva glycoproteins are involved in the cariogenic process (7, 18), and receptors for blood components or extracellular matrix (ECM) could lead to the bacterial colonization of damaged valves (26). Streptococcal receptors for components of the basement membrane (laminin [23], proteoglycans [6], and fibronectin [15]) and platelet proteins (8) have been described elsewhere. In view of increasing evidence for the role of adaptative regulation in bacteria (10), differences between an inhabitant of the oral cavity and the same organism involved in endocarditis might be in the expression of new receptors. Since a general feature of infective endocarditis is the colonization of damaged valves, where proteins of the basement membrane and the ECM are accessible, some information might be transmitted to the bacteria at such sites. We thus addressed the question of a possible role for the ECM proteins in streptococcal regulation. The direct or indirect informative role of the ECM proteins in cellular and tissue regulation is well documented (2, 3), but it has never been described in bacteria. We decided to work with an S. gordonii strain isolated from a patient with infective endocarditis and able to bind laminin. We first selected nonbinding variants of this strain, which might correspond to the status of the bacteria at the oral site. We grew the variants in the presence of laminin or collagen in order to mimic the events occurring when the strain reaches the damaged valves. Finally, we *

MATERIALS AND METHODS

Strains, growth conditions, and sera. Four strains (S. gordonii 2316, Streptococcus oralis 815, Streptococcus mitis 716, and S. oralis 516) considered to be responsible for four cases of infective endocarditis and isolated from blood cultures were used in this study. The strains were identified with the Api-32-Strep galleries (Api System, Montalieu Vercieu, France). Strains were picked from blood agar plates, grown in Todd-Hewitt broth, and stored at -20°C. Staphylococcus aureus Cowan I was donated by P. Speziale, University of Pavia, Pavia, Italy. Strains were maintained in brain heart infusion broth (BHI; Difco Laboratories) and stored at -70°C in BHI supplemented with 20% glycerol. A minimal medium was developed according to Carlsson (5), but RPMI 1640 (Vietech, Saint Laurent de More, France) and Dulbecco's minimal essential medium with 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; GIBCO BRL) were also used. As no differences were observed between the three minimal media tested, we decided to use RPMI supplemented with 5% fetal calf serum (GIBCO BRL) or not supplemented. Preparation of proteins. S. gordonii cell wall-associated proteins were obtained from the washed bacterial pellet by using glass beads (100 Rm; Sigma) in 0.5 M phosphate buffer (pH 6), and extracellular proteins were recovered from the culture supernatant as described elsewhere (18). Human collagen I was obtained from the Boy Institute (Reims, France), and bovine collagen I and II and human collagen III and IV were obtained from SERAD (Lyon, France). Human collagen V and 14C-labeled human collagen I were given by H. Emonard (Institut Pasteur, Lyon, France). Human fibronectin was purified according to the method of Caillot et

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al. (4). Purified laminin from mouse Engelbreth Holm swarm tumor was a generous gift from H. Kleinman (Bethesda, Md.). ECM proteins were conjugated to CNBr-activated Sepharose-4B (Pharmacia) according to the instructions of the manufacturer. Bovine serum albumin (BSA) was obtained from Sigma. Sera and antisera. Sera were collected weekly from four patients with infective endocarditis from their admission to the hospital until the end of treatment. Sera from patients with valvulopathies were also collected. Anticollagen (I, II, III, IV, and V) and antilaminin antibodies were raised in rabbits and purified according to the method of Stocker et al. (21). Each anticollagen antibody was purified by chromatography through two columns of collagens of different isotypes and eluted from the corresponding collagen column; antilaminin antibody was affinity purified from laminin coupled to Sepharose-4B. Each ECM protein antibody was routinely checked for specifity and absence of cross-reactivity against the different collagens, fibronectin, and laminin by enzymelinked immunosorbent assay (ELISA) (21) or immunofluorescence (25), and their contents of immunoglobulin G (IgG) and IgM were determined by ELISA. Peroxidase-conjugated goat anti-human IgG and anti-rabbit IgG antibodies were from Diagnostic Pasteur and were preliminary adsorbed on S. gordonii 2316 cell wall extracts bound to nitrocellulose filters as described elsewhere (19), and peroxidase-labeled goat anti-rabbit IgM antibodies were from Nordic. Electrophoresis. Wall-associated proteins were electrophoresed on 7.5, 10, and 12% polyacrylamide slab gels according to the method of Laemmli (13). Antigens were transferred to nitrocellulose sheets (ECL membranes; Amersham) as described by Towbin et al. (24) or stained for protein with 0.1% Coomassie blue or by the ammoniacal silver procedure (17). Molecular weight protein markers were purchased from Bio-Rad. Scanning analyses were performed by using the Biocom 200 (Les Ullis, France) image-processing station. Immunochemistry. Antigens blotted on nitrocellulose sheets were detected according to the method of Ogier et al. (18) with the following modifications. Briefly, for immunodetection, blots were soaked in 1% BSA in 50 mM Tris HCl (pH 7.2)-0.1 M NaCl-0.05% Tween (THST) and incubated with specific antisera diluted in THST. The immune complexes were labeled with peroxidase-conjugated antiglobulins diluted 1/100 or 1/10,000 in THST, and the peroxidase activity was detected by using 3-amino 9-ethyl carbazole (Orthodiagnostic Systems) or luminol (Amersham), respectively, according to the instructions of the manufacturers. For detection of laminin or collagen binding on the transferred antigens, the sheets were blocked in 1% BSA in THST, washed, and incubated overnight at 4°C with different concentrations of laminin or collagen in THST. After extensive washing, antilaminin or anticollagen antibodies were added during 90 min at 37°C, and the immune complexes were detected as described above. Direct ['4C]collagen I binding was assessed under the same conditions, and the complexes were visualized by autoradiography. Laminin or collagen antigenicity was checked by ELISA according to the method of Stocker et al. (21), with 5 pLg of proteins per well. The S. gordonii wall-associated antigens were similarly studied, but the proteins were coated (maximum of 50 ,ug per well in phosphate-buffered saline) onto microtitration plates (Falcon 3912 microtest III; Becton Dickinson and Co.) overnight at 4°C. Assays were done in triplicate, and the mean values were obtained after subtraction of values for controls obtained with BSA.

S. GORDONII LAMININ-BINDING PROTEIN

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Test for adherence of S. gordonii to immobilized proteins. Laminin and collagen I were coated onto microtitration plates (Linbro tissue culture multiwell plate; Linbro Scientific, Inc.) by adding 50 RI of each protein solution (at 20 jig/ml in RPMI) to each well and incubating the plates for 1 h at 37°C. Plates were washed twice with 100 RI of RPMI, incubated for 30 min with BSA (0.05% in RPMI), and washed again with RPMI. S. gordonii 2316 was grown overnight and washed in RPMI, and 50 RI of the bacterial suspension (diluted to 20,000 CFU/ml in RPMI) was added to the wells. After 1 h at 37°C, nonbinding bacteria were recovered from each well and added to another laminin- or collagen-coated well, where they were allowed to react again with the ECM proteins. After 1 h at 37°C, nonbinding bacteria were recovered and added again to coated wells as before. Nonbinding bacteria were finally recovered, and the last wells were gently washed twice with RPMI. Each wash was added to the first supernatant, and the nonbinding bacteria were spread on BHI plates to be counted. Controls were carried out without ECM protein coated to the wells, and the percentage of nonbinding bacteria was calculated from the difference between results with the assays and the control. Each assay was done in triplicate. A positive control was performed with a good collagen-binding strain (S. aureus Cowan I; 20), resulting in almost 90% binding. Selection of non-laminin- or non-collagen-binding variants. Variants of S. gordonii 2316 were selected on the basis of their inabilities to bind laminin or collagen I in the assay developed above. At the end of the first adherence assay, the nonbinding bacteria were grown overnight in 5 ml of RPMI. Different rounds of selection were accomplished in the same manner, and the resulting bacteria were finally grown in 50 ml of RPMI without any storage step. The cell wall-associated antigens were extracted from the washed bacteria harvested when the concentration reached about 107/ml. The efficiency of the selection was checked by using the assay described above. Protein determination. Protein concentrations were measured by the method of Lowry et al. (16). RESULTS Characterization of inducible proteins from S. gordonji 2316. The reference strain for this study was selected from a collection of streptococcal strains responsible for infective endocarditis and for which we had the corresponding sera. The strain S. gordonii 2316 appeared to be useful because it bound laminin with a reasonable efficiency in our in vitro test (>20% of binding cells) but bound collagen I and fibronectin very weakly (