Nuffield Department of Pathology and Bacteriology1, and Bone Infection Unit, Nuffield Orthopaedic Centre and Nuffield Department of Medicine3, The John Radcliffe Hospital, Headington, Oxford, UK
Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland2
Interdepartmental Academic Unit of Microbiology and Infectious Diseases, Oxford University, Oxford, UK4
Author for correspondence: Sharon J. Peacock. Tel: +44 1865 220538. Fax: +44 1865 220342. e-mail: sharon.peacock{at}ndp.ox.ac.uk
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ABSTRACT |
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Keywords: adherence, endothelium, fibronectin, Staphylococcus aureus
Abbreviations: FnBP, fibronectin-binding protein
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INTRODUCTION |
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An important feature of S. aureus sepsis is the frequency with which it seeds from the bloodstream to other body sites. Bacterial metastasis from blood to tissues such as bones, joints and solid organs is clinically apparent in 153% of individuals with staphylococcal bacteraemia (reviewed by Ing et al., 1997 ) and must involve interactions between circulating bacteria and vascular endothelial cells. S. aureus adheres to human endothelial cells in vitro (Vercellotti et al., 1984
; Ogawa et al., 1985
) but, surprisingly, both the endothelial surface receptor(s) and the bacterial-cell-wall-associated adhesin(s) responsible remain undefined. Once adhesion has occurred S. aureus cells undergo a process akin to phagocytosis (Lowy et al., 1988
; Yao et al., 1995
). This internalization initiates changes in cytokine expression (Yao et al., 1995
, 1996
), and induces hyper-adhesiveness for monocytes and granulocytes (Beekhuizen et al., 1997
). Following endothelial cell uptake, S. aureus has been demonstrated both within vacuoles and free in the cytoplasm (Menzies & Kourteva, 1998
). The subsequent fate of the endothelial cell varies between studies but appears to depend on the secretion of
- toxin, which is cidal to endothelial cells (Vann & Proctor, 1988
). Lack of
-toxin production by small colony variants may explain why these can persist in the intracellular niche without causing endothelial cell death (Balwit et al., 1994
; von Eiff et al., 1997
).
Attempts to define the endothelial receptor for S. aureus adherence have yielded a 50 kDa membrane glycoprotein on human cells (Tompkins et al., 1990 ), and a 130 kDa membrane glycoprotein on bovine cardiac endothelial cells (Johnson, 1993
). Neither has been characterized further. Acidic fibroblast growth factor has been reported to reduce bacterial adherence (Blumberg et al., 1988
), which is also modulated by extracellular matrix heparan sulfate (Alston et al. , 1997
), while human tumour necrosis factor has been shown to enhance adhesion to glutaraldehyde-fixed endothelial cells in the presence of plasma (Cheung et al., 1991a
). In the latter model, fibrinogen has been reported to act as a bridging molecule in adherence (Cheung et al., 1991b
) without definition of the cognate ligands for it on either cell type.
The bacterial determinants that promote adhesion of S. aureus to endothelium have not been elucidated. The majority of clinical isolates produce capsular polysaccharide serotype 5 or 8 (Karakawa & Vann, 1982 ), which when purified has been shown to bind to endothelial cells and result in release of interleukin-6 and interleukin-8 (Soell et al., 1995
). However, this is inhibited by the presence of pooled human serum from healthy blood donors (Soell et al., 1995
). S. aureus also expresses a range of cell-wall-associated proteins that promote adherence to extracellular matrix proteins and/or soluble plasma components (Foster & McDevitt, 1994
). The fibrinogen- binding protein ClfA and collagen-binding protein have been shown to be important in the pathogenesis of experimental endocarditis (Moreillon et al., 1995
; Hienz et al., 1996
). The fibronectin- and fibrinogen-binding proteins of S. aureus promote bacterial attachment to plasma clots (Raja et al., 1990
; Moreillon et al., 1995
), and to plastic coated with these host proteins in vitro and ex vivo (Vaudaux et al., 1989
, 1993
, 1995
; Greene et al. , 1995
). It is possible that these observations have direct relevance to the mechanisms by which S. aureus adheres to host cells, given that endothelial cells synthesize and incorporate fibronectin into the extracellular matrix, and secrete it into the culture medium when grown in vitro (Jaffe & Mosher, 1978
).
The aim of this study was to evaluate the role of a number of bacterial surface structures in adherence of S. aureus to endothelial cells in vitro. The approach taken was to compare the adherence of a range of defective mutants with that of the isogenic parent as a means of identifying candidate adhesins and cognate receptors for further study. Our results indicate that the interaction between the fibronectin-binding proteins (FnBPs) and endothelial-cell- associated fibronectin represents the dominant pathway for the adherence of S. aureus to human endothelial cells in vitro , and that FnBPs are required for subsequent bacterial internalization.
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METHODS |
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Bacterial strains.
The laboratory strains of S. aureus used, and their sources, are listed in Table 1. The clinical strains JR75, JR76, JR77, JR78 and JR80 were isolated in the Oxford microbiology department in 1995 from blood cultures of patients with native valve S. aureus endocarditis. Phillips and PH100 were gifts from Drs Magnus H öök and Jo Patti, Texas. The fnbA::TcR fnbB::Em R mutations were co-transduced from DU5883 to strain P1 and JR80 by phage-85-mediated transduction (Asheshov, 1966
). Transductants resistant to 2 µg tetracycline ml-1 and 10 µg erythromycin ml-1 were selected. The lack of adherence to purified human fibronectin at 10 µg ml-1 (Sigma) was confirmed by microtitre adherence assay using an established method (data not shown) (Hartford et al., 1997
).
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Endothelial cell culture.
Endothelial cells were obtained from human newborn umbilical vein using an adaptation of a previously described method (Jaffe et al. , 1973a ). Both ends of the cord vein were cannulated using a Portex luerlock adaptor (Southern Syringe), after which the vessel was flushed with PBS. Cells were released by instilling M199 supplemented with 50 U penicillin ml-1 (Gibco), 50 µg streptomycin ml-1 (Gibco) and 0·5 mg collagenase type IA ml-1 into the vessel lumen. After incubation for 20 min at 37 °C the cell suspension was collected by centrifugation, resuspended in M199 supplemented with fetal bovine serum (20%, v/v; Gibco), 90 µg heparin ml-1, 5 ng recombinant fibroblast growth factor ml-1, 50 U penicillin ml-1 and 50 µg streptomycin ml-1 and seeded into a 25 cm2 tissue culture flask. Cells were maintained at 37 °C in 5% CO2 and passaged twice on reaching confluence using a 1:3 split, before subculture onto 13 mm Thermanox coverslips (Gibco) in 24-well tissue culture plates. All flasks and coverslips used in cell culture were pre-coated with gelatin (0·2%, v/v) overnight at 37 °C. The identity of the cells was confirmed as endothelial by their cobblestone appearance at confluency, and positive immunofluorescent staining for von Willebrand factor (Jaffe et al., 1973b
).
Endothelial cell adhesion assay.
Sterile 24-well flat-bottomed tissue culture plates were blocked with BSA (1%, w/v) for 1 h and rinsed twice with PBS. A bacterial inoculum of 108 c.f.u. suspended in 500 µl M199 was added to each well. This inoculum was selected on the basis of dose response curves (data not shown). Confluent endothelial cells coating 13 mm Thermanox coverslips were added after dip-washing three times in M199 to remove traces of culture media. The 24-well plates were incubated at 37 °C in CO2 for 1 h. This incubation period was selected on the basis of time-course studies (data not shown). The coverslips were then dip-washed three times in M199 and once in PBS to remove non-associated bacteria. The endothelial cells were fixed with Cytofix (Cellpath), air-dried, and stained with crystal violet (0·5%, w/v) for 5 min. Following dip-rinsing in water, the coverslips were air-dried and mounted on glass slides. The number of endothelial-cell-associated bacteria was quantified using a visual method. The bacterial count included both adherent and internalized bacteria, but for simplicity bacteria are referred to as adherent in the remainder of the text. Bacteria prepared for the adhesion assay as above were shown not to divide over the course of an hour in binding medium (data not shown), making the number of visualized bacteria an accurate representation of those that had adhered. Each coverslip was scanned under low power to ensure confluence and integrity of the monolayer. Using a calibrated graticule under oil immersion and ax100 magnification lens, the number of bacteria associated with 1 mm2 of confluent endothelial cells was enumerated by a standardized counting procedure. Without prior visual inspection at high power, five high-power fields were selected for counting, as follows: (1) the centre of the coverslip; (2) two points between the centre and left edge; and (3) two points between the centre and right edge.
Endothelial cell internalization assay.
Internalization of S. aureus by endothelial cells was evaluated by incubating the monolayer with lysostaphin at the end of the adherence assay. This has been shown to remove S. aureus that is adherent to the monolayer but does not affect the viability of internalized bacteria (Vann & Proctor, 1987 ). The endothelial cell adhesion assay was performed using the method described above, at the end of which coverslips were rinsed three times in M199 and placed into 24-well plates containing lysostaphin at 10 µg ml-1 in M199 or M199 alone. These were incubated for 20 min at 37 °C in CO2 then fixed and stained as before. Crystal violet was shown to penetrate the endothelial cell and stain internalized bacteria. The numbers of internalized bacteria per mm2 in the lysostaphin-treated monolayers were compared with the total bacterial count (adherent plus intracellular) for the controls. Internalization was also examined in a second assay in which 108 c.f.u. were centrifuged onto the surface of the endothelium during a 5 min spin at 1000 r.p.m. The adherence assay was then allowed to continue over 1 h at 37 °C in CO2, followed by lysostaphin treatment as described above. The 60 min time point used to examine internalization in this assay was selected on the basis of time-course studies (data not shown). Coverslips were rinsed, fixed and stained, and the numbers of bacteria per mm2 were counted using the standardized counting procedure.
Competitive endothelial binding inhibition assay with the recombinant form of the ligand-binding domain of FnBP.
Endothelial cell adhesion assays were performed using the method described above, with the exception that the recombinant form of the ligand-binding domain of FnBPB of Streptococcus dysgalactiae (Joh et al., 1994 ) (rFNBD-B; a gift from Dr Magnus Höök, Texas, referred to below as rFNBD protein) was added to the wells immediately prior to bacterial inoculation at a final concentration of 1 µg ml-1, 10 µg ml-1 or 50 µg ml-1. To control for the possibility that recombinant protein non-specifically interfered with bacterial adherence, parallel assays were performed in the presence of a recombinant truncated ClfA protein (Clf41, residues 221559) (OConnell et al., 1998
) at a final concentration of 25 µg ml-1. A second control was bacteria in the absence of recombinant protein. The total volume was maintained at 500 µl for all wells.
Effect of anti-human fibronectin antibodies on adhesion to endothelial cells.
The effect on adherence of anti-human fibronectin antibodies was evaluated using 8325-4 defective in protein A (DU5875) so as to control for the confounding interaction between protein A and the Fc region of IgG. The following antibodies were used at a concentration of 10 µg ml-1: rabbit polyclonal antibody to purified human fibronectin (F3648; Sigma); sheep polyclonal antibody to purified human fibronectin (Serotec UK; Oxford); mAb recognizing an epitope located within the 5th type III repeat of human plasma fibronectin (mouse IgG1 clone IST-4; F 0916, Sigma); and mAb recognizing the N-terminus of fibronectin (mouse IgG3 clone 10B7; Biogenesis). The following control antibodies were used at a concentration of 10 µg ml-1: normal sheep IgG (I 5131, Sigma); normal rabbit IgG (I 5006, Sigma); mouse IgG1, Kappa (MOPC-31C; M 9035, Sigma); and mouse IgG3, Kappa (FLOPC 21; M 3645, Sigma). All antibodies were added to the wells immediately prior to bacterial inoculation.
Bacterial adhesion to solid-phase purified fibronectin.
Purified human fibronectin at 10 µg ml-1 (Sigma) in TBS was spotted onto tissue culture grade plastic Petri dishes and incubated at 37 °C in air for 1 h, then flooded with 1 % BSA in TBS and maintained overnight at 4 °C. Dishes were rinsed twice in M199 immediately prior to use. After bacterial preparation as described above, strains were suspended in M199 with 1% BSA to a final concentration of 5x107 c.f.u. ml-1 and 1·5 ml was added to each dish. Following incubation at 37 °C in air for 1 h, the dishes were rinsed four times with M199, fixed with glutaraldehyde (2%, v/v) in M199 for 2 h and stained with 0·5% crystal violet for 5 min. Adhesion was quantified using a visual method. The purified fibronectin spots were examined using a calibrated graticule under oil immersion with ax100 magnification lens. The number of bacteria adherent to an area of 1 mm2 purified protein were enumerated by a standardized counting procedure. Without prior visual inspection at high power, five high-power fields were selected for counting, as follows: (1) the centre of the protein spot; (2) two points between the centre and left edge; and (3) two points between the centre and right edge.
Endothelial cell adhesion assays after pre-coating with, or in the presence of, fibrinogen and fibronectin.
Adherence assays were performed using one of two modifications. (1) Confluent endothelial cells coating Thermanox coverslips were pre- coated for 30 min at 37 °C in air with either purified human fibronectin at 300 µg ml-1 (Sigma), or purified human fibrinogen at 1 mg ml-1 (Sigma). The coverslips were then dip-rinsed in M199 four times prior to use in the adherence assay. (2) Adherence assays were performed in the presence of either purified human fibronectin at 300 µg ml-1 or purified fibrinogen at 1 mg ml-1, with or without rFNBD protein. Contamination of commercially available human fibrinogen by fibronectin is a potential confounder in endothelial cell adherence assays. The human fibrinogen used in this study was evaluated and purified prior to use, as follows. The presence of contaminating fibronectin was confirmed by Western blotting following fractionation by SDS-PAGE of a 15 µl aliquot of fibrinogen solution, 2 mg ml-1, dissolved in M199, as previously described by McDevitt et al. (1992) (data not shown). Fibronectin was removed by passing the fibrinogen solution through a gelatin-Sepharose column (Pharmacia Biotech) using the procedure described by the manufacturer. The absence of contaminating fibronectin in the eluate was confirmed by Western blot. Western blotting did not demonstrate the presence of contaminating immunoglobulin.
Statistical analysis.
Results for the numbers of bacteria adherent to or internalized by endothelium, or adherent to purified fibronectin, were expressed as the mean count per 1 mm2 surface area. All points were performed in triplicate and each experiment was performed three times. Statistical analysis was carried out using the Statview 4.5 statistical software package (Abacus). Comparison of the mean count between bacterial strains was performed using an unpaired t-test with correction for multiple comparisons.
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RESULTS |
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The recombinant form of the ligand-binding domain of FnBP-B from Streptococcus dysgalactiae inhibits endothelial binding of S. aureus
The ligand-binding activity of the S. aureus FnBP is located in region D, which is composed of three consecutive repeats of 37 or 38 residues (designated D1, D2 and D3), plus one incomplete repeat (D4) (Raja et al., 1990 ; Signas et al., 1989
). D1D3 comprises a high-affinity binding domain, while synthetic peptides representing each motif demonstrate low- affinity binding and the ability to inhibit fibronectin binding to S. aureus in a competitive manner (Huff et al., 1994
). The involvement of this domain in the interaction between S. aureus FnBP and human endothelial cells was evaluated by competitive adherence inhibition assays using the recombinant form of the binding domain of FnBP-B from Streptococcus dysgalactiae. This protein has been shown to inhibit the binding of 125I-labelled intact fibronectin or the N-terminal fibronectin domain to S. aureus (Joh et al., 1994
). We speculated that if the S. aureus FnBPs were interacting with endothelial surface fibronectin, adhesion would be inhibited by rFNBD protein.
The effect of 10 µg rFNBD protein ml-1 on adherence of S. aureus 8325-4, P1 and five recent clinical isolates is shown in Fig. 2. There was a significant reduction in count in the presence of rFNBD protein for all isolates, while the recombinant truncated ClfA protein (Clf41, residues 221559) had no effect on adherence (range 89·695·5% of the control, P >0·05) (data not shown). These results imply that the binding domain (D repeat region) of S. aureus FnBP participates in the interaction between S. aureus and the endothelial monolayer. Although markedly reduced, bacterial adhesion to the monolayer was not abolished at any of the concentrations of rFNBD protein used (data not shown). This concurs with the observation that FnBP-defective mutants can adhere to the monolayer, albeit at a reduced level compared to the parent strain.
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Effect of plasma proteins on adherence of S. aureus to endothelial cells
Previous studies have shown that plasma proteins are involved in bacterial adherence to endothelium. We evaluated the role of such proteins by pre-coating endothelial monolayers with purified human fibronectin or fibrinogen, or by adding these host proteins into the assay.
Pre-coating the monolayer with fibronectin followed by rinsing prior to use in the adherence assay had no effect (Fig. 4). This contrasted with the effect of actually adding fibronectin to the assay, which led to agglutination of bacteria as described previously (Proctor et al., 1984
) and adherence of aggregates to the monolayer (data not shown).
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Endothelial cells do not internalize S. aureus mutants deficient in FnBP
Using the adherence assay in which 108 c.f.u. were incubated with the monolayer for 1 h with no prior centrifugation of bacteria onto the endothelium, the number of bacteria internalized by endothelial cells was 9%, 25% and 21% of the total (intracellular+adherent) for wild-type 8325-4, P1 and JR80, respectively (data not shown). This contrasted with the three isogenic FnBP-deficient mutants, for which no intracellular bacteria were visualized either in the standard 1 mm2 surface area of endothelium examined, or during detailed scanning of the monolayer in multiple fields. It is possible that the apparent lack of internalization of the FnBP-defective mutants resulted from the low number of bacteria adherent to the monolayer rather than interruption of a specific uptake pathway. This was examined by centrifuging 108 c.f.u. onto the monolayer at the start of the adherence assay. The numbers of internalized bacteria per mm2 of endothelium after 60 min incubation at 37 °C were 397, 826 and 790, respectively, for 8325-4, P1 and JR80. This compared with <1 bacterium per mm2 for FnBP-defective mutants of 8325- 4, P1 and JR80. The lack of intracellular bacteria for all three mutants indicates that FnBPs are critical for internalization of S. aureus by endothelial cells in vitro.
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DISCUSSION |
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Our findings are important because they cast light on a key interaction in the pathogenesis of metastatic S. aureus infection, the adherence of bacteria to endothelial cells. Since this organism is able to infect apparently normal bone and joint tissues, a direct interaction with endothelial cells is likely as a first step in the invasion of these deeper tissues. Fibronectin is well placed to act as a receptor in this regard. It is a normal component of the extracellular matrix on the luminal surface of an endothelial monolayer and our findings are consistent with a previous report of the ultrastructural localization of fibronectin between bovine endothelial cells and adherent S. aureus (Vann et al., 1989 ). The observation that S. aureus 8325-4 has two FnBPs, FnBPA and FnBPB, encoded by the closely linked genes fnbA and fnbB (Signas et al., 1989
; Jonsson et al., 1991
) underscores the importance of interactions with fibronectin in the biology of the organism. Given that the defect in endothelial cell adhesion seen with the FnBP- deficient mutant was restored by the presence of a multicopy plasmid encoding one of fnbA or fnbB, either FnBPA or FnBPB alone can mediate adherence of 8325-4. Understanding the relative importance of each protein in the interaction of staphylococci with endothelial cells will require further study.
Adhesion of S. aureus to live endothelial cells is rapidly followed by internalization, a process which requires the presence of bacterial FnBPs. Fibronectin, through its known interactions with integrin receptors present on endothelium (Albelda et al., 1989 ), is an ideal candidate molecule to orchestrate these events. Many other invasive pathogens use integrins as cellular receptors (for review see Berendt & McCormick, 1997
) and Streptococcus pyogenes has already been shown to invade a number of epithelial cell lines through the interaction between fibronectin and epithelial-cell-surface integrins (Ozeri et al. , 1998
). Our observations thus prompt the speculation that S. aureus invades endothelial cells through similar mechanisms.
Under physiological conditions, the interaction of S. aureus with endothelial cells takes place at greatly lower bacterial density, in whole blood and under conditions of flow. The relevant activation status of the endothelial cells in vivo is unknown and their phenotype will vary according to site. We previously showed that there are important differences between microvascular and large vessel endothelium in the expression and function of host receptors for the adhesion of malaria-infected erythrocytes (McCormick et al., 1997 ). Furthermore, for adhesion of both leukocytes and malaria-infected erythrocytes, endothelial receptors show differential adhesion under shear flow conditions, with rolling and static receptors. It therefore remains possible that under conditions of flow and cytokine activation, additional adhesion pathways operate independently of, or alongside, the fibronectin pathway. Elucidating this under the full range of conditions that might prevail in vivo requires further detailed studies.
Plasma proteins are also an important component of these interactions. We were surprised to find no convincing role in adhesion for fibrinogen, which has been previously reported to act as a bridging molecule (Cheung et al., 1991b ). There are a number of differences between the previous study and our own that may explain this, including the bacterial strains used and the method of quantifying bacterial adherence. An additional difference is that the earlier group used an assay where the endothelial cells were fixed with glutaraldehyde. This may have modified the affinity or accessibility of the binding sites on the fibronectin molecules, rendering them unable to interact with the FnBPs on the bacteria. In support of this, we find that glutaraldehyde fixation of purified fibronectin that has been immobilized on plastic reduces the adherence of S. aureus 8325- 4 and the clinical isolate JR80 to 5% and 8%, respectively, of that for the non-treated control. We cannot exclude the possibilities that under different conditions of growth or activation, the fibronectin pathway plays a lesser role or that if it is inoperative, secondary adhesion mechanisms become important. Indeed, in our system, low levels of residual bacterial binding (approx. 1020% of control) are seen when the fibronectin-binding pathway is non-functional due to mutation, rFNBD protein or anti-fibronectin antibodies. The receptor for this secondary pathway remains unidentified.
Finally, it is important to note that FnBPs are not unique to S. aureus, being widely distributed among the streptococci (reviewed by Patti et al., 1994b ). Whether other organisms capable of causing complicated bacteraemias also adhere to endothelial cells by this route remains a matter for speculation and further study.
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ACKNOWLEDGEMENTS |
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Received 18 May 1999;
revised 27 July 1999;
accepted 17 August 1999.