Isolation and characterisation of a 17-kDa staphylococcal heparin-binding protein with broad specificity

A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around 17 kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand factor and, weakly, fibrinogen. The N-terminal sequences of the 17-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins

Interactions between staphylococci, heparin, heparin dependent growth factors and biomaterials

Resident skin micro-organisms such as S. epidermidis and other coagulase-negative staphylococci (CoNS), are by far the most common causes of biomaterial-associated infections. These micro-organisms often exhibit surface adhesins that specifically bind serum and tissue proteins adsorbed to implanted biomaterials. The aim of this study was to extend our knowledge about such adhesins produced by CoNS. This thesis focuses on defining staphylococcal components interacting with sulphated glycosaminoglycans (GAGs) or heparin-binding growth factors. It was found that different CoNS strains (similar to S. aureus) interact with immobilised heparin, chondroitin and dextran sulphate. Moreover binding of the soluble heparin dependent growth factors, basic fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF) was demonstrated and proposed to impair wound healing. Novel 17-kDa heparin binding proteins were isolated from both S. epidermidis strain RP 12 and S. haemolyticus strain SM131, and partially characterised. These proteins showed binding not only to heparin but also to bFGF and thrombospondin. As tested in several human cell lines, the heparin binding ligands appeared to be involved in the adherence of staphylococci to eucaryotic host cells. Finally, a new method, using collagen for immobilising antibiotics to heparinised biomaterials was developed, and the coating was found effective in reducing colonisation of staphylococci to biomaterials. It was concluded that improved combatment of biomaterial-associated infections will require detailed knowledge of the molecular events of colonisation, as well as effective antibiotic bonding of certain materials

The role of glycosaminoglycan binding of staphylococci in attachment to eukaryotic host cells

Attachment of microorganisms to host cells is believed to be a critical early step in microbial pathogenesis. The aim of the study was to determine the role of the known glycosaminoglycan (GAG) binding activity of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in their attachment to six different eukaryotic cell lines. Three staphylococcal species expressing GAG binding capacity-S. aureus, S. epidermidis, and S. hemolyticus-were chosen for investigation. Six different eukaryotic cell lines, endothelial HUVEC and EA. hy 926 cells, epithelial A549 and HeLa S3 cells, fibroblasts HEL Sp 12 and macrophages J774.A1, were included. A modified ELISA with biotinylated bacteria was used for estimating the adhesion of staphylococci to each of the cell lines. Our results showed that staphylococci adhered to each of the cell lines studied, although the binding of CoNS strains to epithelial cells was lower than to the other cells. The attachment to all cell types could be partially decreased by pretreatment of the bacteria with various polysulfated agents (highest inhibition was 60%), as well as by chlorate and heparitinase treatment of the cells. These observations may suggest that at least one mode of staphylococcal attachment utilizes GAG chains present on the surface of virtually all adherent cells

Venous shear stress enhances platelet mediated staphylococcal adhesion to artificial and damaged biological surfaces.

We investigated the role of blood components in the adhesion of staphylococci to biological and artificial surfaces under well-defined flow conditions by using the Cone and Plate(let) Analyzer. An enzyme-linked immunosorbent assay-like binding assay with biotinylated bacteria determined the extent of bacterial adhesion to subendothelial extracellular matrix (ECM), polystyrene (PS) and adult bovine aortic endothelial (ABAE) cell monolayer. The presence of adsorbed plasma proteins on PS and ECM did not increase and in some cases reduced staphylococcal adhesion under flow conditions (200s(-1)). However, their presence on ABAE cells increased bacterial adhesion but to a level still lower than the adhesion to PS and ECM. In contrast, adhered platelets significantly increased staphylococcal adhesion to both PS and ECM, but did not affect the adhesion to ABAE cells. Furthermore, bacterial adhesion to the platelets coated ECM and PS under flow conditions (200s(-1)) was increased by 1.4 to 2.6-fold compare to static conditions. The platelet-enhanced bacterial adhesion was markedly inhibited by blockade of the platelet GPIb receptor. In conclusion, staphylococcal extensive adhesion to ECM and PS surfaces is increased by venous flow and mediated by surface adhered activated platelets via a GPIb dependent mechanism. On the other hand, ABAE cells demonstrated limited bacterial adhesion that is mediated by adsorbed plasma proteins. Our results suggest that under physiological venous flow conditions the intact vessel wall is less prone for bacterial adhesion than damaged vessel wall

Quantitation of bacterial adhesion to polymer surfaces by bioluminescence

Quantitation of microbes adhering to a surface is commonly used in studies of microbial adhesion to different surfaces. We have quantified different staphylococcal strains adhering to polymer surfaces by measuring bacterial ATP (adenosine triphosphate) by bioluminescence. The method is sensitive, having a detection limit of 104 bacterial cells. Viable counting of bacterial cells may yield falsely low results due to the presence of 'dormant' and adherent bacteria. By using bioluminescence, this can be avoided. Cells of different bacterial species and cells of strains of the same species were shown to differ significantly in their basal ATP content (8.7 x 10-13 - 5.2 x 10-22 MATP). The size of adherent and planktonic bacteria decreased with time (0.7 μm → 0.3 μm, 20 days). During incubation in nutrient-poor buffer ('starvation'), the ATP content of adherent bacteria decreased after 24-96 h whereas that of planktonic bacteria was stable over 20 days. The presence of human serum or plasma did not interfere significantly with the test results. Since the ATP concentration of bacterial strains of different species varies and is also influenced by the growth conditions of bacteria (solid or liquid culture medium), a species-specific standard curve has to be established for bacteria grown under the same culture conditions. We conclude that the method is a sensitive tool to quantify adherent bacteria during experiments lasting for less than 6 h and constitutes a valuable method to be used in conjunction with different microscopical techniques