The remarkably multifunctional fibronectin binding proteins of Staphylococcus aureus.

Staphylococcus aureus expresses two distinct but closely related multifunctional cell wall-anchored (CWA) proteins that bind to the host glycoprotein fibronectin. The fibronectin binding proteins FnBPA and FnBPB comprise two distinct domains. The C-terminal domain comprises a tandem array of repeats that bind to the N-terminal type I modules of fibronectin by the tandem ß-zipper mechanism. This causes allosteric activation of a cryptic integrin binding domain, allowing fibronectin to act as a bridge between bacterial cells and the α5ß1 integrin on host cells, triggering bacterial uptake by endocytosis. Variants of FnBPA with polymorphisms in fibronectin binding repeats (FnBRs) that increase affinity for the ligand are associated with strains that infect cardiac devices and cause endocarditis, suggesting that binding affinity is particularly important in intravascular infections. The N-terminal A domains of FnBPA and FnBPB have diverged into seven antigenically distinct isoforms. Each binds fibrinogen by the 'dock, lock and latch' mechanism characteristic of clumping factor A. However, FnBPs can also bind to elastin, which is probably important in adhesion to connective tissue in vivo. In addition, they can capture plasminogen from plasma, which can be activated to plasmin by host and bacterial plasminogen activators. The bacterial cells become armed with a host protease which destroys opsonins, contributing to immune evasion and promotes spreading during skin infection. Finally, some methicillin-resistant S. aureus (MRSA) strains form biofilm that depends on the elaboration of FnBPs rather than polysaccharide. The A domains of the FnBPs can interact homophilically, allowing cells to bind together as the biofilm accumulates.

New insights into xanthan synergistic interactions with konjac glucomannan: A novel interaction mechanism proposal.

The interactions of xanthans containing precise acetate and pyruvate concentration with Konjac glucomannan (KGM) were studied at different sodium chloride and polymer concentrations. A new unified model of the interaction is proposed, taking into account previous models in the literature. This study suggests that the interactions occur by two distinct mechanisms dependent on xanthan conformation. These interactions are not mutually exclusive and may co-exist and hence produce complicated traces. Consequently two types of gel which melt at different temperature ranges can be formed. Depending on the xanthan helix coil transition temperature, one or both of the synergistic states may exist in the hydrocolloid blend. The proposed model has been tested rheologically and using differential scanning calorimetry by varying salt concentration and using samples containing different functional group concentrations.

Staphylococcus epidermidis serine--aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3.

Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVß3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVß3, and that a mutant lacking SdrG failed to bind. Blocking αVß3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.

The effect of ball milling and rehydration on powdered mixtures of hydrocolloids.

In many applications, particularly in food related work, it is assumed that ball milling merely serves as a means of reducing crystallinity by the steady attrition of crystals. In this work mixtures of cellulose with other biopolymers have been co-ball milled in the dry state typically at moisture contents of <12% (w/w) and the effects of recrystallizing these mixtures studied. We have found that recystallizing the mixtures under a humid (97%RH) atmosphere increases the crystallinity of the cellulose fraction in a fashion governed by the other hydrocolloid present in the mixture. Some of the measured effects occur during ball milling of the dry powders. A relative method of fitting mixtures of type I and type II cellulose is described. Progressive transition between these forms with time was discovered for eucalyptus and microcrystalline cellulose at 97%RH. Locust bean gum (LBG) appeared to exert a protective effect on both eucalyptus and microcrystalline cellulose against the destruction of crystallinity by ball milling. For eucalyptus cellulose high levels of type I were produced during recrystallization with LBG under humid conditions. Both cellulose samples crystallized in the type I form in the presence of LBG whereas type II was produced in the presence of other hydrocolloids. Possible mechanisms for these unusual observations are proposed.

On the origin of sharp peaks in the X-ray diffraction patterns of xanthan powders.

A series of xanthans containing different levels of the charged group pyruvate has been examined. The X-ray diffraction patterns of the powders of these materials had different levels of a sharp pattern superimposed on an amorphous background. As the moisture content increased so the intensity of the sharp pattern increased up to a level between 20% and 40% moisture content where the sharp pattern disappeared. X-ray diffraction pattern identification software and an inorganic X-ray diffraction database showed the origin of the sharp peaks to be due to sodium sulphate polymorphs. The behaviour of the xanthans was thought to be due to the differences in charge on the molecule; however, the increase in the crystalline component observed with increased amounts of water was unexpected. The possibility of the drying of samples was considered but the interplay between ions, the charged polymer and the water present was considered necessary to more closely describe the results.

Elucidating the role of Staphylococcus epidermidis serine-aspartate repeat protein G in platelet activation.

BACKGROUND: Staphylococcus epidermidis is a commensal of the human skin that has been implicated in infective endocarditis and infections involving implanted medical devices. S. epidermidis induces platelet aggregation by an unknown mechanism. The fibrinogen-binding protein serine-aspartate repeat protein G (SdrG) is present in 67-91% of clinical strains. OBJECTIVES: To determine whether SdrG plays a role in platelet activation, and if so to investigate the role of fibrinogen in this mechanism. METHODS: SdrG was expressed in a surrogate host, Lactococcus lactis, in order to investigate its role in the absence of other staphylococcal components. Platelet adhesion and platelet aggregation assays were employed. RESULTS: L. lactis expressing SdrG stimulated platelet aggregation (lag time: 2.9 +/- 0.5 min), whereas the L. lactis control did not. L. lactis SdrG-induced aggregation was inhibited by alpha(IIb)beta3 antagonists and aspirin. Aggregation was dependent on both fibrinogen and IgG, and the platelet IgG receptor FcgammaRIIa. Preincubation of the bacteria with Bbeta-chain fibrinopeptide inhibited aggregation (delaying the lag time six-fold), suggesting that fibrinogen acts as a bridging molecule. Platelets adhered to L. lactis SdrG in the absence of fibrinogen. Adhesion was inhibited by alpha(IIb)beta3 antagonists, suggesting that this direct interaction involves alpha(IIb)beta3. Investigation using purified fragments of SdrG revealed a direct interaction with the B-domains. Adhesion to the A-domain involved both a fibrinogen and an IgG bridge. CONCLUSION: SdrG alone is sufficient to support platelet adhesion and aggregation through both direct and indirect mechanisms.

Antibody response in patients with endocarditis caused by Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus expresses a variety of adhesins involved in the colonization of host tissues. This study aimed to evaluate the role of staphylococcal surface proteins in the aetiology of infective endocarditis (IE) and the host immune response to infection. MATERIALS AND METHOD: The ELISA assays were used to assess the adherence of S. aureus isolates recovered from the blood cultures of 19 patients with IE (16 were drug abusers) to subendothelial matrix proteins. Anti-adhesin antibody titre was measured incubating surface-coated bacterial antigens with patients' IgG. S. aureus effects on platelet aggregation were evaluated with an aggregometer. RESULTS: Staphylococcus aureus isolates, from the patients with IE, exhibited a high expression of several surface components recognizing extracellular matrix proteins: clumping factors A and B (ClfA and ClfB) and fibronectin-binding proteins (FnbpA and FnbpB), whereas only four strains expressed the collagen-binding protein CNA. Bacteria also interacted with platelets both in the absence or presence of fibronectin or fibrinogen and some strongly supported platelet aggregation. Almost all patients presented significantly higher antibody reactivity to ClfA, ClfB, FnbpA, CNA and MAP (MHC class II analogous protein) than in sera from healthy individuals. On the contrary, the reactivity to CNA was remarkable only in three patients. The IgG preparations weakly inhibited the binding of bacteria to fibronectin, whereas they exhibited considerable blocking activity on staphylococcal attachment to fibrinogen or collagen. CONCLUSION: Adhesins ClfA, ClfB and FnbpA are produced in vivo and appear important factors both in valve colonization and in promoting host immune responses.

Global regulation of Staphylococcus aureus genes by Rot.

Staphylococcus aureus produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including agr, sar, sigB, sae, and arl, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an agr-negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a rot agr double mutant to that of its agr parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes. Our data also indicate that Rot and agr have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of S. aureus virulence gene expression.

Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus.

The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the approximately 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta-sheets with little or no discernible alpha-helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain N1 appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.

Simulation of endo-PG digest patterns and implications for the determination of pectin fine structure.

Novel stepwise approaches to the calculation of enzyme digest patterns are described and used in the validation of a computer simulation. Results obtained using the simulation show that, while a previously proposed model of endo-PG action captures some of the salient features of this enzymes behaviour, it is not sufficient to successfully predict experimental digest patterns from pectic substrates. Subsequently, it has been shown that a modified model incorporating existing information regarding subsite architecture and speculative site tolerances for esterified residues, goes someway towards improving the situation.

Influence of thermal history on the structural and mechanical properties of agarose gels.

Using a multitechnique approach, two temperature domains have been identified in agarose gelation. Below 35 degrees C, fast gelation results in strong, homogeneous and weakly turbid networks. The correlation length, evaluated from the wavelength dependence of the turbidity, is close to values of pore size reported in the literature. Above 35 degrees C, gelation is much slower and is associated with the formation of large-scale heterogeneities that can be monitored by a marked change in the wavelength dependence of turbidity and visualised by transmission electron microscopy. Curing agarose gels at temperatures above 35 degrees C, and then cooling them to 20 degrees C, produces much weaker gels than those formed directly at 20 degrees C. Dramatic reductions in the elastic modulus and failure strain and stress are found in this case as a result of demixing during cure. An interpretation, based on the kinetic competition between osmotic forces (in favor of phase separation) and elastic forces (that prevent it) is proposed.

Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transcription, shedding and cleavage by metalloprotease.

The fibrinogen-binding protein clumping factor B (ClfB) of Staphylococcus aureus is present on the surface of cells from the early exponential phase of growth in greater amounts than on cells from late exponential phase and is barely detectable on cells from stationary phase. Expression of a clfB-lacZ fusion indicated that transcription stopped before the end of exponential phase. Mutations in the global regulators agr and sar had no effect on clfB transcription. The loss of ClfB protein from cells in stationary phase was due to expression ending before cells stopped growing, combined with shedding of some of the protein into the growth medium and dilution of those molecules remaining on the cell surface during the two to three cell division events leading to stationary phase. Two forms of the protein occurred on the cell surface, the smaller of which was generated by loss of a domain from the N terminus. The proportion of the smaller form increased as the cultures grew. The metalloprotease aureolysin was shown to be responsible for cleavage of ClfB. Cleavage was inhibited by EDTA and o-phenanthroline and did not occur in an aureolysin-deficient mutant. Purified aureolysin promoted cleavage of cell surface-located ClfB as well as the recombinant A domain of ClfB. Cleavage was detected at two sites, one located between residues Ser(197) and Leu(198) and the other between Ala(199) and Val(200). The truncated form of ClfB did not bind fibrinogen.

Gelation behaviour of konjac glucomannan with different molecular weights.

The deacetylation and gelation of konjac glucomannan (KGM) following alkali addition was investigated by Fourier transform infrared, while the rheological properties of KGM with different molecular weights were studied by dynamic viscoelastic measurements in shear mode and penetration force tests. It was found that gelation occurred after significant deacetylation had taken place. Rheometrical studies revealed that KGM with different molecular weights exhibited different gelation characteristics in small amplitude oscillatory shear flow. For the samples of fractionated KGM with lower molecular weights, a decrease in both the storage shear modulus (G') and loss shear modulus (G") was observed during gelation at temperatures above 75 degrees C. It is suggested that the decrease results from the wall slip between sample and measuring geometry owing to a rapid gelation process with syneresis and/or disentanglement of molecular chains adsorbed on the surface of parallel plates from those located in the bulk. Penetration force tests were employed to confirm the occurrence of slippage and thereby no decreases in rigidity of samples were observed during gelation. For the native KGM samples decreases in G' and G" during gelation were not observed, and it is suggested that this is due to the effect of the higher molecular weight and increased solution viscosity of these samples on the gelation kinetics.

Expression of pls, a gene closely associated with the mecA gene of methicillin-resistant Staphylococcus aureus, prevents bacterial adhesion in vitro.

The pls gene, coding for a large surface protein of methicillin-resistant Staphylococcus aureus, was cloned from a strain which adheres poorly to several mammalian proteins. The structure of pls revealed three distinct repeat regions, one of which was a serine-aspartate repeat characteristic of the Clf-Sdr family of surface proteins in staphylococci. The lengths of the repeat regions varied in different clinical strains and could be used as epidemiological markers. pls was found to be closely associated with the mecA gene by pulsed-field gel electrophoresis analysis of SmaI-digested DNA. A pls mutant constructed by allele replacement adhered well to immobilized fibronectin and immunoglobulin G, in contrast to the parental strain, suggesting that Pls could have a role in preventing adhesion at some stages during an infection.

Identification of residues in the Staphylococcus aureus fibrinogen-binding MSCRAMM clumping factor A (ClfA) that are important for ligand binding.

Clumping factor A (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of this pathogen to both soluble and immobilized fibrinogen (Fg). Previous studies have localized the Fg-binding activity of ClfA to residues 221-559 within the A region of this protein. In addition, the C-terminal part of the A region (residues 484-550) has been implicated as being important for Fg binding. In this study, we further investigate the involvement of this part of ClfA in the interaction of this protein with Fg. Polyclonal antibodies generated against a recombinant protein encompassing residues 500-559 of the A region inhibited the interaction of both S. aureus and recombinant ClfA with immobilized Fg in a dose-dependent manner. Using site-directed mutagenesis, two adjacent residues, Glu(526) and Val(527), were identified as being important for the activity of ClfA. S. aureus expressing ClfA containing either the E526A or V527S substitution exhibited a reduced ability to bind to soluble Fg and to adhere to immobilized Fg. Furthermore, bacteria expressing ClfA containing both substitutions were almost completely defective in Fg binding. The E526A and V527S substitutions were also introduced into recombinant ClfA (rClfA-(221-559)) expressed in Escherichia coli. The single mutant rClfA-(221-559) proteins showed a significant reduction in affinity for both immobilized Fg and a synthetic fluorescein-labeled C-terminal gamma-chain peptide compared with the wild-type protein, whereas the double mutant rClfA-(221-559) protein was almost completely defective in binding to either species. Substitution of Glu(526) and/or Val(527) did not appear to alter the secondary structure of rClfA-(221-559) as determined by far-UV circular dichroism spectroscopy. These data suggest that the C terminus of the A region may contain at least part of the Fg-binding site of ClfA and that Glu(526) and Val(527) may be involved in ligand recognition.

Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens.

Previous studies have demonstrated that a proportion of Staphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene (sel) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3' end of the GMP synthase gene (gmps) in the S. aureus chromosome. SaPIbov contains a central region of sequence identity with the previously characterized tst pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527-543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine Vbeta-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.

Investigations towards an efficacious and safe strangles vaccine: submucosal vaccination with a live attenuated Streptococcus equi.

As part of a search for a safe and efficacious strangles vaccine, several different vaccines and different vaccination routes were tested in foals. The degree of protection was evaluated after an intranasal challenge with virulent Streptococcus equi by clinical, postmortem and bacteriological examinations. Inactivated vaccines containing either native purified M-protein (500 microg per dose) or whole S equi cells (10(10) cells per dose) administered at least twice intramuscularly at intervals of four weeks, did not protect against challenge. Different live attenuated S equi mutants administered at least twice at intervals of four weeks by the intranasal route were either safe but not protective or caused strangles. In contrast, a live attenuated deletion mutant administered intramuscularly, induced complete protection but also induced unacceptable local reactions at the site of vaccination. Submucosal vaccination in the inner side of the upper lip with the live attenuated mutant at > or =10(8) colony-forming units per dose, appeared to be safe and efficacious in foals as young as four months of age. The submucosal vaccinations caused small transient swellings that resolved completely within two weeks, and postmortem no vaccine remnants or other abnormalities were found at the site of vaccination.

Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins.

Although Staphylococcus aureus is primarily considered an extracellular pathogen, recent evidence suggests that this bacterium can invade a variety of nonprofessional phagocytic cells. Here we investigate the early stages of cellular invasion by S. aureus and determine the bacterial and host components that are required for this process. S. aureus expresses two cell surface-associated fibronectin (FN)-binding proteins (FnbpA and FnbpB) that mediate the interaction of the bacteria with both soluble and solid-phase FN in vitro. Using a mutant of S. aureus that lacks the expression of both Fnbps, we show that the expression of either protein is necessary for efficient uptake by the mouse fibroblast line GD25beta1A. Invasion could be inhibited by soluble recombinant proteins encompassing either the FN-binding D repeat region or the A region (and B repeats) of FnbpA, suggesting that the activities of both regions are important in this process. We demonstrate that FN is also required for invasion of this cell line. In the presence of FN-depleted fetal bovine serum, the invasion level was reduced by approximately 40% compared to in the presence of whole fetal bovine serum. Invasion could be further reduced by the addition of anti-mouse FN antibodies to the assay. Finally, we utilize a mutant mouse fibroblast line, which lacks beta1 integrin expression, to demonstrate that host cell beta1 integrins are necessary for efficient cellular invasion. The level of invasion of the mutant cell line GD25 was reduced by approximately 97% compared to the beta1-expressing complemented cell line GD25beta1A. In addition, invasion of the GD25beta1A cell line could be inhibited by an RGD-containing peptide, further implicating a role for integrins in this process. Based on these observations, we put forward a model of S. aureus invasion in which host FN forms a bridge between the bacterial Fnbps and host cell beta1 integrins, leading to bacterial uptake.

Protein A is the von Willebrand factor binding protein on Staphylococcus aureus.

Endovascular infection is a highly critical complication of invasive Staphylococcus aureus disease. For colonization, staphylococci must first adhere to adhesive endovascular foci. Von Willebrand factor (vWF) is a large, multimeric glycoprotein mediating platelet adhesion at sites of endothelial damage. Earlier it was demonstrated that vWF binds to and promotes the surface adhesion of S. aureus, prompting this effort to identify the vWF adhesin. In Western ligand assays of S. aureus lysates, staphylococcal protein A (SPA) was recognized by purified vWF. Surface plasmon resonance demonstrated the binding of soluble vWF to immobilized recombinant protein A with a K(d) of 1.49 x 10(-8) mol/L. Using flow cytometry, the binding of fluorescein isothiocyanate-labeled vWF to S. aureus was found to be saturable and inhibitable by unlabeled vWF, antiprotein-A antibodies, or IgG. Isogenic Deltaspa::Tc(r) mutants were constructed by the insertion of a tetracycline resistance cassette into spa using allelic replacement, and it exhibited decreased binding of soluble vWF and decreased adhesion to vWF-adsorbed surfaces. The interaction was restored on complementation of the mutants with spa-containing plasmid pSPA7235. In conclusion, protein A confers interaction of S. aureus with soluble and immobilized vWF in a newly discovered function characterizing protein A as a novel member of the staphylococcal surface protein adhesin superfamily and suggesting its potential role in the pathogenesis of endovascular staphylococcal disease.

Contribution of clumping factor B to pathogenesis of experimental endocarditis due to Staphylococcus aureus.

Staphylococcus aureus Newman with an insertion mutation in clfB, the gene encoding clumping factor B, only marginally decreased infection rate (P>0.05) in rats with experimental endocarditis. In contrast, clfB complementation on a multicopy plasmid significantly increased infectivity (P<0.05) over the deleted mutants. Although clfB could affect endovascular infection, its importance in experimental endocarditis was limited.