Expression of staphylococcal clumping factor A impedes macrophage phagocytosis

Clumping factor A (ClfA), a fibrinogen-binding protein linked to the Staphylococcus aureus cell wall, is an important virulence factor in infection models, e.g., of septic arthritis. However, the mechanism(s) by which ClfA contributes to the virulence of the bacterium is unknown. In the present study, the impact of ClfA expression on the phagocytosis of S. aureus by macrophages was investigated using clfA-positive and clfA-negative isogenic strains. Furthermore, the possible contribution of ClfA to the proinflammatory and immunostimulatory activity of S. aureus was studied. Our results indicate that ClfA expression significantly protects S. aureus against macrophage phagocytosis. This protection does not require the presence of intact fibrinogen, a ligand for ClfA. ClfA expression by S. aureus enhanced the proliferative response of spleen cells. On the other hand, a clfA mutant strain caused more release of proinflammatory mediators by macrophages than its clfA-positive parental strain. Both the protection against phagocytosis and the enhanced immunostimulatory activity provided by ClfA expression are likely to contribute to the in vivo virulence of S. aureus.     

Fibrinogen binding sites P336 and Y338 of clumping factor A are crucial for Staphylococcus aureus virulence

We have earlier shown that clumping factor A (ClfA), a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P(336) and Y(338), were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP(336)Y(338) mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP(336)Y(338) mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP(336) and Y(338) were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP(336)Y(338) mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP(336)SY(338)A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.     

A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen

Plasma fibrinogen participates in several physiological and pathological events thus becoming a useful studying material in biomedical research. Here we report a new convenient method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen. Clumping factor A (ClfA) is a cell wall-anchored surface protein of S. aureus bacteria that binds with a high affinity to the fibrinogen gamma chain C-terminus via a segment encompassing the residues 221-550. This activity of ClfA (ClfA(221-550)) was produced in fusion to the C-terminus of glutathione-S-transferase (GST) with recombinant technology and used as an affinity ligand to capture plasma fibrinogen. GST-ClfA(221-550) fusion protein was immobilized onto the glutathione-conjugated beads packed in a plastic column by its GST part. Then, this affinity column was loaded with citrated and heparinized human plasma. After washing out unbound proteins, column-captured fibrinogen was specifically eluted down with a citrate buffer solution (50mM, pH 5.6). Purified human fibrinogen exhibited the ability to support platelet adhesion and aggregation and formed fibrin clot by thrombin, indicating that ClfA(221-550)-purified human fibrinogen is a functionally active product. We also found that both the rat and mouse fibrinogens could be purified as well as human fibrinogen with this method. By virtue of its simplicity and feasibility, ClfA(221-550)-based method would be very useful to the investigators who need fibrinogen to perform their studies.     

Clumping factor A-mediated virulence during Staphylococcus aureus infection is retained despite fibrinogen depletion

Clumping factor A (ClfA), a fibrinogen-binding protein expressed on the Staphylococcus aureus cell surface, has previously been shown to act as a virulence factor in experimental septic arthritis. Although the interaction between ClfA and fibrinogen is assumed to be of importance for the virulence of S. aureus, this has not been demonstrated in any in vivo model of infection. Therefore, the objective of this study was to investigate the contribution of this interaction to ClfA-mediated virulence in murine S. aureus-induced arthritis. Ancrod, a serine protease with thrombin-like activity, was used to induce in vivo depletion of fibrinogen in mice. Ancrod treatment significantly aggravated septic arthritis following inoculation with a ClfA-expressing strain (Newman) compared to control treatment. Also, ancrod treatment tended to enhance the arthritis induced by a clfA mutant strain (DU5876), indicating that fibrinogen depletion exacerbates septic arthritis in a ClfA-independent manner. Most importantly, the ClfA-expressing strain was much more arthritogenic than the isogenic clfA mutant, following inoculation of fibrinogen-depleted mice. This finding indicates that the interaction between ClfA and free fibrinogen is not required for ClfA-mediated functions contributing to S. aureus virulence. It is conceivable that ClfA contributes to the virulence of S. aureus through interactions with other host ligands than fibrinogen.     

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.     

Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus

The ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign-body and wound Infections. Recently, we reported the cloning and sequencing of the gene clfA encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman and showed that the gene product was responsible for the clumping of bacteria in soluble fibrinogen and for the adherence of bacteria to solid-phase fibrinogen. This was confirmed here by showing that antibodies raised against purified Region A inhibited both of these properties. Also, immunofluorescent microscopic analysis of wild-type Newman and a clfA::Tn917 mutant of Newman with anti-ClfA Region A sera confirmed that Region A is exposed on the bacterial cell surface. Furthermore, polystyrene beads coated with the Region A protein formed clumps in soluble fibrinogen showing that the ClfA protein alone is sufficient for the clumping phenotype. Western immunoblotting with anti-ClfA Region A antibodies identified the native ClfA receptor as a 185 kDa protein that was released from the cell wall of S. aureus by lysostaphin treatment. A single extensive ligand-binding site was located within Region A of the ClfA protein. Truncated ClfA proteins were expressed in Escherichia coli. Lysates of E. coli and proteins that had been purified by affinity chromatography were tested for (i) their ability to bind fibrinogen in Western ligand blotting experiments, (ii) for their ability to inhibit clumping of bacteria in fibrinogen solution and adherence of bacteria to solid-phase fibrinogen, and (iii) for their ability to neutralize the blocking activity of anti-ClfA Region A antibody. These tests allowed the ligand-binding domain to be localized to a 218-residue segment (residues 332-550) within Region A.     

Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A

Staphylococcus aureus is an important cause of infective endocarditis (IE) in patients without a history of prior heart valve damage. The ability to stimulate the activation of resting platelets and their subsequent aggregation is regarded as an important virulence factor of bacteria that cause IE. Clumping factor A is the dominant surface protein responsible for platelet activation by S. aureus cells in the stationary phase of growth. This study used Lactococcus lactis as a surrogate host to study the mechanism of ClfA-promoted platelet activation. Expression of ClfA from a nisin-inducible promoter demonstrated that a minimum level of surface-expressed ClfA was required. Using platelets that were purified from plasma, the requirement for both bound fibrinogen and immunoglobulin was demonstrated. The immunoglobulin G (IgG) requirement is consistent with the potent inhibition of platelet activation by a monoclonal antibody specific for the platelet FcgammaRIIa receptor. Furthermore the IgG must contain antibodies specific for the ClfA A domain. A model is proposed whereby bacterial cells armed with a sufficient number of surface-bound fibrinogen molecules can engage resting platelet glycoprotein GPIIb/IIIa, aided by bound IgG molecules, which encourages the clustering of FcgammaRIIa receptors. This can trigger activation of signal transduction leading to activation of GPIIb/IIIa and aggregation of platelets. In addition, analysis of a mutant of ClfA totally lacking the ability to bind fibrinogen revealed a second, although less efficient, mechanism of platelet activation. The fibrinogen-independent pathway required IgG and complement deposition to trigger platelet aggregation.     

Synthesis and Evaluation of a Conjugate Vaccine Composed of Staphylococcus aureus Poly-N-Acetyl-Glucosamine and Clumping Factor A

The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA) and deacetylated poly-N-β-(1-6)-acetyl-glucosamine (dPNAG). Mice immunized with 1, 5 or 10 μg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA) goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to augment the OPK and protective activities of antibodies to the PNAG cell surface polysaccharide.     

Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.     

The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen

Staphylococcus aureus is an important pathogen capable of causing a wide spectrum of diseases in humans and animals. This bacterium expresses a variety of virulence factors that participate in the process of infection. These include MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that mediate the adherence of the bacteria to host extracellular matrix components, such as collagen, fibronectin (Fn), and fibrinogen (Fg). Two Fn-binding MSCRAMMs, FnbpA and FnbpB, have been previously identified. The Fn binding activity has been localized to the approximately 40-amino acid residue D repeats in the C-terminal part of these proteins. However, no biological activity has yet been attributed to the N-terminal A regions of these proteins. These regions exhibit substantial amino acid sequence identity to the A regions of other staphylococcal MSCRAMMs, including ClfA, ClfB, and SdrG (Fbe), all of which bind Fg. This raises the question of whether the Fn-binding MSCRAMMs can also bind specifically to Fg. In this report, we show that a recombinant form of the A region of FnbpA does specifically recognize Fg. We localize the binding site in Fg for recombinant FnbpA to the gamma-chain, in particular to the C-terminal residues of this polypeptide, the site also recognized by ClfA. In addition, we demonstrate that recombinant FnbpA can compete with ClfA for binding to both immobilized and soluble Fg. By the use of surface plasmon resonance spectroscopy and fluorescence polarization, we determine the dissociation equilibrium constant for the interaction of recombinant FnbpA with intact immobilized Fg and with a synthetic C-terminal gamma-chain peptide, respectively. Finally, by overexpressing FnbpA in a mutant strain of S. aureus that lacks the expression of both ClfA and ClfB, we show that native FnbpA can mediate the interaction of S. aureus with soluble Fg.     

金黄色葡萄球菌凝集因子A的免疫原性

为研究金黄色葡萄球菌(Staphylococcus aureus)凝集因子A(ClfA)免疫原性及免疫保护作用,应用PCR方法扩增出金黄色葡萄球菌Newman、Wood46和HLJ23-1株的clfa基因并进行序列分析,再将Newman株的clfa基因插入到pQE-30载体上,导入宿主菌Escherichia coli M15(pREP4)并诱导表达和纯化ClfA重组蛋白。用纯化的ClfA免疫小鼠,检测血清中抗体和细胞因子水平,首次免疫后35 d时用金黄色葡萄球菌Wood46、HLJ23-1、Newman株对小鼠攻毒。结果发现:clfa基因序列高度保守;ClfA重组蛋白在E.coli M15中获得表达;在首次免疫后35 d时血清抗体效价和细胞因子浓度与对照组相比,均显著升高(P<0.05);攻毒结果为蛋白免疫组小鼠获得一定的免疫保护。由此表明,ClfA重组蛋白有较好的免疫原性和免疫保护力。     

Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus

The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the α- and β-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the γ-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential-phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca²⁺ and Mn²⁺, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.     

The Sortase A Substrates FnbpA, FnbpB, ClfA and ClfB Antagonize Colony Spreading of Staphylococcus aureus

Staphylococcus aureus is an important human pathogen that is renowned both for its rapid transmission within hospitals and the community, and for the formation of antibiotic resistant biofilms on medical implants. Recently, it was shown that S. aureus is able to spread over wet surfaces. This motility phenomenon is promoted by the surfactant properties of secreted phenol-soluble modulins (PSMs), which are also known to inhibit biofilm formation. The aim of the present studies was to determine whether any cell surface-associated S. aureus proteins have an impact on colony spreading. To this end, we analyzed the spreading capabilities of strains lacking non-essential components of the protein export and sorting machinery. Interestingly, our analyses reveal that the absence of sortase A (SrtA) causes a hyper-spreading phenotype. SrtA is responsible for covalent anchoring of various proteins to the staphylococcal cell wall. Accordingly, we show that the hyper-spreading phenotype of srtA mutant cells is an indirect effect that relates to the sortase substrates FnbpA, FnbpB, ClfA and ClfB. These surface-exposed staphylococcal proteins are known to promote biofilm formation, and cell-cell interactions. The hyper-spreading phenotype of srtA mutant staphylococcal cells was subsequently validated in Staphylococcus epidermidis. We conclude that cell wall-associated factors that promote a sessile lifestyle of S. aureus and S. epidermidis antagonize the colony spreading motility of these bacteria.     

Molecular Characterization of the Interaction of Staphylococcal Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) ClfA and Fbl with Fibrinogen

The ligand-binding domain of Fbl (the fibrinogen binding protein from Staphylococcus lugdunensis) shares 60% sequence identity with ClfA (clumping factor A) of Staphylococcus aureus. Recombinant Fbl corresponding to the minimum fibrinogen-binding region (subdomains N2N3) was compared with ClfA for binding to fibrinogen. Fbl and ClfA had very similar affinities for fibrinogen by surface plasmon resonance. The binding site for Fbl in fibrinogen was localized to the extreme C terminus of the fibrinogen γ-chain at the same site recognized by ClfA. Isothermal titration calorimetry showed that Fbl and ClfA had very similar affinities for a peptide mimicking the C-terminal segment of the fibrinogen γ-chain. The peptide also inhibited binding of Fbl and ClfA to fibrinogen. A series of substituted γ-chain variant peptides behaved very similarly when used to inhibit ClfA and Fbl binding to immobilized fibrinogen. Both ClfA and Fbl bound to bovine fibrinogen with a lower affinity compared with human fibrinogen and did not bind detectably to ovine fibrinogen. The structure of the N2N3 subdomains of Fbl in complex with the fibrinogen γ-chain peptide was modeled based on the crystal structure of the N2N3 subdomains of the ClfA-γ-chain peptide complex. Residues in the putative binding trench likely to be involved in fibrinogen binding were identified. Fbl variant proteins with alanine substitutions in key residues had reduced affinities for fibrinogen. Thus Fbl and ClfA bind the same site in fibrinogen by similar mechanisms.     

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.     

Adherence of Staphylococcus aureus is enhanced by an endogenous secreted protein with broad binding activity

A novel mechanism for enhancement of adherence of Staphylococcus aureus to host components is described. A secreted protein, Eap (extracellular adherence protein), was purified from the supernatant of S. aureus Newman and found to be able to bind to at least seven plasma proteins, e.g., fibronectin, the alpha-chain of fibrinogen, and prothrombin, and to the surface of S. aureus. Eap bound much less to cells of Staphylococcus epidermidis, Streptococcus mutans, or Escherichia coli. The protein can form oligomeric forms and is able to cause agglutination of S. aureus. Binding of S. aureus to fibroblasts and epithelial cells was significantly enhanced by addition of Eap, presumably due to its affinity both for plasma proteins on the cells and for the bacteria.     

Binding of human plasma proteins to Streptococcus pyogenes M protein determines the location of opsonic and non-opsonic epitopes

Antibodies directed against a pathogenic microorganism may recognize either protective or non-protective epitopes. Because antibodies elicited by a vaccine must be directed against protective epitopes, it is essential to understand the molecular properties that distinguish the two types of epitope. Here we analyse this problem for the antiphagocytic M protein of Streptococcus pyogenes, using the opsonizing capacity of antibodies to estimate their ability to confer protection in vivo. Our studies were focused on the M5 protein, which has three surface-exposed regions: the amino-terminal hypervariable region (HVR) and the B- and C-repeat regions. We first analysed the role of different M5 regions in phagocytosis resistance under non-immune conditions, employing chromosomal mutants expressing M5 proteins with internal deletions, and demonstrate that only the B-repeat region is essential for phagocytosis resistance. However, only antibodies to the HVR were opsonic. This apparent paradox could be explained by the ability of fibrinogen and albumin to specifically bind to the B- and C-repeats, respectively, causing inhibition of antibody binding under physiological conditions, while antibodies to the HVR could bind and promote deposition of complement. These data indicate that binding of human plasma proteins plays an important role in determining the location of opsonic and non-opsonic epitopes in streptococcal M protein.     

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.     

A Structural Model of the Staphylococcus aureus ClfA–Fibrinogen Interaction Opens New Avenues for the Design of Anti-Staphylococcal Therapeutics

The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) γ-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin α_IIbβ₃ and ClfA bind to the same segment in the Fg γ-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the “Dock, Lock and Latch” mechanism previously described for the Staphylococcus epidermidis SdrG–Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not α_IIbβ₃ may allow the design of novel anti-staphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may have originated from a common ancestor but have evolved to accommodate specific ligand structures.     

Identification and Characterization of a Novel 38.5-Kilodalton Cell Surface Protein of Staphylococcus aureus with Extended-Spectrum Binding Activity for Extracellular Matrix and Plasma Proteins

The ability to attach to host ligands is a well-established pathogenic factor in invasive Staphylococcus aureus disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (srtA) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene emp (1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR, emp was demonstrated in all 240 S. aureus isolates of a defined clinical strain collection as well as in 6 S. aureus laboratory strains, whereas it is lacking in all 10 S. epidermidis strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50, a significantly decreased adhesion of mEmp50 to immobilized fibronectin and fibrinogen, and restoration of these characteristics upon complementation of mEmp50. Emp expression was also demonstrable upon heterologous complementation of S. carnosus. rEmp expressed in Escherichia coli interacted with fibronectin, fibrinogen, and vitronectin in surface plasmon resonance experiments at a K(d) of 21 nM, 91 nM, and 122 pM, respectively. In conclusion, the biologic characterization of Emp suggests that it is a member of the group of secreted S. aureus molecules that interact with an extended spectrum of host ligands and thereby contribute to S. aureus pathogenicity.