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。     

Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus

Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fuily compiemented the ciumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen-coated PMMA. in addition, the cloned clfA gene on a shuttle plasmid aiiowed the weakiy ciumping strain 8325-4 to form clumps with the same avidity as the wild-type strain Newman and also significantly enhanced the adherence of 8325-4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encodes a fibrinogen-binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the ceil surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall-associated proteins. Significant homology was found between the ClfA protein and the fibronectin-binding proteins of S. S. aureus, particularly in the N-and C-termini.     

Serological reactions associated with the clumping factor of Staphylococcus aureus

Rotter, Joan (University of Oklahoma Medical Center, Oklahoma City), and Florene C. Kelly. Serological reactions associated with the clumping factor of Staphylococcus aureus. J. Bacteriol. 91:588-594. 1966.-Evidence that the substance which causes staphylococci to clump in the presence of fibrinogen (clumping factor) is antigenically similar in strains which are serologically diverse according to agglutination reactions has been obtained from fibrinogen-cell clumping inhibition tests. Antisera for clumping factor (CF)-positive strains inhibited the clumping reaction of all strains tested. After adsorption with homologous cells or with cells of other CF-positive strains, the antisera no longer inhibited clumping. When antisera were adsorbed with trypsin-treated, CF-positive cells, or with cells of CF-negative mutants, the ability to inhibit the clumping reaction persisted. Antibody to CF activity was not associated with coagulase. Latex coated with extracts derived from the cells of five CF-positive and six CF-negative strains was, in each instance, agglutinated by sera from rabbits immunized with CF-positive cells. After adsorption with trypsinized, CF-positive cells, antisera still agglutinated latex which had been treated with the CF-positive extracts, but not with the CF-negative extracts. Similar results were obtained after antisera were adsorbed with the cells of CF-negative mutants. Cell agglutination titers of sera from rabbits immunized with CF-negative staphylococci were significantly lower than those produced in response to CF-positive cells, regardless of their coagulase activity. If the CF-inhibiting antibody also functions as an agglutinin, it apparently is not solely responsible for this difference.     

A protein component of the cell surface of Staphylococcus aureus

1. Treatment with trypsin of cells of Staphylococcus aureus strain Cowan I, known to carry a protein component that includes the Jensen protein A, results in an increase in the negative mobility of the cells at pH values greater than 6. 2. Similar treatment of cells of strain Wood 46, which has no surface protein component, causes no change in the electrophoretic mobility. 3. Electrokinetically heterogeneous populations are observed in two strains of S. aureus, one of human and the other of animal origin. Evidence is presented ascribing this to the presence of varying amounts of protein components on the surface of different cells.     

The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface

Clumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative N-terminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.     

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.     

O-Acetyl groups as a component of the bacteriophage receptor on Staphylococcus aureus cell walls

The capacity of Staphylococcus aureus H cell walls to inactivate bacteriophage 52A was lost by removing O-acetyl groups but not by removing ester-linked d-alanine.     

A novel variant of the immunoglobulin fold in surface adhesins of Staphylococcus aureus: crystal structure of the fibrinogen-binding MSCRAMM,clumping factor A

We report here the crystal structure of the minimal ligand-binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This fibrinogen-binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE-variant or the DEv-IgG fold. This subgroup includes the ligand-binding domain of the collagen-binding S.aureus MSCRAMM CNA, and many other structures previously classified as jelly rolls. Structure predictions suggest that the four fibrinogen-binding S.aureus MSCRAMMs identified so far would also contain the same DEv-IgG fold. A systematic docking search using the C-terminal region of the fibrinogen gamma-chain as a probe suggested that a hydrophobic pocket formed between the two DEv-IgG domains of the clumping factor as the ligand-binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues (408)AGDV(411) of the gamma-chain, resulted in proteins with no or markedly reduced affinity for fibrinogen.     

Distribution of protein A on the surface of Staphylococcus aureus

Surface proteins of Staphylococcus aureus fulfill many important roles during the pathogenesis of human infections and are anchored to the cell wall envelope by sortases. Although the chemical linkage of proteins to cell wall cross bridges is known, the mechanisms whereby polypeptides are distributed on the staphylococcal surface have not been revealed. We show here that protein A, the ligand of immunoglobulin, is unevenly distributed over the staphylococcal surface. Upon removal with trypsin, newly synthesized polypeptide is deposited at two to four discrete foci. During subsequent growth, protein A appears to be slowly distributed from these sites. When viewed through multiple focal planes by laser scanning microscopy, protein A foci are arranged in a circle surrounding the bacterial cell. This pattern of distribution requires the LPXTG sorting signal of protein A as well as sortase A, the transpeptidase that anchors polypeptides to cell wall cross bridges. A model is presented whereby protein A deposition at discrete sites coupled with cell wall synthesis enables distribution of protein A on the staphylococcal surface.     

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.     

Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions

Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg α-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel β-sheet complementation as observed for the clumping factor A·γ-chain peptide complex. The β-sheet complementation is shorter in the ClfB·Fg α-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.     

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.     

Key role for clumping factor B in Staphylococcus aureus nasal colonization of humans

Background

Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor B (ClfB) promotes adhesion to squamous epithelial cells in vitro and might be a physiologically relevant colonization factor.

Methods and Findings

We define the role of the staphylococcal cytokeratin-binding protein ClfB in the colonization process by artificial inoculation of human volunteers with a wild-type strain and its single locus ClfB knock-out mutant. The wild-type strain adhered to immobilized recombinant human cytokeratin 10 (CK10) in a dose-dependent manner, whereas the ClfB⁻ mutant did not. The wild-type strain, when grown to the stationary phase in a poor growth medium, adhered better to CK10, than when the same strain was grown in a nutrient-rich environment. Nasal cultures show that the mutant strain is eliminated from the nares significantly faster than the wild-type strain, with a median of 3 ± 1 d versus 7 ± 4 d (p = 0.006). Furthermore, the wild-type strain was still present in the nares of 3/16 volunteers at the end of follow-up, and the mutant strain was not.

Conclusions

The human colonization model, in combination with in vitro data, shows that the ClfB protein is a major determinant of nasal-persistent S. aureus carriage and is a candidate target molecule for decolonization strategies.     

Optimization of recombinant clumping factor of Staphylococcus aureus expression in Escherichia coli XLI-BLUE

Experiments for optimization of the clfA gene expression in E. coli XLI-Blue cells were conducted. The reducing of the strain efficiency and the elimination of the plasmid during the cultivation of a producer have been shown. Several methods for raising the amount of plasmid-containing cells and increasing the yield of recombinant protein were proposed. Bacterial growth medium was selected to make this process more efficient. In such conditions after IPTG induction of the recombinant cells we have received high level of a soluble protein (about 30% of total cellular protein). The recombinant protein containing the His tag on N-terminus was purified on IDA-Sepharose column with high yield.     

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.     

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection 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.     

Localization on the cell surface of streptococcal protein antigen A

Abstract The cellular localization of the major surface protein SpaA of Streptococcus sobrinus 6175 was examined by immunoelectron microscopy with rabbit polyclonal antibodies directed against purified SpaA protein. Immunoelectron microscopic analysis of thin sections of S. sobrinus cells revealed that the SpaA protein is associated with the fibrillar fuzzy coat of S. sobrinus cells and appears to be distributed over the entire surface of S. sobrinus cells.