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Biomedical subjects

J I Flock

Publications and source records attributed to J I Flock.

At least 19 recordsLinked to original sources
PubMed2000

Antibody responses in patients with staphylococcal septicemia against two Staphylococcus aureus fibrinogen binding proteins: clumping factor and an extracellular fibrinogen binding protein.

We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens, and cutoff levels (upper 95th percentile) against these antigens were determined. No correlation was seen between serum antibody levels against Clf and Efb. In acute-phase samples 27% of patients showed positive antibody levels against Clf and 10% showed positive levels against Efb, while in convalescent-phase samples 63% (26 of 41) showed a positive serology against Clf and 49% (20 of 41) showed a positive serology against Efb. Antibody levels against Efb were significantly lower in the acute-phase sera than in sera from healthy individuals (P = 0. 002). An antibody response against Clf was most frequent in patients suffering from osteitis plus septic arthritis and from endocarditis (80% positive). The antibody response against Efb appeared to develop later in the course of disease. A possible biological effect of measured antibodies was demonstrated with the help of an inhibition ELISA, in which both high-titer and low-titer sera inhibited the binding of bacteria to fibrinogen. In conclusion, we have demonstrated in vivo production of S. aureus fibrinogen binding proteins during deep S. aureus infections and a possible diagnostic and prophylactic role of the corresponding serum antibodies in such infections.

Adolescent↗
PubMed1999

Immunogenicity of peptides derived from a fibronectin-binding protein of S. aureus expressed on two different plant viruses.

The D2 peptide derived from an S. aureus fibronectin-binding protein (FnBP) was expressed on the surface of the icosahedral cowpea mosaic virus (amino acids 1-30 of D2) or on the rod-shaped potato virus X (amino acids 1-38 of D2), termed CPMV-MAST1 and PVX-MAST8, respectively. Mice and rats were immunized subcutaneously with CPMV-MAST1 and mice with PVX-MAST8 in adjuvant and high titres of FnBP-specific antibody were obtained. The mouse IgG was predominantly of the IgG2a and IgG2b isotypes, which strongly bound complement component C1q, suggesting a TH1-bias in the peptide-specific responses. Sera from mice and rats immunized with CPMV-MAST1 and from mice immunized with PVX-MAST8 were shown to completely inhibit the binding of fibronectin to immobilised recombinant FnBP and rat sera against CPMV-MAST1 were able to block adherence of S. aureus to fibronectin. These studies demonstrate that the D2 peptide is highly immunogenic when expressed on 2 different plant viruses and highlight the potential of plant virus-based vaccines to protect against S. aureus infections.

Adhesins, Bacterial↗
PubMed1999

Extracellular-matrix-binding proteins as targets for the prevention of Staphylococcus aureus infections.

Staphylococcal infections cause a number of serious diseases, ranging from acute septicaemia to chronic problems such as osteomyelitis and septic arthritis. Resistance to antibiotics is a growing problem and has re-ignited interest in vaccines and in passive immunization with antibodies. Natural infections and vaccines based on whole bacteria lead to poor antibody responses, but recent research using animal models of several staphylococcal diseases reveals that vaccines based on recombinant staphylococcal extracellular-matrix-binding proteins are much more protective. Passive immunization with antibodies against one of these proteins (collagen-binding protein) also shows promise in a mouse model of sepsis.

Adhesins, Bacterial↗
PubMed1999

Functional studies of a fibrinogen binding protein from Staphylococcus epidermidis.

A gene encoding a fibrinogen binding protein from Staphylococcus epidermidis was previously cloned, and the nucleotide sequence was determined. A portion of the gene encompassing the fibrinogen binding domain has now been subcloned in an expression-fusion vector. The fusion protein can bind to fibrinogen in a capture enzyme-linked immunosorbent assay and can be purified by fibrinogen affinity chromatography. This protein can completely inhibit the adherence of S. epidermidis to immobilized fibrinogen, suggesting that the adherence of S. epidermidis to fibrinogen is mainly due to this protein. Antibodies against this fibrinogen binding protein were also found to efficiently block the adherence of S. epidermidis to immobilized fibrinogen. Despite homology with clumping factors A and B from S. aureus (cell surface-associated proteins binding to fibrinogen), binding involved the beta chain of fibrinogen rather than the gamma chain, as in clumping factor A.

Animals↗
PubMed1999

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.

Bacterial Adhesion↗
PubMed1999

Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice.

The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or in the presence of ISCOM matrix, primed CPMV-specific T cells and generated high titers of CPMV- and FnBP-specific immunoglobulin G (IgG) in sera. Furthermore, CPMV- and FnBP-specific IgA and IgG could also be detected in the bronchial, intestinal, and vaginal lavage fluids, highlighting the ability of CVPs to generate antibody at distant mucosal sites. IgG2a and IgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal IgA was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.

Adhesins, Bacterial↗
PubMed1998

Coexisting members of HIV-1 p17 gene quasispecies represent proteins with distinct antigenicity and immunogenicity.

BACKGROUND: Despite the comparatively conserved nature of the HIV-1 gag gene, countless quasispecies of the p17 gene coexist in HIV-1-infected patients. It is not known if the minor genetic differences in quasispecies will affect immune recognition. OBJECTIVE: To characterize the antigenicity and immunogenicity of three different members of HIV-1 p17 quasispecies. METHODS: Three members of HIV-1 p17 gene quasispecies, one from patient A (clone 9; qsA9) and two from patient E (clones 5 and 8; qsE5 and qsE8), were expressed and purified from Escherichia coli. The antigenicity of the p17 proteins was analysed using sera from HIV-1-infected individuals, and the immunogenicity was evaluated using sera and lymphocytes from primed mice of three different haplotypes. RESULTS: The antigenicity of the qsE5 and qsE8 p17 recombinant proteins were distinct when tested for reactivity with human p17 antibodies. The qsE5 and qsE8 p17 were equally immunogenic in H-2d mice, but not in H-2b and H-2k mice. In H-2b mice the qsE8 protein induced higher levels of anti-p17 IgG2a, IgG2b and IgG3 than the qsE5 protein. Corroborating the IgG subclass pattern, H-2b-restricted qsE5-specific T cells produced higher in vitro levels of interferon-gamma, but not of interleukin (IL)-4, IL-5 and IL-6, than qsE8-specific T cells, suggesting a more pronounced T-helper (TH)1-like response. CONCLUSIONS: The p17 gene quasispecies coexisting in the same patient at the same time may represent antigenically and immunogenically distinct proteins despite sequence homologies of above 90%. Subsequently, subtle differences between two p17 protein quasispecies are enough to prime different TH1/TH2 subsets. These findings will have implications for therapeutic HIV-1 immunizations.

Amino Acid Sequence↗
PubMed1998

Identification of functional domains in Efb, a fibrinogen binding protein of Staphylococcus aureus.

Staphylococcus aureus produces and secretes a protein, Efb, that binds to fibrinogen, seems to be required for virulence, and may benefit the microorganism by delaying wound healing. Interactions of Efb with fibrinogen are influenced by divalent metal cations, including Ca2+. Increasing concentrations of Ca2+ increased the binding of fibrinogen to immobilized Efb, whereas binding of Efb to immobilized fibrinogen was decreased with increasing Ca2+ concentration. Studies with synthetic peptides showed that peptides from the carboxyl terminal half of Efb bound to soluble fibrinogen and enhanced the binding of fibrinogen to Efb. A peptide corresponding to a repeated sequence in the amino terminal half of the protein also bound fibrinogen and inhibited binding of fibrinogen to Efb. These results may provide clues to the biological function of Efb and aid in the rational design of agents to block the Efb fibrinogen interaction.

Amino Acid Sequence↗
PubMed1998

Multiple binding sites in the interaction between an extracellular fibrinogen-binding protein from Staphylococcus aureus and fibrinogen.

Efb (previously Fib) is a fibrinogen-binding protein secreted by Staphylococcus aureus. It has previously been shown that it plays a role in a wound infection model in the rat and that antibodies against Efb reduce the number of recovered bacteria from the mammary glands in a mouse mastitis model. Efb binds to the alpha-chain of fibrinogen and does not participate in bacterial adherence to fibrinogen. The binding of Efb to fibrinogen is divalent, with one binding site within the two repeat regions in Efb at the N terminus and one binding site at the C terminus. The divalent binding nature leads to precipitation of Efb-fibrinogen complex when the proteins are added to each other at a 1:1 molar ratio. The interaction between Efb and fibrinogen is strongly enhanced by Ca2+ or Zn2+ but not by Mg2.

Animals↗
PubMed1998

A fibrinogen-binding protein of Staphylococcus epidermidis.

The present study reports on fibrinogen (Fg) binding of Staphylococcus epidermidis. Adhesion of different S. epidermidis strains to immobilized Fg was found to vary significantly between different strains, and the component responsible was found to be proteinaceous in nature. To further characterize the Fg-binding activity, a shotgun phage display library covering the S. epidermidis chromosome was constructed. By affinity selection (panning) against immobilized Fg, a phagemid clone, pSEFG1, was isolated, which harbors an insert with an open reading frame of approximately 1.7 kilobases. Results from binding and inhibition experiments demonstrated that the insert of pSEFG1 encodes a specific Fg-binding protein. Furthermore, affinity-purified protein encoded by pSEFG1 completely inhibited adhesion of S. epidermidis to immobilized Fg. By additional cloning and DNA sequence analyses, the complete gene, termed fbe, was found to consist of an open reading frame of 3,276 nucleotides encoding a protein, called Fbe, with a deduced molecular mass of approximately 119 kDa. With a second phage display library made from another clinical isolate of S. epidermidis, it was possible to localize the Fg-binding region to a 331-amino-acid-long fragment. PCR analysis showed that the fbe gene was found in 40 of 43 clinical isolates of S. epidermidis. The overall organization of Fbe resembles those of other extracellular surface proteins of staphylococci and streptococci. Sequence comparisons with earlier known proteins revealed that this protein is related to an Fg-binding protein of Staphylococcus aureus called clumping factor.

Amino Acid Sequence↗
PubMed1997

Expression of collagen-binding protein and types 5 and 8 capsular polysaccharide in clinical isolates of Staphylococcus aureus.

In vitro collagen binding of 216 Staphylococcus aureus isolates from patients with various diagnoses was studied. Polymerase chain reaction was used to examine these isolates regarding the existence of the corresponding cna gene. Distribution of capsular polysaccharide (CP) types was examined. Fifty-six (57%) of 99 S. aureus isolates from patients with endocarditis or bacteremic bone or joint infection were cna-positive compared with 65 (56%) of 117 isolates from bacteremic patients without signs of bone or joint infection (P = .99). There was a good correlation between in vitro collagen binding and presence of the cna gene. These data suggest that collagen binding is not a prerequisite for the development of endocarditis, osteomyelitis, or septic arthritis. There was no significant difference in the distribution of CP types among various patient groups, although there was a strong association between CP type 8 and the existence of the cna gene.

Antibodies, Bacterial↗
PubMed1997

Fibronectin binding by Propionibacterium acnes.

Strains of Propionibacterium acnes, isolated from different kinds of orthopaedic and biomaterial-associated infections and from skin flora were shown to express binding of soluble as well as immobilized fibronectin. Among these 7 strains isolated from orthopaedic infections, 2 from breast prostheses, and 9 skin isolates, 2, 2, and 5 strains respectively bound immobilized fibronectin. The fibronectin binding was sensitive to protease and heat treatment, and was inhibited by a cell surface extract from one of the binding strains. In SDS-PAGE and autoradiography of cell surface extracts, a band corresponding to a MW of about 80 kD reacted with fibronectin and the 150 kD fragment of fibronectin. Binding to fibronectin and the 150 kD fragment of fibronectin could be inhibited with heparin. We thus present a first Fn binding protein of P. acnes, a surface exposed protein of 80 kD. None of the strains bound soluble collagen, and only one strain expressed weak binding of vitronectin and bone sialoprotein II.

Adhesins, Bacterial↗
PubMed1996

Streptococcus mutans major adhesion surface protein, P1 (I/II), does not contribute to attachment to valvular vegetations or to the development of endocarditis in a rat model.

Streptococcus mutans P1 antigen functions as an adhesion factor for binding to salivary pellicle on tooth surfaces. It induces increased antibody titres in patients with Strep. mutans endocarditis. A mutant of Strep. mutans deficient in the function of the gene (spa P) encoding the surface antigen P1, and its isogenic parental strain, were used in a rat endocarditis experiment. Absence of P1 did not decrease adhesion to vegetations determined l h after intravenous infection. The number of bacteria recovered from valvular vegetations after 48 h from animals with manifest endocarditis did not differ between the strains. Consequently, the Pl antigen appears to be unimportant both for adhesion and virulence in endocarditis caused by Strep. mutans.

Animals↗
PubMed1996

Collagen binding of Staphylococcus aureus is a virulence factor in experimental endocarditis.

The role of Staphylococcus aureus collagen binding in the development of experimental endocarditis was studied. Two isogenic strains of S. aureus, 1 carrying an insertional inactivation of the gene encoding collagen-binding protein, were compared in a rat model of catheter-induced infective endocarditis (i.e.). Separate groups of rats with traumatized aortic valves were intravenously challenged with 1 of the strains. In rats sacrificed 24 h after inoculation, the collagen-binding strain significantly outnumbered the mutant strain (P < .001); however, 1 h after challenge, there was no difference in numbers of the 2 strains. The results were substantiated, using a 1:1 mixture of the parent strain and the mutant as an inoculate. Our findings suggest that collagen binding of S. aureus is important in the sustenance of experimental IE and plays a limited role during the initial attachment of the microorganism to traumatized aortic valves.

Animals↗
PubMed1996

Lack of the extracellular 19-kilodalton fibrinogen-binding protein from Staphylococcus aureus decreases virulence in experimental wound infection.

A mutant deficient for the 19-kDa extracellular fibrinogen-binding protein (Fib) from Staphylococcus aureus has been constructed. The gene was inactivated by allele replacement. A 2.0-kb fragment from transposon Tn4001 carrying the gene for gentamicin resistance was inserted into the gene encoding Fib (fib). The genotype was verified by PCR analysis, and the loss of Fib was demonstrated by Western blotting (immunoblotting). The mutation has not altered the ability of the strain to bind to fibrinogen or fibronectin compared with that of the isogenic parental strain, FDA486. The mutant, designated K4.3, was compared with strain FDA486 in a wound infection model in rats. Sixty-eight percent of the rats challenged with parental strain FDA486 developed severe clinical signs of wound infection, whereas only 29% of the animals challenged with isogenic mutant K4.3 showed severe symptoms (P < 0.01). The weight loss of animals infected with the wild type was also significantly different from that of animals infected with the mutant strain. The result demonstrates that the extracellular 19-kDa fibrinogen-binding protein from S. aureus contributes to the virulence in wound infection and delays the healing process.

Alleles↗
PubMed1996

Reconsideration of the role of fibronectin binding in endocarditis caused by Staphylococcus aureus.

The adherence characteristics in vivo and virulence of two isogenic strains of Staphylococcus aureus differing in fibronectin binding were compared in a rat model of catheter-induced infective endocarditis. No differences were found between the two strains. The results strongly point to the multifactorial nature of bacterial adherence to damaged heart valves and suggest that other binding functions can compensate for the lack of fibronectin binding in S. aureus.

Animals↗
PubMed1996

Insertional inactivation of the gene for collagen-binding protein has a pleiotropic effect on the phenotype of Staphylococcus aureus.

This report describes phenotypical changes caused by the insertional inactivation of the gene for the collagen-binding protein in Staphylococcus aureus PH100. Insertional inactivation resulted in reductions in the amount of fibronectin-binding protein in PH100 and the ability of intact cells to aggregate in the presence of fibronectin. However, the capacity of PH100 to adhere to immobilized fibronectin remained the same.

Adhesins, Bacterial↗