Biomedical subjects

J I Flock

Publications and source records attributed to J I Flock.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1995

Isolation and characterisation of a vitronectin-binding surface protein from Staphylococcus aureus.

In a previous study we demonstrated that cells of Staphylococcus aureus strain V8 bind 125I-labelled vitronectin in a receptor-ligand type of interaction, and a protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein (Liang, O.D. et al. (1993) Biochim. Biophys. Acta 1225, 57-63). In the present communication we report on the isolation and preliminary characterisation of the 60 kDa vitronectin-binding protein. The bacterial cell surface proteins were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h and separated on an FPLC Mono-Q column with a gradient of 0-0.5 M NaCl in 20 mM Tris buffer at pH 9.0. Fractions containing vitronectin-binding activity, assayed on microtiter plates with immobilised human vitronectin, were collected and SDS-PAGE analysis showed the content to be a single protein band at the 60 kDa position. In Western blot experiments the protein transblotted onto nitrocellulose membranes could bind soluble vitronectin. Its amino-terminal amino acid sequences showed a striking similarity with those of a 60 kDa heparan sulfate-binding protein from the same staphylococcal strain (Liang, O.D. et al. (1992) Infect. Immun. 60, 899-906), suggesting that they are identical molecules. This was supported by ligand blotting experiments where both vitronectin and heparan sulfate were shown to bind to the same protein band in parallel strips.

Amino Acid Sequence↗

PubMed1995

Development and characterization of a new model of hematogenous osteomyelitis in the rat.

Hematogenous osteomyelitis was produced in the tibia or the mandible of rats by drilling a hole into the bone, injecting sodium morrhuate, and inoculating Staphylococcus aureus Phillips into the femoral vein. Animals were sacrificed after 2 weeks and examined. The infection was characterized grossly and radiographically by bone deformation, histopathologically by a characteristic suppurative reaction, and microbiologically by the recovery of S. aureus Phillips from the infected tissue. These findings indicate that the model mimics human osteomyelitis with respect to its inflammatory bone changes. In contrast to earlier rat models in which bacteria were injected directly into the bone, this new experimental model allows study of the initiating events of osteomyelitis such as bacterial attachment and might assist as a model for both prophylactic and therapeutic trials.

Animals↗

PubMed1995

Typing of coagulase-negative staphylococci from peritonitis in CAPD-patients by the PhP-CS system and REA.

Coagulase-negative staphylococci (CNS) were the most common bacteria causing peritonitis in patients treated with continuous ambulatory peritoneal dialysis (CAPD). In order to investigate if the same clone was responsible for the peritonitis in the different patients and if the exit site was the source of infection we followed 68 patients on CAPD for 2 years. During this period 9 patients had 12 episodes of peritonitis caused by CNS. Cultures were taken from exit site and peritoneal fluid in all patients at peritonitis and during the first study year at monthly intervals. In each culture up to 10 isolates of CNS were randomly collected and frozen. All 437 CNS isolates from the patients with CNS peritonitis were typed using a biochemical typing method and 41 isolates identical by this method were further discriminated by a DNA fingerprinting method. Identical strains were in no case isolated from different patients, indicating that no virulent strain was spread between the patients. The isolates causing the peritonitis were never found at the exist sites before the first day of the peritonitis in any patient. In only two patients was the same strain found at the exit site and in the peritoneal fluid on the first day of peritonitis. It thus seems that no virulent clone of CNS was infecting the patients and we found no evidence of CNS at the exit site causing the peritonitis.

Adult↗

PubMed1995

Incidence of the highly conserved fib gene and expression of the fibrinogen-binding (Fib) protein among clinical isolates of Staphylococcus aureus.

We have recently described a 19-kDa fibrinogen-binding protein, termed Fib, which is secreted into the extracellular medium by Staphylococcus aureus. In this study, the presence of the Fib protein and the fib gene among clinical isolates of S. aureus and among other staphylococcal species known to interact with fibrinogen was investigated. This task was pursued at the DNA, mRNA, and protein levels. It was found that the fib gene was unique to S. aureus and highly conserved at the nucleotide level. The Fib protein was produced by all S. aureus strains investigated but was not detected in all bovine mastitis strains, because of proteolytic degradation by simultaneously produced staphylococcal proteases. It was concluded that the fib gene was unique to S. aureus and that it could be used in the identification of S. aureus.

Animals↗

PubMed1994

Vaccination against Staphylococcus aureus mastitis: immunological response of mice vaccinated with fibronectin-binding protein (FnBP-A) to challenge with S. aureus.

Mice were immunized with fusion proteins encompassing the fibronectin-binding domain of a staphylococcal fibronectin-binding protein (FnBP-A). A specific antibody response against the fibronectin-binding part of the fusion proteins was detected in the serum of all vaccinated animals. The protective potential of these vaccinations was evaluated in a mouse mastitis model, using Staphylococcus aureus, strain SA113, for challenge. The mice vaccinated with FnBP fusion proteins showed a decreased number of bacteria recovered from the mammary glands and significantly reduced cases of severe mastitis. Histopathological examination of tissue from challenged glands of vaccinated mice revealed either no pathological reactions or disseminated inflammatory reactions with focal necrosis whereas four of six examined tissues from challenged glands of non-vaccinated animals showed total necrosis. A combination of FnBP fusion protein with staphylococcal alpha-toxoid did not increase the efficacy of the vaccination and animals vaccinated with alpha-toxoid alone were as sensitive to challenge as those from the non-vaccinated control group. Thus vaccination of mice with recombinant FnBP resulted in significant protection against challenge with S. aureus.

Adhesins, Bacterial↗

PubMed1994

Evidence that the heparin-binding consensus sequence of vitronectin is recognized by Staphylococcus aureus.

Binding of heparin-binding form of vitronectin to Staphylococcus aureus was inhibited completely by heparin or by the same form of vitronectin. The binding was inhibited only to about 50% by the non-heparin-binding form of vitronectin, indicating an apparent involvement of the heparin-binding properties in the interaction between vitronectin and S. aureus. This was supported by experiments in which a synthetic peptide (Ala347-Arg361, comprising heparin-binding consensus sequences) was found to partly inhibit bacterial adherence to immobilized vitronectin. A bacterial cell surface protein could bind to the quinquedecapeptide, but not to the highly charged peptides consisting entirely of arginine or lysine, immobilized on microtiter plates and the binding could be competitively inhibited by an excess of soluble peptide. Direct binding of radiolabeled peptide to bacterial cells was also demonstrated, which was rapid, saturable, and pH-dependent. Furtherly a bacterial surface protein having molecular mass of 60 kDa was isolated by affinity chromatography on a quinquedecapeptide-HiTrap-NHS column. Our data suggest that the heparin-binding properties of vitronectin play a role in bacterial recognition.

Amino Acid Sequence↗

PubMed1994

Cloning and characterization of a gene for a 19 kDa fibrinogen-binding protein from Staphylococcus aureus.

Staphylococcus aureus has been shown to interact specifically with fibrinogen. Three different extracellular fibrinogen-binding proteins, two of which have coagulase activity, are produced by S. aureus strain Newman. The role of these fibrinogen-binding proteins during staphylococcal colonization and infection has not yet been fully elucidated. Here we describe the cloning, sequencing and expression of a gene for a 19 kDa fibrinogen-binding protein. This gene, called fib, encodes a 165-amino-acid polypeptide, including a 29-amino-acid signal sequence. The recombinant protein, which has an estimated molecular mass of 15.9 kDa, bound fibrinogen and was recognized by a polyclonal antiserum against the native Fib protein. Homologies between the Fib protein and the fibrinogen-binding domain of coagulase suggest that amino acids within this domain are involved in the binding to fibrinogen.

Amino Acid Sequence↗

PubMed1994

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model.

A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus. The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibrinogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus.

Animals↗

PubMed1993

Multiple interactions between human vitronectin and Staphylococcus aureus.

Multiple interactions between human vitronectin and Staphylococcus aureus strain V8 were observed. An upward-curved Scatchard plot indicated both high-affinity binding (Kd1 = 7.4 x 10(-10) M) with 260 binding sites per bacterial cell and moderate-affinity binding (Kd2 = 7.4 x 10(-8) M) with 5240 copies per cell. Negative cooperativity of this binding was characterized by its Hill coefficient of less than unity (0.70 +/- 0.08). Up to 60% of the vitronectin-bacteria interaction was unaffected by high ionic strength (i.e., 2.4 M NaCl), and was not inhibited by highly-charged heparin oligosaccharides. Various oligosaccharides (4-20 monosaccharide units) generated by partial deaminative cleavage of heparin were found to affect vitronectin binding to S. aureus. Short-chain-length oligosaccharides increase and long oligosaccharides inhibit vitronectin binding, in accordance with direct association of these saccharides with multimeric vitronectin. A protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein. These results indicate that interaction of multimeric vitronectin, mostly present at extracellular matrix sites with multiple recognition sites on the S. aureus surface, may contribute to bacterial colonisation.

Binding Sites↗

PubMed1993

Immunization with fibronectin binding protein from Staphylococcus aureus protects against experimental endocarditis in rats.

Rats were immunized with a fusion protein (gal-FnBP) encompassing beta-galactosidase and the domains of fibronectin binding protein from Staphylococcus aureus responsible for binding to fibronectin. Antibodies against gal-FnBP were shown to block the binding of S. aureus to immobilized fibronectin in vitro. Endocarditis in immunized and non-immunized control rats was induced by catheterization via the right carotid artery, resulting in damaged aortic heart valves which became covered by fibrinogen and fibronectin. The catheterized rats were then infected intravenously with 1 x 10(5) cells of S. aureus. The number of bacteria associated with aortic valves was determined 1 1/2 days after the challenge infection and a significant difference in bacterial numbers between immunized and non-immunized groups was then observed (p < 0.05).

Adhesins, Bacterial↗

PubMed1993

High yield of active STb enterotoxin from a fusion protein (MBP-STb) expressed in Escherichia coli.

Maltose binding protein (MBP) fused to STb, a heatstable enterotoxin of Escherichia coli, was secreted into the periplasm. A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb. The gene fusion is under the control of the strong and inducible Ptac promoter. Three hours after induction with IPTG, cells were harvested. Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin. The fusion protein purified in this way was biologically active in ligated intestinal segments of rats. Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column. The toxin was eluted at approximately 38% acetonitrile. The 5000-Da toxin was shown to be pure by SDS-PAGE and immunoblotting. The recovered enterotoxin was active in the rat loop assay. Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb. The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.6. Typically, 8 liters of bacterial culture resulted in 2.2 mg of pure STb. This genetic construction provides a readily obtainable source of biologically active STb toxin.

ATP-Binding Cassette Transporters↗

PubMed1993

Evaluation of new enzyme-linked immunosorbent assay based on a supernatant containing Staphylococcus aureus alpha-toxin produced by Bacillus subtilis.

The gene encoding alpha-toxin from Staphylococcus aureus was cloned into a Bacillus subtilis expression vector (pEF 231/alpha-Tox). The protease-deficient B. subtilis strain DB 104 transformed with pEF 231/alpha-Tox expressed and secreted 5 mg of alpha-toxin per liter into the growth medium. The alpha-toxin-containing supernatant was diluted 200-fold and used as coating antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of septicemia and endocarditis caused by S. aureus. Paired sera from patients in acute and convalescent stages of S. aureus and non-S. aureus infections were used to evaluate this ELISA. To evaluate the effectiveness of the crude preparation, the results were compared with those of an ELISA based on a commercially available alpha-toxin. Similar rises in serum titers were obtained with either type of alpha-toxin preparation. This is the first time a crude supernatant without any further purification has been used as an ELISA coating antigen. We therefore conclude that B. subtilis is a suitable host organism for cheap and simple production of prokaryotic recombinant antigens to be used in serodiagnosis.

Antibodies, Bacterial↗