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T J Foster

Publications and source records attributed to T J Foster.

At least 55 records · Page 3Linked to original sources
PubMed1996

Host-bacteria interactions in foreign body infections.

Persistent staphylococcal infections are a major medical problem, especially when they occur on implanted materials or intravascular catheters. This review describes some of the recently discovered molecular mechanisms of Staphylococcus aureus attachment to host proteins coating biomedical implants. These interactions involve specific surface proteins, called bacterial adhesins, that recognize specific domains of host proteins deposited on indwelling devices, such as fibronectin, fibrinogen, or fibrin. Elucidation of molecular mechanisms of S aureus adhesion to the different host proteins may lead to the development of specific inhibitors blocking attachment of S aureus, which may decrease the risk of bacterial colonization of indwelling devices.

Adhesins, Bacterial↗
PubMed1996

Chorismate synthase from Staphylococcus aureus.

The aroC gene encoding chorismate synthase and the ndk gene encoding nucleoside diphosphate kinase (Ndk) were cloned from Staphylococcus aureus. DNA sequencing suggests that aroC is located in an operon with aroB and aroA and encodes a protein of 388 amino acids with 61% identity to the aroF gene product of Bacillus subtilis. The ndk gene of S. aureus encodes a protein of 149 amino acids which exhibits a high degree of identity to other bacterial Ndk proteins. The 3' end of the S. aureus gerCC gene was also identified by sequencing and was located immediately upstream of ndk. The gerCA and gerCB genes were found to be located upstream of gerCC by Southern hybridization analysis. This observed linkage of the gerC genes with the ndk, aroC and aroB genes has been similarly observed in B. subtilis. The S. aureus chorismate synthase was overexpressed to a high level in Escherichia coli using a T7 promoter plasmid construct, the enzyme was purified to near homogeneity in two steps and found to be a homotetramer with a subunit molecular mass, estimated by electrospray mass spectrometry, of 43024 Da. The properties of S. aureus chorismate synthase are compared with those of the B. subtilis and E. coli enzymes.

Amino Acid Sequence↗
PubMed1996

A low-fibronectin-binding mutant of Staphylococcus aureus 879R4S has Tn918 inserted into its single fnb gene.

A low-fibronectin-binding mutant of Staphylococcus aureus strain 879R4SSp generated by transposon Tn918 mutagenesis is attenuated in a rat endocarditis model (J.M. Kuypers & R.A. Procter, 1989, Infect Immun 57, 2306-2312). PCR and Southern hybridization analysis with primers and probes, respectively, for the fnbA and fnbB genes of strains 8325-4 showed that strain 879R4SSp possesses a single fnb gene which is homologous to fnbA. This was confirmed by sequencing 41 bp of 5' non-coding and 237 bp of 5' coding DNA, which showed 97% base identity to fnbA. Southern hybridization and sequencing showed that Tn918 was inserted 41 bp 5' to fnbA in the mutant 879R4SSp/1536, between the promoter and initiation codon. Reduced adherence of the mutant to surface-bound fibronectin correlated with lower expression of a 180 kDa wall-associated fibronectin-binding protein.

Adhesins, Bacterial↗
PubMed1996

Influence of agr on fibrinogen binding in Staphylococcus aureus Newman.

The ability of Staphylococcus aureus to bind fibrinogen is believed to be important in promoting bacterial adherence to both intravascular catheters and host tissues during infection. We investigated the influence of the global regulator agr on the fibrinogen binding capacity and its relationship to the expression of coagulase (encoded by coa) and clumping factor (encoded by clfA) in strain Newman. Strains were obtained by transducing site-specific mutations of clfA, coa, and agr into strain Newman to obtain single, double, and triple mutants of the respective genes. As expected, the clfA mutant bound less soluble 125I-labeled fibrinogen than the corresponding coa mutant in agr+ strains; however, with agr mutant strains, the upregulation in fibrinogen binding capacity correlated mostly with the increased expression and transcription of coagulase as shown by Western (immunoblot) and Northern (RNA) blot analysis. In particular, the coa agr double mutant resulted in a significant reduction in fibrinogen binding compared with that of the agr mutant. The contribution of clfA to fibrinogen binding in agr-negative strains was less than that of coa (32,740 +/- 1,189 versus 18,141 +/- 334 and 38,919 +/- 1,021 cpm for clfA agr, coa agr, and the single agr mutant, respectively). Thus, coagulase is a major binding protein for soluble fibrinogen in the agr-negative background. In in vitro microtiter and catheter adherence assays with solid-phase fibrinogen, clumping factor, but not coagulase, plays a major role in binding to immobilized fibrinogen. coa transcription was negatively modulated by agr and occurred mainly during the exponential growth phase. In contrast, clfA transcription was agr independent and was strongest during the postexponential phase. Although an agr coa clfA triple mutant bound less soluble fibrinogen than the agr coa double mutant (8,504 +/- 831 versus 18,141 +/- 334 cpm), significant residual fibrinogen binding capacity remained in the triple mutant, thus suggesting an additional fibrinogen binding component. By using direct ligand affinity blotting with 125I-fibrinogen, we could identify coagulase and an additional unidentified 52-kDa protein as a fibrinogen binding component in cell extracts. This band was absent in the extract of the coa clfA double mutant.

Bacterial Proteins↗
PubMed1996

A live (delta aroA) Aeromonas salmonicida vaccine for furunculosis preferentially stimulates T-cell responses relative to B-cell responses in rainbow trout (Oncorhynchus mykiss).

We have previously described (L. M. Vaughan, P. R. Smith, and T. J. Foster, Infect. Immun. 61:2172-2181, 1993) the construction of a kanamycin-resistant aromatic-dependent mutant of Aeromonas salmonicida, the causative agent of furunculosis, and its use as a live vaccine. Here we describe the construction of an unmarked aroA deletion mutant and examine the nature and magnitude of immune responses in rainbow trout (Oncorhynchus mykiss) to this vaccine strain. Good proliferative and antibody responses were elicited by using a range of vaccine doses from 2 x 10(6) to 2 x 10(9) live bacteria per fish, and a clear vaccine dose effect was apparent. A significant positive effect of using live bacteria to prime for lymphocyte proliferation and antibody production was apparent. However, the problem of directly comparing the vaccine doses of live and killed bacterial preparations is discussed, since some replication of live bacteria in vivo is expected. Most importantly, the live vaccine preferentially stimulated enhanced T-cell responsiveness, as evidenced by significantly greater increases in T-cell proliferation than in B-cell proliferation, compared with responses by the respective cell populations from fish given a killed vaccine. The manner in which live vaccines elicit strong cell-mediated immune responses and the relevance to fish vaccine design are discussed.

Aeromonas↗
PubMed1995

Variation in the size of the repeat region of the fibrinogen receptor (clumping factor) of Staphylococcus aureus strains.

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, the gene encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman was cloned and sequenced. The ClfA protein possesses a highly unusual 308 residue dipeptide repeat region composed predominantly of Asp and Ser. Polymerase chain reaction analysis of seven different strains showed that the size of the clfA repeat coding region varies from 580 bp to 1320 bp. In contrast, the clfA region A is the same size in each strain. The size of the clfA repeat region did not correlate with the ability of these strains to form clumps in a solution of fibrinogen. Indeed, the strain with the smallest repeat size of 580 bp clumped almost as well as strain Newman. Each strain of S. aureus examined contained several high molecular mass proteins that reacted with anti-ClfA region A antibody. In some cases the molecular mass of the major protein varied in accordance with the length of the coding sequence for the repeat region R.

Bacterial Proteins↗
PubMed1995

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes.

Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB. A double fnbAfnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbAfnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo.

Adhesins, Bacterial↗
PubMed1995

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.

Bacterial Adhesion↗
PubMed1995

Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.

Animals↗
PubMed1995

Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts.

We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization.

Animals↗
PubMed1995

Quantitative comparison of clumping factor- and coagulase-mediated Staphylococcus aureus adhesion to surface-bound fibrinogen under flow.

The contributions of clumping factor and coagulase in mediating Staphylococcus aureus adhesion to surface-adsorbed fibrinogen have been quantified by using a new methodology and analysis. The attachment or detachment kinetics of bacteria were directly observed in a radial flow chamber with a well-defined laminar flow field and a spatially varying shear rate and were quantified by recursively scanning the chamber surface and counting cells via automated video microscopy and image analysis with a motorized stage and focus control. Intrinsic rate constants for attachment or detachment were estimated as functions of shear rate for the wild-type Newman strain of S. aureus and for mutants lacking clumping factor, coagulase, or both proteins on surfaces coated with plasma, fibrinogen, or albumin. Clumping factor, but not coagulase, increased the probability of attachment and decreased the probability of detachment of S. aureus on plasma-coated surfaces; however, both clumping factor and, to a lesser extent, coagulase increased the probability of attachment on the purified-fibrinogen-coated surface. All mutants were resistant to detachment on the purified-fibrinogen-coated surface, suggesting the possibility of an additional adhesion mechanism which was independent of coagulase or clumping factor and effective only for fully attached cells. Together, these results suggest that the presence of clumping factor plays the primary role in enhancing adhesion to surfaces with adsorbed fibrinogen, not only by enhancing the probability of cell attachment but also by increasing the strength of the resulting adhesion.

Bacterial Adhesion↗
PubMed1995

Effects of Staphylococcus aureus toxins on the growth of bovine mammary epithelial cells (MAC-T) in culture.

The effects of Staphylococcus aureus M60 culture supernatant on growth of mammary epithelial cells were tested in vitro. Exposure of MAC-T cells to S. aureus culture supernatant reduced cell proliferation and colony-forming ability because of reduced ability to adhere to plastic. Growth-inhibiting effects of S. aureus culture supernatant were abolished by pretreatment with trypsin. Lysis of S. aureus cells with lysostaphin demonstrated that the inhibition was also present in cell lysates. Treatment of MAC-T cells with culture supernatant from isogeneic mutants of S. aureus M60 that produced either alpha or beta toxins implicated alpha toxin as the main factor inhibiting cell proliferation.

Animals↗
PubMed1994

Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence.

A class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact with the host by binding to soluble host proteins, to matrix proteins or to host cells. They probably have important roles in pathogenicity by allowing bacteria to avoid host defences and by acting as adhesins. Four such proteins of Staphylococcus aureus have been characterised: protein A (immunoglobulin binding protein), fibronectin binding proteins, collagen binding protein and the fibrinogen binding protein (clumping factor). Site-specific mutants are being used to define their roles in pathogenesis in in vitro and in vivo models of adherence and infection.

Bacterial Adhesion↗
PubMed1994

Virulence of coagulase-deficient mutants of Staphylococcus aureus in experimental endocarditis.

A sensitive rat endocarditis model which employed relatively small inocula (< or = 10(4) cfu) was used to examine the role of coagulase in the pathogenesis of infection. Rats with indwelling, intracardiac catheters were challenged intravenously with three strains of Staphylococcus aureus. The virulence of a coagulase-positive parental strain DU5808 was compared in terms of its ID50 for resected vegetations and catheters to that of two coagulase-negative mutant strains (DU5809 and DU5814) which had undergone site-specific mutagenesis. Confidence intervals of infection rates were calculated and comparisons were performed of weights of infected vegetations, bacterial concentrations in vegetations and early mortality rates. From these virulence parameters, it was concluded that there were no differences in virulence among the three strains. The results from this investigation support the previous findings in mouse models of subcutaneous infection and mastitis, which indicated that coagulase production by S. aureus does not appear to function as a virulence factor. Together, these data refute the longheld belief that coagulase is important in the pathogenesis of infection and indicate that other markers of virulence must be operative in diseases caused by the enduring pathogen S. aureus.

Analysis of Variance↗
PubMed1994

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, fully complemented the clumping 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 allowed the weakly clumping 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 cell 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. aureus, particularly in the N- and C-termini.

Amino Acid Sequence↗
PubMed1993

Molecular analysis and expression of the lipase of Staphylococcus epidermidis.

Lipase of Staphylococcus epidermidis 9 was purified from culture supernatant fluid. Two polypeptides (51 and 43 kDa) were detected by SDS-PAGE, of which the 43 kDa polypeptide reacted with anti-lipase serum. The S. epidermidis 9 lipase gene (gehC) was cloned in Escherichia coli and localized to a 2.1 kb sequence by subcloning and transposon mutagenesis. The nucleotide sequence of gehC (2064 nucleotides) was determined and the predicted amino acid sequence of the encoded lipase (77 kDa) identified. A 97 kDa lipase was detected in extracts of E. coli harbouring gehC and in post-exponential-phase culture supernatant fluids of S. epidermidis 9. Data presented indicate that the lipase behaves anomalously during SDS-PAGE and that a pro-lipase is proteolytically processed in cultures of S. epidermidis 9 during growth.

Amino Acid Sequence↗
PubMed1993

Recombination at the coagulase locus in Staphylococcus aureus: plasmid integration and amplification.

The integrating plasmid pCOA18, comprising pUC18 linked to a mutated coagulase (coa) gene from Staphylococcus aureus, and constructed by substituting coa sequences with a tetracycline (Tc)-resistance marker (delta coa::Tcr), was transformed into Staphylococcus aureus RN4220, where it underwent recombination with the chromosomal coa locus. Allele-replacement mutants were recovered at a low frequency directly after transformation. The majority of transformants carried pCOA18 integrated in the chromosome by a single Campbell-type recombination event. The majority of integrants contained tandem repeats of pCOA18 and expressed high levels of resistance to Tc (> 30 micrograms ml-1) compared to the single-copy integrants and allele-replacement mutants (15 micrograms ml-1). Transduction of a single-copy integrant to a Coa+ recipient allowed the cointegrant to be resolved and allele-replacement recombinants to be selected. In addition, growth of a single-copy integrant on high concentrations of Tc (> 30 micrograms ml-1) selected for amplified derivatives at a frequency of 10(-5). It was estimated that up to 19 copies of pCOA18 could occur in a tandem array in the chromosome.

Alleles↗