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D McDevitt

Publications and source records attributed to D McDevitt.

36 records · Page 2Linked to original sources
PubMed1995

Staphylococcus aureus expresses a major histocompatibility complex class II analog.

Staphylococcus aureus expresses various surface proteins which specifically recognize and bind to different host molecules. We have previously identified a bacterial protein that exhibits a broad specificity and binds to several mammalian extracellular proteins. The gene encoding this bacterial component has now been cloned and sequenced. The deduced protein consists predominantly of six repeated domains of 110 residues. Each of the repeated domains contain a subdomain of 31 residues that share striking sequence homology with a segment in the peptide binding groove of the beta chain of the major histocompatibility complex (MHC) class II proteins from different mammalian species. The purified recombinant bacterial protein bound several mammalian proteins, including recombinant osteopontin, suggesting a protein-protein interaction and also specifically recognized a 15-amino acid residue synthetic peptide. Taken together, these results suggest that the bacterial protein resembles mammalian MHC class II molecules with respect to both sequence similarities and peptide binding capabilities.

Amino Acid Sequence↗
PubMed1995

Typing of Legionella pneumophila serogroup 1 isolates by degenerate (D-)RAPD fingerprinting.

A new method to identify clonal strains of pathogenic bacteria has been developed recently in this laboratory. The method utilizes degenerate random amplified polymorphic DNA primers (D-RAPD) to amplify random fragments in crude bacterial lysates, generating reproducible DNA banding profiles or fingerprints. We use this method to type outbreak and non-outbreak isolates of Legionella pneumophila serogroup 1 from four hospitals near to, and affiliated with the University of Pittsburgh Medical Center. Patient isolates from a large outbreak, and nearly half of the contemporaneous environmental isolates showed the same DNA profile. Other isolates derived from non-outbreak patients showed easily distinguishable profiles. Other Legionella isolates collected between 1984 and 1994 were also analysed by this method. Our studies demonstrate that four strains were common among patient and environmental isolates at the four hospitals. These strains were also found to be different from a limited number of isolates from outside the Pittsburgh area. Because of its speed, simplicity and powerful discriminating ability, we believe that the D-RAPD approach provides epidemiologists and hospital infection control teams with a powerful tool in their efforts in analysing and terminating infection outbreaks.

Bacterial Typing Techniques↗
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↗
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

Coagulase deficiency in clinical isolates of Staphylococcus aureus involves both transcriptional and post-transcriptional defects.

The molecular basis of the non-expression of coagulase was investigated for 14 coagulase-negative isolates of Staphylococcus aureus obtained from different clinical samples. These isolates had typical S. aureus characteristics such as production of clumping factor, DNAase and protein A, but, with one exception, failed to produce detectable amounts of alpha-haemolysin. All 14 strains had DNA homologous to the coagulase gene (coa), but a coa-specific transcript was found in only seven of them. alpha-Haemolysin mRNA was detected in only eight strains without direct correlation to coa-mRNA expression. Thus, coagulase and alpha-haemolysin deficiencies in S. aureus may involve either transcriptional or post-transcriptional alterations although additional regulatory factors may influence the expression of both genes.

Bacterial Toxins↗
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

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↗
PubMed1992

Genetic evidence that bound coagulase of Staphylococcus aureus is not clumping factor.

Staphylococcus aureus Newman cells carry a surface receptor for fibrinogen called clumping factor. The bacteria also express coagulase, an extracellular protein that binds to prothrombin to form a complex with thrombinlike activity which coverts fibrinogen to fibrin. We have confirmed a recent report (M. K. Bodén and J.-I. Flock, Infect. Immun. 57:2358-2363, 1989) that coagulase can bind to fibrinogen as well as to prothrombin and also that a fraction of coagulase is firmly attached to the cell. A mutant with a deletion in the chromosomal coa gene was isolated by allelic replacement. Allelic replacement either was directly selected by electrotransformation of S. aureus R3N4220 with a nonreplicating suicide plasmid, pCOA18, carrying the delta coa::Tcr mutation or occurred after transduction of the integrated pCOA18 plasmid. The coa mutant was completely devoid of coagulase activity but interacted both with soluble fibrinogen and with solid-phase fibrinogen with the same avidity as the parental strain. This strongly suggests that the bound form of coagulase is not clumping factor and is not responsible for the adherence of S. aureus Newman to solid-phase fibrinogen. The fibrinogen binding determinant of coagulase was located in the C terminus of the protein, by analyzing truncated fusion proteins, in contrast to the prothrombin-binding region which was located in the N terminus.

Bacterial Adhesion↗
PubMed1988

Esophageal duplication cyst as a cause of choke in the horse.

A mare was examined for episodic choking and an abscess in the perilaryngeal region. One month before referral, the mass progressively enlarged, causing esophageal choking. An extraluminal compression of the esophagus 90 cm from the external nares was found on endoscopy and contrast radiography. Serosanguinous fluid aspirated from the mass contained keratinized squamous cells and neutrophils, compatible with an esophageal cyst. Surgical exploration was performed, with incomplete surgical excision. The remaining portion of the cyst was marsupialized and treated locally with a 2% inorganic iodine solution. Further episodes of esophageal choke in this horse have not been reported.

Animals↗