Strain Wood Research Articles

Effect of metalloproteinase from Staphylococcus aureus on in vitro stimulation of human lymphocytes

Metalloproteinase (MP) produced by the majority of Staphylococcus aureus strains exerts, in a wide concentration range (0.1-100 micrograms/ml), no cytotoxic action on mononuclear leukocytes of human peripheral blood. The enzyme itself does not appreciably stimulate proliferation and differentiation of lymphocytes in culture, but affects the stimulation of both T and B lymphocytes by polyclonal activators. The action is dose-dependent. High doses of MP (100 micrograms/ml) lower the blastic transformation after stimulation with Con A, SpA, NDCM, S. aureus strain Wood 46 and with suboptimal doses of PWM. Optimal concentrations of the enzyme potentiate the stimulation of lymphocytes by PWM, PHA, S. aureus strains Cowan 1 and Wood 46, and by NDCM. The same potentiation effect was achieved whether the enzyme was added concurrently with the mitogen or 18 h later. This implies that the beginning of cell activation is not affected. A high MP concentration decreases the production of Ig in culture after stimulation with PWM whereas lower concentrations of MP enhance this production. Production of Ig after stimulation with NDCM and Wood 46 is decreased by MP. The possible action of exoproteinase from S. Aureus on the immune response during infection is discussed.

Cryptic α‐toxin gene in toxic shock syndrome and septicaemia strains of Staphylococcus aureus

The majority of clinical isolates of Staphylococcus aureus that produce toxic shock syndrome toxin-1 (TSST-1) fail to express alpha-toxin, despite having a copy of the hla gene in the chromosome. The hla gene was cloned from an Hla- TSST-1+ strain, Todd 555, which had been isolated from a case of toxic shock syndrome in the USA. Of the 630 bases of the Todd 555 gene sequenced, 46 differed from the hla gene sequence of strain Wood 46. The defect in alpha-toxin expression was shown to be due to a nonsense mutation which converted a CAG glutamine codon in the equivalent position in the functional Wood 46 sequence to a TAG stop codon. The same mutation was present in the hla gene cloned from a human septicaemia strain (V37) isolated in Dublin. The nonsense mutation of Todd 555 was suppressed by the supE44 mutation in Escherichia coli resulting in haemolytic activity in cell lysates. Hybrid hla genes were formed by splicing fragments of hla from Todd 555 and Wood 46. Expression of one such chimaeric hla gene in S. aureus demonstrated that the Todd 555 hla gene has a functional agr-regulated promoter. The silent hla gene may be a cryptic gene in S. aureus.

Staphylococcus aureus α-Toxin Crystals on Lipid Layers: Effects of Trypsin Treatment

α-toxin is a lethal, dermonecrotic, cytotoxic, and hemolytic protein which is secreted by most strains of Staphylococcus aureus . The biological effects are brought about by pertubation of membrane structures making them permeable to ions and small molecules. The structural basis for the mode of action is not known in detail. One hypothesis is that an oligomeric form of the toxin, observed both biochemically and by electron microscopy, circumscribe a hydrophilic pore through which transport can occur. Recent functional studies have demonstrated that oligomerization is a necessary but not a sufficient criterion for permeabilization. For example, trypsin treated α-toxin was, despite its lack of membrane damaging activity, able to form 200 kDa aggregates on mouse adrenocortical (Yl) tumor cells. In this work we have compared the structure of trypsin treated α-toxin with that of the intact toxin.α-toxin was purified from S. aureus strain wood 46 by isoelectric focussing and gel filtration. Tryptic toxin fragments (18 and 17 kD as determined by SDS-PAGE) were generated by digestion of native α-toxin (1 mg in 1.5 ml Tris buffered saline) with 30μg trypsin for 4 h at 37°C, pH 8.0. The reaction was terminated by the addition of 30μg lima beam trypsin inhibitor. Toxin specimens were applied to lipid layers pre-formed on carbon support films. Electron microscopy was performed after negative staining either with 1% Na-PTA (pH 7.0) or with a mixture of Na-PTA and glucose. Subsequent image processing of large coherent crystalline arrays produced correlation averaged projection maps. A three-dimensional model was obtained by collecting and combining data from tilted views.

In vitro studies of haemolysis by some staphylococci grown in chemically defined media.

Staphylococcus aureus (five strains) and Staph. epidermidis (one strain) have been evaluated for comparative growth and haemolysin titre in both brain heart infusion (BHI) and in developed, nutritionally adequate, chemically defined media (CDMs) varying only in amino acid composition. The ability to show a particular haemolytic profile was strain-dependent and the haemolytic titre (HU50/ml) was both strain- and medium-dependent. Highest titres of both alpha and beta type haemolysins were obtained in BHI. Maximum titres were in general detected in the late exponential phase in both CDMs and BHI. Titres declined during the stationary phase in CDMs. Staphylococcus epidermidis produced a delta-type haemolysis profile on BHI-based blood agars, but only rabbit blood was sensitive in agars based on a developed, chemically defined medium (CDM/A; 13 amino acids) in which all six staphylococci grew. The addition of yeast extract to CDM/A increased alpha haemolysin titre, but suppressed beta haemolysin formation; beta haemolysin was, however, detected in yeast extract/phosphate-buffered saline. Strain Wood 46 degraded haemoglobin, but only in (initially) whole blood; red blood cell-free haemoglobin-rich plates (BHI) were unaffected during growth. A novel haemolytic profile is described for Staph. aureus NCTC 8532 growing on blood agars based on CDM/A and may relate to the production of methaemoglobin during haemolysis.

Inhibition by Immunoglobulins ofStaphylococcus aureusAdherence to Fibronectin-Coated Foreign Surfaces

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. Using a previously described in vitro assay, we show that promotion of S. aureus adherence by surface-bound fibronectin, adsorbed on polymethylmethacrylate (PMMA) coverslips, is antagonized by antistaphylococcal antibodies present in immunoglobulin G (IgG) purified from human plasma. Among the different organisms tested, the protein A-deficient strain Wood 46 of S. aureus was the most strongly inhibited by purified IgG or whole serum dose-dependently. Bacterial adherence was not influenced by preincubating fibronectin-coated PMMA with either purified IgG or whole serum. However, inhibition of bacterial adherence was directly related to the extent of IgG binding to S. aureus Wood 46. When F(ab')2 fragments of purified IgG were tested in the adherence assay, they could also reduce the interaction between S. aureus Wood 46 and fibronectin-coated PMMA. Two other staphylococcal strains were also tested in the adherence inhibition assay: Whereas the protein A-rich strain Cowan I of S. aureus was moderately inhibited by purified IgG or whole serum, S. epidermidis KH 11 was not at all inhibited by IgG which bound poorly to the bacterial cells. This study has demonstrated that bacterial coating by humoral factors, and specifically IgG, may influence significantly subsequent adherence of S. aureus to surface-bound fibronectin.

Study of staphylococcus aureus teichoic acid immunodominant site by help of synthetic haptens

The β ribitol teichoic acid was extracted and purified from Staphylococcus aureus strain Wood 46 and chemically and immunologically characterised. Rabbit antiserum was prepared against formalin killed Staphylococcus aureus cells. Liquid phase immunoprecipitation of the β ribitol teichoic acid-anti- Staphylococcus aureus serum system was studied by laser nephelometry. Various mono- and disaccharides (N-acetyl-glucosamine-ribitol with α- or β-linkage and N-acetyl-glucosamine-ribitol-phosphate with β-linkage) were prepared by organic synthesis, reproducing part of the ribitol teichoic acid molecule. Inhibition by those mono- or disaccharides of the precipitation of the β-ribitol teichoic acid- Staphylococcus aureus antibodies system was studied quantitatively by determining inhibitory ratio of each inhibitor. Glucose, ribitol and glucosamine were weak inhibitors whereas N-acetyl-glucosamine was a better one, stronger than disaccharide with an α-linkage. The β linked disaccharide and β-methyl-N-acetyl-glucosamine gave comparable inhibition and both compounds were effective inhibitors. The most potent inhibitor was phosphorylated β-linked disaccharide which inhibited 25% more than the same disaccharide without phosphorus. Thus, the function of phosphorus in Staphylococcus aureus β ribitol teichoic acid recognition by antibodies was demonstrated.

Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb + and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.

Synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity

Several strains of Staphylococcus aureus have been found to secrete proteases that activate infectivity of influenza virus by proteolytic cleavage of the hemagglutinin. The enzymes of the bacterial strains Wood 46 and M 86/86 have been characterized in some detail and were found to be serine proteases. In their substrate specificities and inhibitor sensitivities they proved to be similar to, but not identical with trypsin and plasmin. The hemagglutinin of an individual virus strain could be cleaved by the proteases of some but not all staphylococcal strains, and a given enzyme could cleave only some but not all hemagglutinins analyzed. When mice were coinfected intranasally with the appropriate strains of influenza virus and S. aureus, the hemagglutinin was readily activated allowing multiple cycles of virus replication in the lung. Under these conditions, the animals came down with a fatal disease exhibiting extended lesions in the lung tissue. In contrast, after infection with virus or bacteria alone, there were no significant pathological changes. When the staphylococcal strain did not contain a protease that was able to activate the hemagglutinin of the coinfecting virus strain, the animals did not exhibit disease. These observations demonstrate that coinfecting bacteria can play an essential role in the development of influenza pneumonia by providing a protease suitable for cleavage activation of the hemagglutinin.

Role of bacterial exopolymers and host factors on adherence and phagocytosis of Staphylococcus aureus in foreign body infection.

Using a previously developed guinea pig model of foreign body infection, we examined ultrastructural and functional surface alterations of Staphylococcus aureus strain Wood 46 during the early phase of infection. Exopolymer-free bacteria were prepared and inoculated into subcutaneously implanted tissue cages. After three hours, the bacteria showed abundant capsular and intercellular exopolymers, which were visualized by transmission electron microscopy. Exopolymers were also produced by S. aureus exposed in electron microscopy. Exopolymers were also produced by S. aureus exposed in vitro to fluid from the tissue cage. In contrast, human serum albumin prevented exopolymer production by S. aureus. The influence of exopolymers on the susceptibility of S. aureus to ingestion and phagocytic killing by neutrophils was tested in vitro and found to be negligible. Furthermore, adherence of S. aureus to fibronectin-coated surfaces was unaffected by the presence or absence of exopolymers. Thus, in our experimental model, exopolymers are produced early during the onset of infection, but they have little impact on adherence and phagocytosis.

Specific attachment of Staphylococcus aureus to immobilized fibronectin.

Staphylococcus aureus cells have been shown to possess surface-associated proteins with affinity for soluble fibronectin. We have investigated the ability of these surface proteins to mediate attachment to immobilized fibronectin and collagen. Attachment was quantified by determination of bacterial ATP in a bioluminescence assay. The ability to attach to fibronectin- or collagen-coated plastic surfaces was investigated for four S. aureus strains: Cowan 1, Newman, SA113(83A), and Wood 46. Cells from the different strains varied in their attachment properties, but all cells except those of strain Wood 46 attached readily to substrates coated with fibronectin. Only cells from strain Cowan 1 attached reproducibly to collagen-coated substrates in the absence of fibronectin. The attachment of cells from strain SA113(83A) to fibronectin-coated surfaces was shown to be dependent on time, fibronectin concentration, and bacterial growth phase. Soluble fibronectin or NH2-terminal fibronectin fragment (Mr, 29,000) disturbed the attachment to surfaces coated with fibronectin bound to denatured collagen type I. The attachment process to such substrates was also effectively inhibited by preincubating the substrate with fibronectin-binding proteins isolated from S. aureus Newman and SA113 (83A) and purified with affinity chromatography.

Escherichia coli strains of serogroup O139 isolated from oedema disease of swine bind fibronectin

15Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with125I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding thanStaphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding thanS. aureus Cowan 1 cells.E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells ofS. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.

Escherichia colistrains of serogroup O139 isolated from oedema disease of swine bind fibronectin

15 Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with 125I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding than Staphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7 E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding than S. aureus Cowan 1 cells. E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells of S. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.

Staphylococcus aureus-induced suppression of contact sensitivity in mice: Suppressor cells elicited by polyclonal B-cell activation are regulated by idiotype-anti-idiotype interactions

Staphylococcus aureus strain Cowan I, a strong polyclonal B-cell activator (PBA), inhibited contact sensitivity to oxazolone in mice when administered 24 hr before sensitization. This suppression was mediated by idiotype-positive (Id +) B lymphocytes, which arose very early during the sensitization process and induced anti-Id B cells. These cells were found at Day 3 of the sensitization process and exerted their effect by activating antigen-specific suppressor T lymphocytes, which affected the efferent phase of the immune response. S. aureus strain Wood 46, which lacks of the ability to act as a PBA, was unable to inhibit contact sensitivity. These results indicate that PBA may play an important role in the regulation of cell-mediated immune reactions.

Immunoglobulins in the hyperimmunoglobulin E and recurrent infection (Job's) syndrome. Deficiency of anti-Staphylococcus aureus immunoglobulin A.

Patients with the hyperimmunoglobulin E and recurrent infection syndrome (HIE) characteristically have frequent skin and respiratory infections caused by Staphylococcus aureus. We have developed a set of enzyme-linked immunosorbent assays that use whole S. aureus (Wood's strain) immobilized on 0.22-micrometers filters and highly specific, affinity-purified enzyme conjugates of goat anti-human IgE, anti-human IgD, anti-human IgG, anti-human IgA, and anti-human IgM. These reagents were used to determine S. aureus-specific immunoglobulin (Ig) levels. As previously published, 10 patients with HIE had markedly higher levels of anti-S. aureus IgE than did 5 patients with eczema and recurrent superficial S. aureus infections (P less than 0.001). The HIE patients were also found to have a deficit of anti-S. aureus serum IgA as compared with 12 normal subjects, 12 patients with chronic granulomatous disease, 5 patients with chronic eczema and recurrent superficial S. aureus infections, and 3 patients with the Chediak-Higashi syndrome (P less than 0.01 for each comparison). In addition the HIE patients had an excess of anti-S. aureus IgM as compared with normal subjects (P less than 0.01). An expected excess of anti-S. aureus IgG was absent. These abnormalities cannot be explained by variations of total serum Ig levels or by a general inability to produce antigen-specific IgA because levels of naturally occurring IgA antibody against Escherichia coli lipopolysaccharide and the antigens of the pneumococcal vaccine are normal. Parotid saliva from patients with HIE contained less salivary IgA per milligram of protein (P less than 0.01) and less salivary anti-S. aureus IgA per milligram of protein (P less than 0.05) than did normal controls. The incidence of infection at mucosal surfaces and adjacent lymph nodes correlated inversely with serum anti-S. aureus IgA (r = -0.647, P = 0.034), serum anti-S. aureus IgE (r = -0.731, P = 0.016), total serum IgE (r = -0.714, P = 0.020), and total serum IgD (r = -0.597, P = 0.049). These findings are evidence of a previously undescribed immunoregulatory defect in patients with HIE, which may contribute to the increased susceptibility to infection in this syndrome.

Characterization of immunoglobulin G binding to Staphylococcus aureus strain Wood 46

Protein A (PA)-activity was detected in Staphylococcus aureus strain Wood 46 which had been considered to be PA-negative. This staphylococcal strain bound 28% of 125I-labelled IgG, compared with 89% by strain Cowan I. The binding activities of both S. aureus strains were saturable, time-dependent and specific. The dissociation constants of 1.6 × 10 −9 M for Wood 46 and 9.3 × 10 −8 M for Cowan I indicated a similar affinity for human IgG in both strains. The number of IgG-binding sites were estimated to be 16,970 for Wood 46 and 41,200 for Cowan I. Exposure to heat and ultrasonication reduced PA-activities of strain Cowan I, but not that of strain Wood 46. Extraction of the staphylococci with guanidine and formic acid resulted in a reduction of IgG-binding activities only in strain Wood 46. Photooxidation, trypsinization and lysozyme treatment also diminished IgG-binding of strain Wood 46 to a larger extent than that of strain Cowan I. Extracellular PA from S. aureus strains Wood 46 and Cowan I could be purified by affinity chromatography on IgG-sepharose. The purified PA preparations gave single protein bands upon SDS-polyacrylamide gel electrophoresis. Their molecular weights were 42,000 and their isoelectric points approximated 5.0.

Foreign body infection: role of fibronectin as a ligand for the adherence of Staphylococcus aureus.

Foreign bodies made of polymethylmethacrylate coverslips were implanted subcutaneously into guinea pigs, were explanted four weeks later, and were tested for in vitro adherence of Staphylococcus aureus strain Wood 46. In the presence of serum, the level of staphylococcal adherence to explanted coverslips was 20 times higher than that of adherence to unimplanted coverslips. Adherence to explanted coverslips was caused by fibronectin deposits on the foreign body surface and was inhibited in a dose-related fashion by specific antibodies to fibronectin.

Inhibition of secretion of staphylococcal alpha toxin by cerulenin.

Secretion of alpha toxin by Staphylococcus aureus strain Wood 46 was preferentially inhibited by cerulenin, an antibiotic that stops fatty-acid synthesis by inhibiting beta-keto acyl acyl carrier-protein synthetase. At the concentrations used, cerulenin had a negligible effect on cell growth and total protein synthesis, but reduced lipid synthesis by 50%. Extracellular and membrane-associated alpha toxin was absent in cultures treated with cerulenin, but toxin formation was resumed after either removal of the antibiotic or addition of exogenous fatty acids. The apparent absence of toxin precursor in membranes of inhibited cells favours inhibition at an earlier stage in toxin synthesis.

Generation of human C3a, C4a, and C5a anaphylatoxins by protein A of Staphylococcus aureus and immobilized protein A reagents used in serotherapy of cancer.

Protein A (SpA) alone or immobilized on bacteria (e.g., Cowan strain I), collodion charcoal, or on Sepharose have been used in serotherapy of cancer in humans and experimental animals. Because SpA forms complexes with IgG that can activate complement, and the physiologic response during treatment often involves hypocomplementemia and reactions that are similar to those induced by anaphylatoxins, we used sensitive and specific radioimmunoassays to test the ability of SpA reagents to generate C3a, C4a, and C5a from human serum. The yield of anaphylatoxins depended on the dose of SpA, with the maximum generation of C3a (47 to 55 micrograms/ml) and C5a (1.4 to 1.9 micrograms/ml) being produced with levels of SpA that were maximally precipitated from serum. Maximum C4a levels (up to 15 micrograms/ml) were obtained at concentrations of SpA equal to or greater than the dose required to give optimal precipitation. The maximum concentrations of anaphylatoxins correspond to essentially quantitative conversions of C3 to C3a, C4 to C4a, and 40% of C5 to C5a after correction for levels found in serum incubated in pyrogen-free saline. Preformed insoluble complexes prepared from either serum or monomeric IgG also were capable of generating anaphylatoxins in fresh whole serum up to levels approximately equal to those observed in serum treated directly with an optimal amount of SpA. The preformed complexes from serum or IgG generated similar high concentrations of anaphylatoxins when carried through four sequential incubations with fresh serum, and complexes that contained approximately 1 microgram SpA were still active. Preincubating the insoluble complexes with chicken anti-SpA serum did not alter their activity. Incubation of serum with collodion charcoal coated with SpA, in a system that models the perfusion technique used to treat cancer, produced complexes that generated significant levels of C3a compared with levels found in serum passaged over albumin charcoal or in untreated serum. The C3a levels in serum from the albumin collodion charcoal were not significantly different from those found in untreated serum. Similar amounts of C3a, C4a, or C5a were observed in serum incubated with differing numbers of bacteria representing a strain of S. aureus rich in cell bound SpA (Cowan strain I) or a strain (Wood 46) deficient in SpA. This suggests that in intact bacteria, cell wall factors other than SpA (e.g., peptidoglycan) are predominantly responsible for generating anaphylatoxins.(ABSTRACT TRUNCATED AT 400 WORDS)

Complexity of lectin-mediated reactions in bacteria-induced histamine release.

We have earlier suggested that bacteria-induced histamine release is caused by different mechanisms, including allergic and non-immunological mechanisms, and that the latter probably depends on lectin-mediated reactions. Two possibilities of lectin-mediated reactions were examined in this study, bacterial surface lectins bind to sugars on the basophil cell membrane leading to histamine release, and the reverse reaction where bacterial aminosugars react with lectins on the basophil cell surface. In the bacterial histamine release caused by the Staph. aureus strain Wood 46 it was possible to demonstrate a reverse reaction, but not a bacterial lectin-mediated reaction. The reaction seems to be complex, as lower concentrations of sugars might potentiate the release of histamine by binding to the target cell or bacteria, while the release is inhibited by higher concentrations.

Cell surface hydrophobicity ofStaphylococcus aureus measured by the salt aggregation test (SAT)

Well-defined laboratory strains as well as 72 clinical strains ofStaphylococcus aureus isolated from bovine mastitis were investigated for surface hydrophobicity by the salt aggregation test (SAT).Staphylococcus aureus strain Cowan 1, rich in protein A and fibronectin-binding surface proteins, was found to show high surface hydrophobicity, whereas strain Wood 46, deficient in these surface proteins, showed low surface hydrophobicity. SAT showed a significant difference in surface hydrophobicity (P<0.001) between protein A-positive and A-negative strains measured by ϰ2-test analysis. Comparison of SAT values with results obtained from hydrophobic interaction chromatography (HIC) showed a good correlation (P<0.025). A high-level protein-A-producing mutant (SA 113prA-3) showed increased surface hydrophobicity as compared with the parent strain (SA 113), whereas ten protein-A-negative mutants showed low surface hydrophobicity in SAT. Of the 72 clinical isolates tested by SAT, 47 (65%) showed autoaggregating properties, i.e., the strains aggregated even in isotonic buffers. Tween 80 (1% vol/vol) and ethylene glycol (50% vol/vol) prevented autoaggregation of some hydrophobic strains aggregating in phosphate-buffered saline. However, 2M of a chaotropic agent (NaSCN) was more efficient in preventing autoaggregation of the strains tested. Heating of cell suspensions to 80°C or 100°C as well as trypsin andStreptomyces griseus protease treatment generally caused a decrease in the cell surface hydrophobicity. This indicates that protein A, fibronectin-binding proteins, and probably other as yet unidentified proteins contribute to the high surface hydrophobicity of most strains isolated from bovine mastitis.