Yukio Usui

Biochemical Properties of Fibrinogen Binding Protein (Clumping Factor) of the Staphylococcal Cell Surface

The staphylococcal fibrinogen binding protein of a strain of coagulase-negative Staphylococcus aureus was purified 229 fold in terms of the haemagglutination unit compared to the starting material by affinity chromatography on fibrinogen-Sepharose. By polyacrylamide gel electrophoresis in both reduced and unreduced gels, the protein showed one major band and minor bands with relative molecular masses of 62,000, 61,000, and 59,000, respectively. The isoelectric point was between 10.2 and 10.8 determined by isoelectric focusing in polyacrylamide gel. By the agar diffusion test one band was obtained against anti-whole cell rabbit serum. Amino acid analysis of fibrinogen binding protein showed glycine, glutamic acid, lysine, alanine, aspartic acid, and arginine as the major components. Protein A or teichoic acid extracted from the homologous strain did not show fibrinogen binding activity assayed by haemagglutination and anti-fibrinogen binding activity of anti-whole cell Fab. The amount of the fibrinogen binding protein to absorb the activity of anti-whole cell Fab was decreased to 1/60 of that of the starting material. Sheep red blood cells coated with the fibrinogen binding protein agglutinated in 0.001% (w/v) fibrinogen solution.

Platelet aggregation by group B streptococci.

Forty-six strains of group B streptococci (GBS), including various serotypes and non-serotypable strains, were tested for their ability to induce platelet aggregation in human platelet-rich plasma; four strains, all belonging to type III, showed a positive reaction. The characteristics of the reaction were investigated in these four positive strains. Aggregation was dependent on the ratio of bacteria to platelets, being maximal at a ratio of 4.3. Platelet aggregation was inhibited by EDTA (100% inhibition at 3.1 mM), indomethacin (100% inhibition at 10 mM), acetylsalicylic acid (93-100% inhibition at 5.0 mM) and quinacrine (100% inhibition at 0.25 mM). Thus the reaction was cation-dependent and required cyclooxygenase activity. Assays for cytosolic lactate dehydrogenase did not indicate platelet lysis. GBS induced the release of [3H]serotonin, which was maximal (68-78%) at 10 min after the reaction was started. Experiments with gel-filtered platelets suggested that GBS-induced platelet aggregation required both fibrinogen and heat-resistant (56 degrees C, 30 min) serum factors. Type-specific antisera prevented the platelet aggregation activity of heat-killed bacteria, but not of live bacteria. Trypsin digestion of the bacterial cells caused an almost complete loss of the platelet aggregation activity.

Platelet Aggregation by Strains of Enterococci

The platelet aggregation capability of whole cells of Enterococcus faecalis, E. faecium and E. avium was tested. The optimum ratios of bacteria to platelets in E. faecalis (strain SMU‐37), E. faecium (strain SMU‐138) and E. avium (strain SMU‐197) were 1.0, 1.2 and 2.0, respectively. During the platelet aggregation induced by the three strains of enterococci, 65–69% of total serotonin was released. The aggregation was totally inhibited by ethylenediaminetetraacetate (10 mM) and apyrase (1 mg/ml), while no effect was shown by aspirin (10 mM), indomethacin (10 mM) and quinacrine (1 mM). By pretreatment of platelet‐poor plasma with heat (56 C, 30 min) or zymosan, the reactivities with platelets of each strain of species were markedly diminished. These results suggest that enterococci‐induced platelet aggregation was an ion‐dependent, cyclooxygenase‐insensitive event, and plasma component(s) was (were) required for the reaction.

Platelet Aggregation Induced by Strains of Various Species of Coagulase-Negative Staphylococci

Major species of coagulase‐negative staphylococci (CNS) were tested for their ability to induce platelet aggregation in rabbit platelet‐rich plasma (PRP). Among 11 species of CNS tested, a majority of the strains of 10 species of CNS (S. epidermidis, S. simulans, S. capitis, S. hyicus, S. sciuri, S. cohnii, S. xylosus, S. hominis, S. haemolyticus, S. warneri) caused induction of the platelet aggregation and serotonin release, while S. saprophyticus did not show such activity. The addition of aspirin (10 mM) or quinacrine (1 mM) to PRP resulted in no remarkable effect on the platelet aggregation induced by these strains and it was shown that the platelet aggregation did not require arachidonate pathways. Complement system components were shown to be one of the plasma factors required for platelet aggregation by ten strains of each species of CNS. The bacterial substance participating in the platelet aggregation by ten species of CNS tested was indicated to be heat‐stable and trypsin‐resistant, while the activity of a strain of S. epidermidis was susceptible to trypsin.

Characterization of partially purified group B streptococcal clumping factor

Using a strain of group B Streptococci, characteristics of the streptococcal clumping factor was investigated. In contrast to that of Staphylococcus, the streptococcal clumping factor reacted with plasmas from guinea-pig and sheep although staphylococcal clumping factor did not react with them. In addition, streptococcal clumping factor was resistant to heat, sonication, and chemical reagents; it was also degradable by proteolytic enzyme and galactose oxidase. Hydrochloric acid extract of the whole cell was subjected to Sephadex column chromatography and fraction containing streptococcal clumping factor was obtained. This fraction reacted selectively with fibrinogen in human plasma, caused paracoagulation, and the factor was presumed to be an acidic proteinaceous substance.

Human fibrinogen gel formed by the action of a cell surface polysaccharide obtained from a Staphylococcus.

An alkali-stable polysaccharide (called compact-colony forming active substance; substance 1) obtained from the cell surface of a strain of Staphylococcus epidermidis caused gel formation of human fibrinogen, with no release of fibrinopeptides. Substance 1 possessed neither esterase nor caseinolytic activities; no inhibition of gel formation was shown by dinitrofluorophosphate. Heparin and galactose prevented gel formation of fibrinogen with substance 1. With the addition of early- and late-fibrinogen or fibrin degradation products into the fibrinogen sample, no prolongation of the gel formation time was observed. This substance is, therefore, assumed to nonenzymatically induce gel formation with fibrinogen, a process resembling paracoagulation.

Biochemical and Immunological Properties of Cell Surface Teichoic Preparations from Encapsulated Strains of Staphylococcus aureus

Summary The biochemical and immunological properties of cell surface teichoic acid preparations were investigated by using strains Smith diffuse, NS58D, NS41D, and NS68D of Staphylococcus aureus, capsular-types A, B, C, and D, respectively, as determined by the serum-soft agar technique, d-fucosamine, d-galactosamine, taurine, and hydroxyproline were detected in these preparations. The surface teichoic acid preparations from strains Smith diffuse, NS58D and NS41D were of the ribitol-type but the glycerol-type was found in strain NS68D. The major acetylglucosaminyl and acetylgalactosaminyl residues of surface teichoic acid preparations from strains Smith diffuse and NS41D were attached to the polyribitol phosphate by the β-linkage, but attachment for strain NS58D was by the α-linkage, and for strain NS68D the attachment to the polyglycerol phosphate was by both the acetylgalactosaminyl α-and β-linkages. In the agar diffusion test, the teichoic acid preparations of all strains produced a single precipitin line only against hyperimmune rabbit sera prepared from corresponding homologous capsular-type strain; however, no precipitin line was produced against heterologous antisera. These results confirmed the serological classification of the capsular-types determined by the serum-soft agar technique.

Some biochemical properties of the components of Staphylococcus aureus binding to human platelets

The binding properties of Staphylococcus aureus in relation to human platelets were investigated. Protease digestion (pronase E, proteinase K, trypsin), heat treatment (80 degrees C, 30 min), and sonication for 5 min significantly reduced the binding abilities of the staphylococcal cells to 0% (p < .01), 50 +/- 5% (p < .05), and 38 +/- 9% (p < .05), respectively, while mixed glycosidases did not. Inhibition experiments indicated that protein A and various sugars were ineffective. A binding study using biotinylated cell surface fractions extracted from the whole cells of S. aureus indicated that the proteins having apparent molecular weights of 14400 and 16500 estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis were involved in the binding between S. aureus and human platelets.

Extracellular product(s) of Staphylococcus aureus stimulate their own growth

The effect of extracellular products obtained from culture supernatant of Staphylococcus aureus strain Cowan I on the bacterial growth was studied in a synthetic medium. Addition of the extracellular products to a fresh medium stimulated growth already after 2 h of incubation, with an approximately two-fold increase in cell density as compared to an unsupplemented medium, probably by promoting an initiation of growth accompanied by a reduction of the initial lag phase. The growth-stimulating effect was also monitored as an increase of adenosine triphosphate (ATP) in the bacterial culture during the different phases of growth.