Isolation and characterization of hyaluronidase from Streptococcus uberis.

All tested cultures of Streptococcus uberis produced free hyaluronidase. Hyaluronidase could be isolated by ammonium sulfate precipitation and was further purified by chromatography on DEAE-cellulose, gelfiltration on ultragel ACA44 and isoelectric focusing. The purification factor was estimated to be 1689. The purified hyaluronidase had an isoelectric point at pH 4.9 and a molecular weight of approximately 54000 D. It showed maximal enzyme activity at pH 6.0 and 45 degrees C. The Michaelis constant was estimated to be 7.0 X 10(-2) mg/ml. Hyaluronidase activity was stimulated by Ca++, Mg++, Mn++, Co++, Li+, and K+ and inhibited by Zn++ and Cd++ at final concentrations of 10 mmol/l, respectively.

Purification of oligomeric staphylococcal alpha-toxin by affinity chromatography on digitonin-sepharose.

An effective concentration of alpha-toxin from Staphylococcus aureus Wood 46, directly from the culture supernatant, could be achieved by adsorption on digitonin-sepharose and elution with 3 mol/l sodium thiocyanate (NaSCN). The toxin was further purified by gelchromatography. The purified product yielded 1 single protein band upon SDS-polyacrylamide electrophoresis. It was nonhemolytic, but reacted with anti-alpha-toxin under complement fixation. Dialysis against 0.14 mol/l NaCl with hydrophobic amino acids partially reactivated the alpha-hemolytic activity of the toxin. Ultracentrifugal analysis yielded sedimentation coefficients for the purified toxin of approximately 3,7 S when dissolved in 3 mol/l NaSCN and of about 12 S after dialysis against 0.14 mol/l NaCl (Table 1). The spontaneous oligomerization of the alpha-toxin during dialysis against 0.14 mol/l NaCl possibly resulted from a change in configuration induced by its adsorption to digitonin-sepharose.

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 X 10(-9) M for Wood 46 and 9.3 X 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.

[Clumping-factor-reactions of Staphylococcus aureus of different origin in plasma- and fibrinogen-preparations(author's transl)]. / "Clumping-Factor"-Reaktionen von Staphylococcus aureus verschiedener Herkunft in Plasma- und Fibrinogenpräparationen.

The clumping-factor (CF) test in microtiter-plates proved suitable for the preliminary identification of Staphylococcus aureus, provided a "susceptible" plasma- or fibrinogen-preparation had been applied (Table 1). Thus, all of 100 S. aureus-cultures from humans gave strongly positive CF-reactions with plasma from humans, rabbits, pigs, cattle, and dogs as well as with fibrinogen-solutions from humans and cattle. Equally, all of 100 S. aureus-cultures from cattle clumped in plasma from pigs, cattle and humans. All of the 50 S. aureus-cultures from dogs were CF-positive in plasma from dogs, humans and horses. Only part of the CF-positive S. aureus-culture reacted with plasma from sheep and goats.

[Production of coagulase from Staphylococcus aureus of different origin (author's transl)]. / Koagulasegewinnung von Staphylococcus aureus-Kulturen unterschiedlicher Herkunft.

Cultivation of Staphylococcus aureus in a fermenter significantly improved production of coagulase (Fig. 1,2,3). Optimal coagulase-activity was reached in the culture supernatant after inoculation with 8 x 10(9) colony-forming S. aureus per ml of brain heart infusion broth and 20 min at 37 degrees C. At this time most of the other enzymes and toxins (particularly protease and staphylokinase) had not been formed or only in small amounts. Characterization of coagulases from respectively 5 S. aureus-cultures from humans, cattle and dogs revealed molecular weights between 56800 and 71000, and isoelectric points between 5.4 and 6.4 (table 1).

[Antigenic effects of Staphylococcus aureus with modified surface-structures (author's transl)]. / Antigene Wirkungen von Staphylococcus aureus mit modifizierten Oberflächenstrukturen.

Antigenic effects of preparations from Staphylococcus aureus ATCC 31243 and Rd 4 (Table 1) with partially different activities of "clumping factor" (CF) and protein A (PA) were compared (Table 2). The (3 h, 60 degrees C) heat-inactivated (hi) as well as the guanidinium-chloride-extracted (gu) staphylococci retained full activity of CF and PA. Acetylation with N-acetylimidazole inactivated completely CF and partially PA. Treatment of the staphylococci with pronase (pron) removed CF and PA. The hi-, gu- and pron-preparations of both S. aureus strains had similar antigenic properties which differed from those of the acetylated staphylococci, when antisera against all preparations were examined in the agglutination (Table 3) and "growth"-agglutination-tests (Fig 1). Acetylation apparently affected the staphylococcal surface to a larger extent than the other procedures applied in these studies.

[Protein A-activity of Staphylococcus hyicus in comparison to protein A of Staphylococcus aureus (author's transl)]. / Protein A-Aktivität von Staphylococcus hyicus im Vergleich zu Protein A von Staphylococcus aureus.

Protein A (PA)-Activity was demonstrated in 44 (93.6%) of 47 Staphylococcus hyicus-cultures. PA from S. hyicus, as well as PA from S. aureus, could be released by extraction with concentrated formic acid or by treatment of the staphylococci with lysostaphin. PA was also demonstrable in the culture medium. Purification of PA from S. hyicus and S. aureus could be achieved by affinity-chromatography on IgG-Sepharose. In Ouchterlony-tests both PA-preparations gave single lines of identity with normal sera from man, pig, dog and guinea pig (Fig. 1). Immunoelectrophoretic analysis indicated a significantly faster migration towards the anode for PA from S. hyicus than PA from S. aureus (Fig. 2). Isoelectric focusing revealed maximal activity for PA from culture supernatant of S. hyicus about pH 4.3 and for that of S. aureus at pH 5.0 (Fig 3). Gelchromatographic experiments indicated a lower molecular weight for PA from S. hyicus than for PA from S. aureus (Fig. 4).

[Lipase and phospholipase C from Staphylococcus aureus of different origin. I. Determination and occurrence (author's transl)]. / Lipase und Phospholipase C von Staphylococcus aureus verschiedener Herkunft.I. Nachweis und Vorkommen.

Lipase and phospholipase C from Staphylococcus aureus of different origin were demonstrated qualitatively by agar diffusion on tributyrin- and lecithin agar. On test media with either 0,3% Na-azide or 0,3% KCN lipase-activity was not inhibited, phospholipase C, on the other hand, completely blocked (Table 1, Fig. 2). In this manner a tentative differentiation was possible between lipase and phospholipase C. For the quantitative determination of lipase the hydrolysis of p-nitrophenyl palmitate proved to be most useful (Fig. 1). S. aureus-cultures of human origin produced more often and more actively lipase and phospholipase C than those from cattle (Table 2).

[Lipase and phospholipase from Staphylococcus aureus of different origin. II. Purification and characterization (author's transl)]. / Lipase und Phospholipase C von Staphylococcus aureus verschiedener Herkunft. II. Reinigung und Charakterisierung.

Lipase and phospholipase C from Staphylococcus aureus could be isolated by gel filtration on Sephacryl S 200 (Fig. 1a, b) and completely separated by refiltration under the same conditions. Isoelectric focusing gave maximal enzyme-activities for lipase at pH 8.6 and 9.5 and for phospholipase C at pH 7.4 (Fig. 2). Thin-layer chromatography revealed that the reaction products in lecithin agar of the phospholipase C-preparations from S. aureus and Bacillus cereus were identical (Table 1).

[Demonstration of species-specific teichoic acids in staphylococcal species with reference to protein A activity (author's transl)]. / Nachweis artspezifischer Teichonsäuren bei Staphylokokken unter Berücksichtigung der Protein A-Aktivität.

Serologically different teichoic acids could be demonstrated as polysaccharide antigens in staphylococcal species by immunodiffusion (Fig. 1) and counterimmunoelectrophoresis (GSE, Fig. 2). Staphylococcus aureus contained polysaccharide A, S. epidermidis polysaccharide B, S. saprophyticus polysaccharide A beta C, and S. hyicus polysaccharide C (Table 2). These polysaccharides were specific for staphylococcal species and could not be found in micrococci. The antigen preparations for the GSE were autoclaved suspensions of the staphylococcal and micrococcal cultures. The specific antisera (Table 1) were obtained after absorption with pronase-treated staphylococcal reference strains. Treatment with pronase removed protein A from the absorbing staphylococci. In this manner the "nonspecific" loss of specific antibodies was prevented. This would have occurred by the attachment of the Fc-component of immunoglobulin G to protein A of S. aureus. The precipitin-lines contained the polysaccharide-antigens and not protein A.

[Improved method for the demonstration of protein A of Staphylococcus aureus (author's transl)]. / Verbessertes Verfahren zum Nachweis von Protein A bei Staphylococcus aureus.

Protein A (PA) could be extracted completely from Staphylococcus aureus by treatment with concentrated formic acid. This led to the development of a semi-quantitative determination of PA by hemagglutination (fig. 1). The treatment with formic acid yielded PA more effectively than the commonly used extraction by boiling (table 1). It could be conducted directly on a loopfull of staphylococci obtained from blood agar. It required no additional cultivation in a fluid medium. Most suitable for the hemagglutination was a commercial preparation of Rh-positive human erythrocytes, blood group O, loaded with Rh-antibodies from humans. This relatively stable preparation had also a higher susceptibility for PA in the slide-test and served for a better detection of PA-positive staphylococci (table 2).

[A method for production of Coxiella burnetii antigen and cell walls by guanidiniumchloride extraction (author's transl)]. / Eine Methode zur Gewinnung von Antigen bzw. Zellwänden von Coxiella burnetii mittels Guanidiniumchlorid-Extraktion.

By use of 6-molar guanidinium chloride a potent Coxiella burnetii antigen could be produced for diagnostic purposes from infected yolk sacs, with little technical expense. This treatment did not only a cause remarkably purifying effect (Tab. 1) but also the extraction of soluble cytoplasmic substance. From guanidinium chloride-treated suspensions a highly purified and uniform suspension of cell walls could be separated by Saccharose Density Gradient centrifugation (Fig. 2). Guanidine extracted organisms retained their full antigenic potential with respect to Phase I and Phase II and lacked anticomplementary activity. Such preparations can be used for serological tests like complement fixation reaction or Enzyme Linked Immunosorbent Assay and are particularly suitable for biochemical studies of Phase antigens of Coxiella burnetii.

Determination of IgG, IgM, and IgA antibodies to Mycoplasma pneumoniae by an indirect staphylococcal radioimmunoassy.

An indirect staphylococcal radioimmunoassay (SRIA) has been developed for determination of M. pneumoniae antibodies. This test allows the detection of antibodies in various immunoglobulin (Ig) classes similar to the previously described radioimmunoprecipitation test (RIP). SRIA has two advantages over RIP: first, it uses 100-fold less anti-Ig reagents than RIP; second, bound can be separated from unbound antigen more easily by the relatively heavy staphylococci. SRIA antibodies, belonging to the IgA class of Ig, could be detected in nasal secretions of volunteers infected intranasally with ts H43 of M. pneumoniae. In sera of patients with M. pneumoniae pneumonia antibodies to the IgG or the IgM class of Igs could be determined separately. This is especially important for an early diagnosis of M. pneumoniae disease.

A radioimmunoassay for tetanus antibodies using protein A - containing Staphylococcus aureus.

To measure tetanus antibodies a trace amount of 125I-labeled tetanus toxin is mixed with appropriate dilutions of human serum or blood. The labeled antigen-antibody complexes are adsorbed to heat-killed staphylococci (Cowan I) via their surface protein A. The radioactivity of the washed solid phase is a function of the initial antibody concentration. The test allows the measurement of 6 X 10(-0) U of tetanus antitoxin in a volume of 0.03 ml. In order to avoid possible interferences, serum has to be diluted 20-fold before use. Taking that into account, the real border limit of sensitivity is 4 X 10(-3) U/ml serum. Antibodies may be measured in serum, in plasma, and even in heparinized blood. As to its sensitivity, the test compares well with the toxin neutralization procedure. It is superior to the previous radioimmunologic, enzymoimmunologic, and hemagglutination techniques with respect to sensitivity and reproducibility. It reflects the values obtained in the toxin neutralization test better than the other in vitro procedures, as shown by parallel assays of 17 sera.

[Purification of protease from staphylococcus aureus (author's transl)]. / Reinigung einer Protease von Staphylococcus aureus.

380 (80%) of 475 Staphylococcus aureus cultures isolated from humans, cattle and dogs were proteolytically active either on casein or gelatin or both (table 1). Protease-activity could also be demonstrated in experimental body-cavities of rabbits (fig. 1). The enzyme-activity was estimated with azocasein. Protease from S. aureus, M 135 precipitated from the culture supernatant with ammonium sulfate at 65% saturation (table 2). It was purified by 2 filtrations on Ultrogel AcA 44 (fig. 2,3) and subsequent isoelectric focusing between pH 3.5-7.0 (fig. 4). The purified protease yielded only 1 line in the SDS-polyacrylamidegel-electrophoresis, in the gelatin-polyacrylamidegel-electrophoresis and in the double immuno-diffusion test (fig. 5). Its isoelectric point was at pH 4.6, and its highest proteolytic activity between pH 7.5-8.3. The molecular weight was estimated by SDS-polyacrylamidegel-electrophoresis to be near 29.000. The protease-activity was completely inhibited in the presence of EDTA, partially inhibited by Cu2+ and Zn2+ and increased by Mn2+ (table 3).