Binding of radiolabeled Staphylococcus aureus delta-toxin to human erythrocytes.

The addition f [3H]isoleucine to a chemically defined medium resulted in the production of delta-toxin (DT) with a high specific radioactivity (0.47 microCi/mg). The purified tritium-labeled toxin ([3H]DT) was found to migrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with a molecular weight of 1,600. Upon electrofocusing, [3H]DT yielded one major peak (pI = 5.90) and two minor peaks (pI = 5.10, 6.95) of radioactivity. The percentage of [3H]DT associated with pelleted fractions of intact erythrocytes or erythrocyte ghosts remained fairly constant over a 100-fold range of toxin concentrations. Erythrocyte ghosts, however, bound more [3H]DT than did intact erythrocytes when exposed to the same concentration of toxin. Erythrocytes maintained in isotonic sucrose were more susceptible to toxin than erythrocytes suspended in saline, but did not bind more [3H]DT. The binding of [3H]DT to erythrocyte ghosts was found to be temperature dependent from 0 to 20 degrees C but was constant from 20 to 50 degrees C.     

Binding of heparan sulfate to Staphylococcus aureus.

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.     

Determination of non-immune binding of immunoglobulin G to Staphylococcus aureus by enzyme-linked immunosorbent assays.

Non-immune binding of human IgG to Staphylococcus aureus was studied by enzyme-linked immunosorbent assays using whole bacteria or bacterial cell walls as the solid phase. Two types of anti-human IgG peroxidase conjugates each with a low affinity for protein A, were used: F(ab')2-fragments of goat IgG and chicken IgY.     

Passive protection by human serum in mice infected with encapsulated Staphylococcus aureus.

The occurrence and nature of passive protective antibody in 100 samples of human serum was investigated in mice challenged with strains of Staphylococcus aureus capsular types A (Smith diffuse strain) and B (strain NS58D). Sixty of the sera passively protected mice against the capsular type-A strain, three against type B, and one against both types. Rabbit antisera against human IgG, IgA and IgM could remove the protective activity from a human serum of high potency, and the activity was also sensitive to 2-mercaptoethanol. Absorption with Smith surface antigen removed protective activity and reduced the concentration of IgG 7-fold, IgA 2.7 fold and of IgM 3-fold more than in a non-protective serum. Consequently, the protective activity of human serum is believed to be associated with antibodies to the S. aureus capsular antigen in the three immunoglobulin classes.     

Phagocytic killing of encapsulated and microencapsulated Staphylococcus aureus by human polymorphonuclear leukocytes.

Phagocytosis by human polymorphonuclear leukocytes (PMNs) is an important host defense against infections caused by Staphylococcus aureus. Using an in vitro assay, we compared the opsonic requirements for phagocytic killing of prototype strains of encapsulated (type 1) and microencapsulated (type 5 and type 8) S. aureus by human PMNs. More than 85% of broth-grown, logarithmic-phase type 5 and 8 S. aureus organisms were killed by PMNs incubated with fresh normal human, rabbit, or guinea pig serum with complement activity. Under similar conditions, the highly encapsulated type 1 strain was not killed. Both encapsulated and microencapsulated strains were opsonized for phagocytosis by heat-inactivated serum raised in rabbits to killed bacteria. Opsonization by homologous serum was required for phagocytosis of the type 1 strain. In contrast, microencapsulated type 5 and 8 S. aureus organisms were killed by heat-inactivated rabbit serum raised to type 5, type 8, or nonencapsulated isolates; this result suggested that antibodies to the capsule or to cell wall components other than the capsule could opsonize these organisms for phagocytosis. The specificity of the assay was confirmed with capsule type 5-specific monoclonal antibodies, which were opsonic only for the type 5 S. aureus isolate. These studies indicate that, unlike the highly encapsulated type 1 strain, broth-grown microencapsulated S. aureus strains do not resist opsonophagocytic killing in vitro by normal serum.     

Microassay for immunoglobulin G antibodies to Treponema pallidum with radioiodinated protein A from staphylococcus aureus: immunoglobulin G response in experimental syphilis in rabbits.

Radioiodinated staphylococcal protein A (SpA) was used to detect the immunoglobulin G (IgG) antibody response to Treponema pallidum in experimental syphilis. This solid-phase assay is based on the principle that SpA binds avidly to the Fc portion of mammalian IgG. The optimal number of organisms for detection of antibody was 10(5) per microwell. Of eight fixatives, 10% ethanol gave an optimum immune binding ratio of infected to normal rabbit serus at a 1:100 serum dilution. Kinetic studies demonstrated maximum binding and the highest immune binding ratio (15:1) with a 60-min incubation each for antibody and (125)I-SpA, respectively. The IgG response in rabbits intratesticularly infected with live T. pallidum and bled at -1, 9, 30, 90, 180, and 480 days was detected first at 9 days, reached a peak at 30 days, and remained elevated for 480 days. Absorption studies with an extract of T. phagedenis biotype reiterii demonstrated that 65 to 85% of the total antitreponemal IgG response was specific for T. pallidum throughout the course of infection. The microassay was quantitative and detected less than 2 ng of antibody.     

Identification of a human lactoferrin-binding protein in Staphylococcus aureus.

Human lactoferrin (HLf) is an iron-binding protein with antimicrobial activity that is present in high concentrations in milk and various exocrine secretions. HLf is also an acute-phase protein secreted by polymorphonuclear leucocytes, and its binding to a large number of clinical isolates of Staphylococcus aureus has been described recently from our laboratory. We have now characterised the HLf-staphylococcal interaction in S. aureus strain MAS-89. The binding of 125I-HLf to strain MAS-89 reached saturation in less than 90 min and was maximal between pH 4 and 9. Unlabelled HLf displaced 125I-HLf binding. Various plasma and subepithelial matrix proteins, such as IgG, fibrinogen, fibronectin, collagen and laminin, which are known to interact specifically with S. aureus, did not interfere with HLf binding. A Scatchard plot was non-linear; this implied a low affinity (1.55 x 10(7) L/mol) and a high affinity (2.70 x 10(8) L/mol) binding mechanism. We estimated that there were c. 5700 HLf binding sites/cell. The staphylococcal HLf-binding protein (HLf-BP) was partially susceptible to proteolytic enzymes or periodate treatment and was resistant to glycosidases. An active HLf-BP with an apparent Mr of c. 450 Kda was isolated from strain MAS-89 cell lysate by ion-exchange chromatography on Q-sepharose. In SDS-PAGE, the reduced HLf-BP was resolved into two components of 67 and 62 Kda. The two components demonstrated a positive reaction with HLf-HRPO in a Western blot. These data establish that there is a specific receptor for HLf in S. aureus.     

Staphylococcus aureus antigens reactive with milk immunoglobulin G of naturally infected dairy cows.

A 14- to 26-kilodalton fraction of Staphylococcus aureus exoproteins isolated by molecular sieve chromatography and electroelution from polyacrylamide gels was shown to specifically react with antibodies in milk of naturally infected dairy cows. Silver staining of the antigen preparation electrophoresed in polyacrylamide gels showed the strongest reactivity in the 24- to 26-kilodalton region with lesser staining at lower apparent molecular sizes. An enzyme-linked immunosorbent assay was developed to differentiate infected from uninfected cows for diagnostic purposes. Samples from S. aureus-infected cows reacted in the assay, and samples from uninfected cows did not. There was no correlation between numbers of somatic cells in the samples and reactivity to the antigens. Samples from cows infected with coagulase-negative staphylococci did not react with the antigens. It was found, however, that some samples from uninfected cows that were recently postpartum or producing low amounts of milk contained antibodies which bound the antigens. This was believed to be due to transport from blood to the mammary gland of antibodies which were generated by previous intramammary infections or infections at other sites.     

Binding of human immunoglobulin M to Staphylococcus aureus bearing protein A

Human immunoglobulin M binds to protein A-bearing Staphylococcus aureus. This binding is specific for protein A since it is inhibited by rabbit Fab against Staphylococcal protein A. Analysis of the IgM binding data gives an apparent K_D of 0.94 nM and a maximum capacity of approximately 2 × 10³ binding sites of IgM per bacteria.     

Binding of immunoglobulin G to peripheral blood lymphocytes

The specific and saturable binding of FITC conjugate of aggregated goat IgG to goat peripheral blood lymphocytes was studied in PBS containing 1% BSA. The polar nature of the specific interaction of heterologous aggregated IgG, IgG monomer and its fragment F(ab'2) with the cells was studied by ELISA using the peroxidase conjugated F(ab'2) of anti-human IgG under different conditions of pH and ionic strength.     

Biological properties of the encapsulated Staphylococcus aureus M

Strain M, classified as a Staphylococcus aureus, behaves like the other rare encapsulated staphylococcal strains. It was clumping-factor negative, grew in diffuse-type colonies in serum-soft agar, and produced rapidly fatal disease in mice. Strain M was highly resistant to phagocytosis by human or mouse leukocytes and required both specific antibody and heat-labile serum factor(s) for efficient ingestion by human polymorphonuclear leukocytes. Electron micrographs confirmed the presence of a large capsule. Agglutination studies, active or passive mouse protection experiments, and opsonic studies revealed that strain M represents a new, immunologically distinct strain of encapsulated staphylococcus. Strain M differs from other known encapsulated staphylococci in several other respects: its cellular and colonial morphology is atypical; its LD(50) in the mouse peritoneal model is 100 times less than that of other mouse lethal strains; it is poorly opsonized by normal human serum; and, finally, it possesses an unusually large capsule as seen in electron micrographs.     

Binding of Staphylococcus aureus by human serum spreading factor in an in vitro assay.

Human serum spreading factor, an animal cell adhesion-promoting glycoprotein, bound Staphylococcus aureus in an assay in which association of bacteria to protein-precoated microtiter wells was quantitated. Interaction of human serum spreading factor with S. aureus suggests that this protein may play a role in host defenses and in the invasiveness and pathogenicity of microbial infections.     

Occurrence of protein A in Staphylococcus aureus and closely related Staphylococcus species.

All but 1 of 143 strains of Staphylococcus aureus were positive for protein A, whereas all 34 strains of Staphylococcus hyicus and 123 of 127 strains of Staphylococcus intermedius were devoid of this cell wall component.     

Lymphocyte stimulation by protein A of Staphylococcus aureus.

Protein A from Staphylococcus aureus (SpA) is known to bind to the Fc region of most mammalian IgG classes. In the present article data are presented showing that SpA is a highly efficient mitogen for human peripheral B lymphocytes, with no detectable activity for T lymphocytes. In order to achieve optimal stimulating conditions SpA should be presented to the lymphocytes on an insoluble matrix, such as the SpA-positive bacteria themselves or SpA covalently attached to Sephadex or Sepharose beads. Using such conditions SpA is equivalent with regard to stimulatory capacity for B lymphocytes as phytohemagglutinin is for the human T lymphocytes. Specificity controls proved beyond doubt that SpA and not any other contaminating product is the B cell mitogen. It is concluded that SpA as an inducer of human B lymphocyte division might serve as a highly useful assay in the clinical assessment of B lymphocyte function. It should also be a suitable tool in the fine analysis of B lymphocyte activation via the specific interactions with surface IgG molecules.     

Staphylococcus aureus binding to human nasal mucin.

Colonization of human nasal mucosa with Staphylococcus aureus sets the stage for subsequent systemic infection. This study characterizes S. aureus adhesion to nasal mucosa in vitro and investigates the interaction of S. aureus with human nasal mucin. S. aureus binding to cell-associated and cell-free mucus was greater than to nonmucin-coated epithelial cells. Scanning electron microscopy of S. aureus incubated with human nasal mucosal tissue showed minimal binding to ciliated respiratory epithelium. In a solid-phase assay, S. aureus bound to purified human nasal mucin-coated wells significantly more than to bovine serum albumin-coated microtiter wells. Binding to mucin was saturable in a dose- and time-dependent fashion. Staphylococcal adherence to human nasal mucin was inhibited by bovine submaxillary mucin but not by fibrinogen. Pretreatment of mucin with periodate but not with pronase reduced adherence. Trypsin treatment of the bacteria significantly reduced adherence to mucin. 125I-labelled nasal mucin bound to two surface proteins (138 and 127 kDa) of lysostaphin-solubilized S. aureus. Binding to human nasal mucin occurs in part via specific adhesin-receptor interactions involving bacterial proteins and the carbohydrate moiety in mucin. These experiments suggest that S. aureus binding to mucin may be critical for colonization of the nasopharyngeal mucosa.     

Activation of human B and T lymphocytes by protein A of Staphylococcus aureus.

Protein A from Staphylococcus aureus, in soluble form or coupled to Sepharose beads, acts as a polyclonal B cell activator (PBA) for human lymphocytes in blood and spleen. PBA activity was demonstrated in spleen cells by the ability of protein A to induce the formation of intracellular immunoglobulin synthesis and to activate polyclonal antibody secretion demonstrated against fluorescein isothiocyanate-coupled sheep erythrocytes in a modified hemolysis in gel assay. More plaqueforming cells (PFC) were seen in unseparated cells than in purified B cells. In blood lymphocytes, only few PFC were activated by soluble protein A. Protein A increased DNA synthesis in blood and spleen cells. At a concentration of 100 microgram/ml the peak response was on day 4 or 5, but at 1 microgram/ml the peak response occurred later. On day 4 of culture, high mitogenic activity was seen in unseparated lymphocytes or mixtures of separated B and T cells, whereas in enriched B and T cell suspensions activity was low. On day 7, however, DNA synthesis in both the enriched B and T cells was higher than in mixtures of B and T cells. Protein A stimulated DNA synthesis in thymus cells with a peak response on day 6. It is concluded that protein A alone or as an IgG complex can activate both B and T cells, though the mechanism of activation is not known and may be different for B and T cells.     

Stimulation of human lymphocyte subpopulations by protein A from Staphylococcus aureus

Protein A from Staphylococcus aureus is known to stimulate human lymphocytes. Using 3H-thymidine incorporation, virus plaque assay and induction of cytotoxic T lymphocytes (CTL), this study showed that soluble or insoluble protein A stimulated different lymphocyte subpopulations. Soluble protein A is highly mitogenic to T lymphocytes. In both 3H-thymidine incorporation and virus plaque assay, its maximum stimulation was as high as the stimulation by the nonspecific mitogens phytohemagglutinin and concanavalin A, and a higher CTL response than that induced by phytohemagglutinin or concanavalin A was induced. Mitogenic activity to B lymphocytes was negligible. S. aureus (Cowan I strain) is itself considered to be an insoluble form of protein A and is 3--4 times more mitogenic to B lymphocytes than pokeweed mitogen without any increase in virus plaque-forming cells. No mitogenicity was noted to T lymphocytes. Sepharose CL-4B-protein A, also known as insoluble protein A, stimulated both T and B lymphocytes effectively, but its mitogenicity to T lymphocytes was considered to be due to the soluble protein A released from the Sepharose CL-4B beads.     

Determination of immunoglobulin classes of antibodies against Coxiella burnetii by protein A from Staphylococcus aureus

Protein A from Staphylococcus aureus was used for differentiating antibodies against Coxiella burnetii according to immunoglobulin classes. Interaction of immune serum with protein A resulted in complete (within the sensitivity limits of the methods used) removal of IgG antibody and had practically no effect on the level of IgM antibody.     

A radioimmunoassay of human leukocyte interferon using protein A-containing Staphylococcus aureus

We report the establishment of a radioimmune assay for human alpha-interferon using highly purified alpha-interferon labeled with iodine-125, a purified rabbit immunoglobulin directed against human interferon and the Cowan strain of Staphylococcus aureus. Radiolabeling conditions used do not change the antigenicity of interferon molecules. The regression of counts bound upon log. dose was found to be linear down to 10 units/ml of alpha-interferon (6-7 X10-12 M). This assay was specific for alpha-interferons derived from human peripheral blood leukocytes and from a continuous line of lymphoblastoid cells. No cross-reaction was found with either human beta-interferon or murine interferon. Neither human serum nor plasma interfered with the assay. Correlation between biological assay and radioimmune assay was found to be significant.     

Immunological response to a Staphylococcus aureus fibronectin-binding protein.

A protein (gal-FnBP), constructed by fusion of the genes encoding beta-galactosidase of Escherichia coli and the binding domains of fibronectin-binding protein (FnBP) of Staphylococcus aureus was used. FnBP is a surface protein responsible for attachment of bacteria to extracellular matrix of various host tissues. Gal-FnBP is more stable and can be produced in larger quantities than native FnBP. The binding specificity of this fusion protein was established in a Western blot analysis. Treatment of gal-FnBP with formalin inactivated the binding capacity of the protein but immunogenicity was retained. Immunisation of mice with formalin-treated gal-FnBP resulted in high antibody titres against the fibronectin-binding part of this fusion protein. These antibodies were measured by their ability to block the specific binding of fibronectin to gal-FnBP in a blocking assay. Sera raised against formalin-treated gal-FnBP and non-treated gal-FnBP blocked this binding to 40 and 25% respectively, thereby indicating the usefulness of gal-FnBP as a vaccine component.