Staphylococcus aureus peptidoglycan studied by electron microscopy of immunoperoxidase-treated sections of cell walls.

The immunoperoxidase technique in combination with electron microscopy was used for demonstration of antigenic determinants of Staphylococcus aureus peptidoglycan. Isolated antibodies against four synthetic peptides with amino-acid sequences similar to those found in native staphylococcal peptidoglycans were used. A homogeneous staining of the cell wall from the outer surface to the cell membrane was demonstrated.

Detection of Yersinia enterocolitica O-antigens by double diffusion in agar and chemical characterization of lipopolysaccharides.

Double diffusion in agar showed that yersinia enterocolitica O-serotypes 3, 7, 8 and 9 each contained a type-specific precipitinogen. Serotype 6 contained several specific precipitinogens and was heterogeneous. The type-specific precipitinogens were shown to be lipopolysaccharides (LPS). Acid hydrolysis of the isolated LPS followed by gel filtration separated the material into two peaks, the first of which contained the serologically active fractions. The active fraction of serotype 3 contained relatively large amounts of a terminally linked deoxyhexose, probably 6-deoxy-L-altrose. Fucose was found to be present in serotype 7.

Immunochemical analysis of the teichoic acid from Staphylococcus simulans.

The wall teichoic acid of Staphylococcus simulans has been characterized as a glycerol phosphate polymer with glycosidically linked N-acetylglucosamine. Susceptibility to beta-N-acetylglucosaminidase and serological similarity to poly C beta from Staphylococcus saprophyticus, showed that the amino sugar is in the beta-configuration.

Immunoperoxidase and electron microscopy studies of staphylococcal lipoteichoic acid.

Immunoperoxidase technique together with electron microscopy shows that lipoteichoic acid (LTA) of Staphylococcus aureus Cowan I is attached to the membrane and penetrates the whole cell wall. A diffuse zone outside and no peroxidase reaction product inside the wall when whole cells were treated with antibody prior to embedding may indicate (i) that LTA is exposed to reaction with antibodies outside the wall, and (ii) that all or most of the anti-LTA antibodies used are of the IgM class and thus unable to penetrate the wall. Thin sections of strain Wood 46 showed the same picture as Cowan I, but treatment of whole cells before embedding gave no diffuse zone outside the wall. This may be due to a thicker wall as found by electron microscopy and/or shorter LTA-chains of strain Wood 46 than those present in the wall of Cowan I.

Antibodies in rabbits immunized with staphylococcal lipoteichoic acid.

Fractionated rabbit antiserum to staphylococcal lipoteichoic acid (LTA) was tested for reaction with homologous antigen by precipitation in agar gel, countercurrent immunoelectrophoresis, immunoperoxidase technique and electron microscopy. The antibodies to LTA present in the rabbit antisera examined were found to be of the IgM class.

Immunochemical analysis of the teichoic acid from Staphylococcus hyicus.

The wall teichoic acid of Staphylococcus hyicus has been isolated and characterized. The teichoic acid is a glycerol phosphate polymer with glycosidically linked N-acetylglucosamine. Interaction with concanavalin A and susceptibility to alpha- but not to beta-N-acetylglucosaminidase showed that the sugar is in the alpha-configuration.

Induction of leukochemotaxis by peptidoglycan of Staphylococcus aureus.

Leukochemotactic activity of staphylococcal peptidoglycan and isolated, specified fragments has been studied. The D-Ala-D-Ala group of the pentapeptide was found to be the major cytotaxigen, and C5a to be the dominant cytotaxin. A molecular weight of about 2,000 appeared to be a critical lower limit for inducing chemotactic response, the highest effect being observed with a fragment having a molecular weight of 3,000. The effect seems to depend on the kind of available antigenic determinants in the fragments and the proportion of specific antibodies in the complement source.

Induction of leukochemotaxis by protein A of Staphylococcus aureus.

The mechanism of the leukochemotactic activity of staphylococcal protein A (pA) has been studied by in vitro and in vivo experiments. Protein A alone or mixed with heat-inactivated serum induced no migration of polymorphonuclear leukocytes, demonstrating that pA is not a cytotaxin, but a cytotaxigen. Protein A, activating both pathways of the C system, induced chemotaxis in a C4 deficient serum, but not in a C5 deficient serum. This shows that the chemotactic mediator elaborated is a split product of C5, i.e. C5a. The chemotactic activity of pA observed in the presence of normal sera (in vitro) and in wound chambers (in vivo) was exclusively caused by C activation via reaction with Fc.

Isolation of enzymatically derived fragments of guinea pig IgG and an examination of their reactivity against Staphylococcal protein A.

Papain digest of guinea pig IgG2 was separated by ion-exchange chromatography into five fractions, one containing mainly Fab and four Fc fractions. Covalently linked Fc (cFc) and non-covalently linked Fc fragments (nFc) were present, both with a molecular weight (Mw) of about 56,000. nFc was split into half fragments by gel filtration under dissociating condition (Mw 25,000 to 27,000). An incomplete Fc fragment (iFc) was also isolated (Mw 36,000), which consisted of a Fc chain (Mw 26,000) non-covalently linked to a piece of another chain. These preparations all reacted with protein A. In addition, the Fc' fragment (Mw 22,000), which is the C-terminus of the Fc fragment, was isolated. This fragment did not react with protein A. The cFc was treated with acid and then digested with trypsin at pH 7.8 for 45 s. The digest were separated into four fractions (I-IV) by gel filtration on Sephadex G-100. Fraction I was indistinguishable from intact Fc, fraction II contained an incomplete covalent Fc fragment (Mw 34,000 to 38,000). Both these fragments reacted with protein A. Fraction IV consisted of fragments belonging to the C-terminus, and was protein A non-reactive. Fraction III was shown to contain a mixture of fragments belonging to fractions II and IV.

Immunochemical studies on the specific agglutinogens of Staphylococcus aureus. I. Isolation and characterization of antigen h1.

The specific Staphylococcus aureus agglutinogen h1 has been purified and shown to be a protein with a molecular weight of about 95,000. Chemical analysis revealed all the common amino acids, except tyrosine and the sulphur-containing ones. The purified h1 antigen was strongly immunogenic in rabbits. The antiserum produced one precipitation line on double diffusion in agar against a suspension of bacteria. It also agglutinated bacteria of the h1-containing strains, as well as tanned sheep erythrocytes sensitized with h1, in high dilutions. Antibodies to other known staphylococcal antigens were not detected. The identity between h1 and Pillet's type 9 antigen was confirmed.

Immunochemical studies on Staphylococcus aureus plasma membrane. 2. Antigenic properties.

Cytoplasma membrane and lipoteichoic acid (LTA) isolated from S. aureus Cowan I were examined serologically. LTA contains both alpha- and beta-glucosyl substituents at glycerol and most probably ester-linked alanine as well, all being antigenic determinants. In addition to LTA, the membrane contains a glycoprotein exhibiting antigenic determinant(s) in both the protein and sugar moieties.

Immunochemical studies on Staphylococcus aureus plasma membrane. I. Isolation and chemical characterization.

Cytoplasma membrane and lipoteichoic acid (LTA) were isolated from S. aureus Cowan I and analysed chemically. Pure membrane was obtained by using human IgG coupled to Sepharose, which then absorbed all cell wall fragments due to the interaction between IgG and protein A on the wall. LTA, shown to be a glucosylglycerol teichoic acid containing ester-linked alanine and pentadecanoic acid as the major fatty acid, was also present in the isolated membrane but only as a minor component. Other carbohydrate, protein and lipid components which were present as a chemical complex, dominated the membrane preparations.

A human IgA myeloma protein interacting with staphylococcal alpha-toxin and protein A.

High anti-staphylolysin activity was found in the serum from a patient with multiple myeloma. The activity was confined to the monoclonal IgA protein and was dependent on an intact Fc part of the molecule. An Fc-dependent interaction with protein A was also demonstrated. In addition, a part of the monoclonal IgA was found to be complexed with alpha2-macroglobulin. The results imply an unusual Fc-reactivity of the IgA protein.

Immunochemical analysis of an unusual cell wall polysaccharide from animal coagulase-positive staphylococci. 1. Fragments obtained after hydrolysis in hydrofluoric acid and alkali.

Polysaccharide P (poly P) of canine coagulase-positive staphylococci contains glycerol, glucose, glucosamine, muramic acid, phosphate, and the usual peptidoglycan amino acids, but does not cross-react serologically with standard teichoic acids. Products from hydrolyses in hydrofluoric acid and alkali contained phosphates of glycerol and glucose as well as combinations of these, but neither glucosyl-glycerol units nor glucosamine-phosphates were observed. The teichoic acid of poly P is probably a polymer of a repeating unit consisting of alternating glycerol, phosphate and glucose.

The effect of specific antibodies on the inhibition of leucocyte migration caused by staphylococcal peptidoglycan.

Specific antibodies to the various antigenic determinants of staphylococcal peptidoglycan are tested for neutralization of the inhibiting effect of peptidoglycan on leucocyte migration. Antibodies to the C-terminal D-Ala-D-Ala group of pentapeptides and to the C-terminal of the glycine bridge showed high neutralizing effect, whereas that of antibodies to the tetrapeptide and to the glycan chain was negligible. The observed neutralization of antibodies against the outermost parts of peptide chains may be due to the inhibition of contact between peptidoglycan and cells.

Immunochemical analysis of an unusual cell wall polysaccharide from animal coagulase-positive staphylococci. 2. Probable structure based on chemical and serological studies.

The teichoic acid of polysaccharide P (poly P) contains glycerol, glucose and phosphate. Hydrofluoric acid and alkali hydrolysates contain glycerol 1-phosphate, glycerol diphosphate, and glucose 1-phosphate, but no glucosyl-glycerol fragments. Glucose and the serological activity of poly P were destroyed by periodate oxidation. Interaction with concanavalin A showed that the glucose is in alpha-configuration and that the hydroxyl groups at positions 3, 4 and 6 are unsubstituted. Most probably, the poly P teichoic acid is a polymer containing a repeating unit in which glycerol 1-phosphate is attached to the 2-position on alpha-D-glucose 1-phosphate.

Further characterization of protein A reactive and non-reactive subfragments of Fc from human IgG.

Tryptic digests of acid-treated Fc from normal human IgG were separated into four peaks (I-IV) by gel filtration on Sephadex G-100. The second peak was further divided into two fractions (II and II'). Peak I was indistinguishable from intact Fc on electrophoresis, immunodiffusion, and reactivity to protein A. The protein A reactive fragments of fractions II, II', and III were shown to contain antigenic determinants of both the CH2 and CH3 domains, to interact with the anti-Gm (1) specific rheumatoid factor, and to fix complement. These results, together with SDS-electrophoresis, showed that protein A reactive fragments are all composed of an intact Fc chain with shorter chains covalently linked to it. The protein A non-reactive fragments of fractions II' and III were homogeneous, fixed complement and showed no interaction with the Gm (1) rheumatoid factor. These results, in addition to the observed antigenic determinants, localized the fragments to the CH2 region.

An examination of the "star-phenomenon", a three component immunoprecipitation involving staphylococcal protein A.

The co-precipitation called "star-phenomenon" occurred between the three component system: protein A, an IgG forming soluble complexes with protein A and F (ab')2-preparations of human IgG, guinea pig IgG or rabbit anti-staphylococcal IgG. Co-precipitation also occurred if the IgG was replaced by normal human Fc fragments. On a protein A column the human F (ab')2-preparation was separated into a major non-reactive and a minor reactive fraction. Only the latter contained Fc-structures, these being isolated on an anti-Fc column and found to belong to undigested IgG. The "star"-forming protein A reactive F (ab')2-fragments were washed through the anti-Fc column. The F (ab')2 fraction from rabbit anti-staphylococcal IgG contained no Fc-structures and only a small portion containing anti-protein A activity was active in co-precipitation.

Influence of IgG, F(ab')2 and IgM on the phagocytic and bactericidal activities of human neutrophil granulocytes.

The phagocytosis and intracellular killing of Staphylococcus aureus by humans granulocytes in the presence of immunoglobulin preparations have been examined. Isolated IgG from pooled human serum induced phagocytosis and intracellular killing of bacteria. F(ab')2 fragments had no significant effect, indicating that the Fc piece of the IgG molecule is of importance not only for the phagocytosis but also for the intracellular killing of bacteria. Isolated IgM stimulated the phagocytosis to a minor extent, with no enhancement of the bactericidal activity.

Human colostral IgA interacting with staphylococcal protein A.

Human colostral IgA from three apparently healthy mothers were all divided into protein A reactive and protein A non-reactive fractions on a Sepharose-protein A column. The reactive fractions, giving no direct precipitation but co-precipitations, were found to interact with protein A through the Fc-region.