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).

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.

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 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.

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.

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).

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

Inhibition of anaphylactic reaction in guinea pigs by antibodies to fragments of Fc from autologous IgG.

The effect of antibodies to Fc subfragments of guinea pig IgG on the anaphylactic reaction in guinea pigs has been studied. Control animals were immunized with guinea pig IgG and Fab, human IgG and albumin. All animals with antibodies to fragments of autologous Fc survived challenge with horse serum, whereas the control animals died in anaphylactic shock.

Estimation of peptidoglycan antibodies by an immunoperoxidase technique.

An immunoperoxidase technique developed for the detection and measurement of anti-peptidoglycan antibodies is described. The technique is based on the Mancini single radial immunodiffusion test with the addition of a reaction step with peroxidase-labelled anti-immunoglobulins. The dark-brown colour produced by the enzyme and a H2O2-diaminobenzidine solution increased the sensitivity considerably.

Studies on polysaccharide C of Staphylococcus epidermidis. 1. Isolation and chemical characterization.

Polysaccharide C (poly C) has been isolated from two strains of S. epidermidis and characterized chemically. The results suggest that poly C is a wall N-acetylglucosaminylglycerol teichoic acid, linked through 1:3-phosphodiester linkages.

Studies on polysaccharide C of staphylococcus epidermidis. 2. Antigenic properties.

The antigenic properties of polysaccharide C (poly C) of S. epidermidis strongly support the analytical indications that it is an N-acetylglucosaminylglycerol teichoic acid with 1:3-phosphodiester linkages. The major antigenic determinant is N-acetyglucosamine, predominantly present in the beta-configuration.

Localization of antigens in thin sections of bacteria by the immuno-peroxidase technique.

The immunoperoxidase technique together with electron microscopy has been examined for the ultrastructural localization of staphylococcal protein A, the immunoferritin method being included for comparison. The results show that protein A is uniformly distributed in the whole cell wall. Both the direct and indirect methods, Fab- as well as Fc-reactions, showed identical results.