Staphylococcal antibodies and hypersensitivity to teichoic acids in man.
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Guinea pigs sensitized with washed, Formalin-killed cells of Staphylococcus aureus, strains 263 or Copenhagen, were skin-tested with various antigens from these strains including washed viable and heat-killed whole cells, cell walls, the peptidoglycan complexes of the walls, teichoic acid, teichoic acid-peptidoglycan fragments, and peptidoglycan fragments. In nonsensitized control animals, all antigens but teichoic acid elicited acute inflammatory reactions which decreased in size after 10 hr. In animals sensitized with the Copenhagen strain, the reactions to all antigens but teichoic acid and peptidoglycan fragments from either strain remained erythematous and indurated for at least 30 hr and were interpreted as hypersensitivity of the delayed type. Responses in animals sensitized with strain 263 generally resembled those in controls, although in some experiments there was evidence of hypersensitivity.
Solublie teichoic acid could not stimulate the formation of antibodies either as an aqueous solution or in an emulsion with Freund incomplete adjuvant. However, when given as precipitates with methylated bovine serum albumin, it stimulated strong anti-teichoic acid responses. High levels of anti-teichoic acid antibodies usually resulted in allergic symptoms upon secondary challenge either with immunogenic teichoic acid-methylated bovine albumin complexes or with non-immunogenic soluble teichoic acid. These symptoms suggested anaphylatic shock, but probably also included the formation of complement-consuming immune complexes.
Lipoteichoic acid (LTA) is a component of nearly all gram-positive membranes and recently has been found to be excreted into growth media by certain lactic acid bacteria. Cell-free extracts of LTA are usually contaminated with proteins, polysaccharides, and nucleic acids, thus causing problems to investigators studying the true biological function(s) of LTA. This report describes the preparation of purified extracellular LTA of Streptococcus mutans BHT and intracellular LTA of S. mutans AHT by three techniques: gel filtration, hydrophobic interaction chromatography, and adsorption to phospholipid vesicles. Gel filtration, the most commonly employed method for LTA purification, was found to remove nucleic acids, teichoic acids, and much polysaccharide while greatly concentrating LTA. But gross amounts of antigenic carbohydrate and protein remained associated with the LTA preparation. Hydrophobic interaction chromatography employing octyl Sepharose-4B allowed the separation of protein but not polysaccharide from partially purified BHT LTA preparations. By means of a new technique described in this paper, synthetic membranes (vesicles) were found to effectively separate all contaminants from the intracellular (AHT) and extracellular (BHT) LTA of S. mutans. This rapid method, on a comparative basis, proved to be the most effective approach for the purification of LTA from two widely differing sources.
The development of delayed hypersensitivity (DH) to Staphylococcus aureus in Swiss mice was evaluated by the footpad (FP) assay. In order to determine which component of the bacteria was responsible for the in vivo immune reactivity, purified Staphylococcal cell wall, cell membrane, protein A, lipoteichoic acid, teichoic acid, as well as lipid-free membrane proteins were isolated. The immune responses of mice receiving one to eight S. aureus injections indicated that the first DH peak, following three injections, was primarily dependent upon protein antigens associated with the bacterial membrane. Increased bacterial injections gave rise to a second DH peak following seven injections which was dependent upon multiple bacterial components including cell wall, protein A, and membrane proteins.
A cell wall component that bound to HeLa cells (HeLa cell-binding CWC) was isolated from a clinical isolate of Staphylococcus aureus. The HeLa cell-binding CWC was resistant to heat (100 C, 1 hr) and proteases, did not stain with Coomassie Brilliant Blue R-250 on SDS-PAGE but stained as a broad band with antiserum against the strain on Western blots. These data suggest that the HeLa cell-binding CWC is not a protein, and may be teichoic acid. Purified teichoic acid bound to HeLa cells, whereas fractions without teichoic acid did not. In Western blots, HeLa cell-binding CWC appeared as a broad band of less than 35 kDa, similar to that of purified teichoic acid. These data suggest that the HeLa cell-binding CWC obtained in this study is teichoic acid. Teichoic acid inhibited S. aureus adherence to HeLa cells and bound to the cells time and dose dependently, in a saturable and reversible manner, and therefore appears to be an adhesin of S. aureus to HeLa cells.
A Staphylococcus aureus-agglutinating lectin, capable of binding to N-acetyl-D-glucosamine, was isolated from the serum of Limulus polyphemus. The monosaccharide alone was incapable of inhibiting bacterial agglutination by this lectin. Quantitative precipitation studies with purified cell wall-derived teichoic acids, either devoid of or containing N-acetyl-D-glucosamine, confirmed the carbohydrate-binding specificity of the lectin and suggested that secondary, non-specific interactions contribute to binding biomolecules containing this sugar. The agglutination pattern with various S. aureus strains having N-acetyl-D-glucosamine-associated teichoic acid, teichoic acid without this sugar, and no teichoic acid indicated that this cell wall component is not the sole binding site for the lectin on intact S. aureus cells. Affinity gel chromatography, using N-acetyl-D-glucosamine-associated teichoic acid as the specific absorbent, has been used to isolate this lectin from Limulus serum.
Structural studies were carried out on the linkage units in the teichoic-acid--glycopeptide complexes isolated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1366. On treatment with 47% hydrogen fluoride, the complexes gave a disaccharide characterized as glucosyl(beta 1----4)N-acetylglucosamine together with major fragments, galactosyl(alpha 1----2)glycerol. By means of Smith degradation and partial acid hydrolysis, the teichoic acid chain was shown to be composed of the repeating units, galactosyl(alpha 1----2)glycerol-3(1)-phosphate, which were joined by phosphodiester bonds at C-6 of the galactose residues. The mild alkaline hydrolysis of teichoic-acid-linked glycan fragments yielded teichoic acid chains and disaccharide-linked glycan fragments, from which the disaccharide, glucosyl(beta 1----4)N-acetylglucosamine, was liberated by mild acid hydrolysis, whereas the same disaccharide linked to the teichoic acid chain was obtained by direct heating of the cell walls at pH 2.5. In addition, the presence of specialized glycerol phosphate units in the linkage region was shown by the isolation of tris(glycerol phosphate)3-glucosyl(beta 1----4)N-acetylglucosamine from the products of the Smith degradation of the teichoic-acid--glycopeptide complexes. Thus, it is concluded that the poly(galactosylglycerol phosphate) chain in the cell walls of B. coagulans AHU 1366 is linked to peptidoglycan through a novel linkage unit, bis(glycerol phosphate)-3-glucosyl(beta 1----4)N-acetylglucosamine.