Teichoic acids: antigenic determinants, chain separation, and their location in the cell wall.

Teichoic acids are linear polymers of glycerophosphate or ribitolphosphate carrying sugar or D-alanine substituents.1 Their chain length varies between 8 and 50 units and is dependent upon the isolation technique. Some occur in bacterial cell walls where they can make up between 8 and 50 per cent of the wall's dry weight; some, the so-called "intracellular" ones, in an ill-defined area between cell wall and cell membranes. From the time of their discovery they were known to be antigenic.2 Thus the group antigens of the streptococcal group D3 and the type antigens of Staphylococcus aureus were shown to be teichoic acids.4 Antisera, however, could be obtained only with whole cells, cell walls, or crude extracts; or they were probably a consequence of previous infections as was the case for human sera against Staphylococcus aureus.5 Pure teichoic acid preparations do not act as immunogens.6' I It will be shown that precipitation and the preparation of a particulate suspension of teichoic acids render them immunogenic. Antisera thus obtained were used to elucidate the antigenic determinants of several teichoic acids. Furthermore, they provided the tool to answer the following two questions: (1) Do teichoic acids from a single bacterial strain exist as a mixed population of completely substituted and completely unsubstituted molecules, or is each molecule substituted in a quantitatively or qualitatively different manner? (2) Can teichoic acids be located within the architecture of the bacterial cell wall? With regard to the first question, it has been almost tacitly assumed up to now that different types of sugar substituents are present on the same polymer or, in the case where only one type of sugar substituent occurs, that each molecule is only partially substituted. Polymers of the two organisms examined here (B. subtilis 3610 and B. licheniformis) appeared to be mixtures of molecules which were either completely devoid of substituents or carried a complete set of substituents. No single macromolecule seemed to contain more than one type of sugar. In answer to the second question, it was demonstrated that teichoic acids in the intact wall are not readily available to antibodies. It was also shown that treatment of the cell walls with lysozyme increased the availability of antigenic sites, thereby suggesting that teichoic acids occur in a somewhat deeper layer of the wall. Materials and Methods.-Antisera: Particulate suspensions of teichoic acids were obtained by the slow addition at 40C of either 5 mg of cetylpyridinium chloride (10 mg/ml) or 5 mg methylated bovine serum albumin (MBSA) (10 mg/ml) to 1 mg purified8 teichoic acid in 1.0 ml of 0.154 M NaCl. Various particle sizes were produced by altering both concentration of the reactants and temperature of the reaction and selected with Millipore filters. After mixing the suspension with an equal volume of Freund's adjuvant, 1/3 of the resulting emulsion was injected intramuscularly, and the remainder in equal amounts into each footpad of a male New Zealand rabbit (7-9 lb).