Structural studies were carried out on the teichoic acids in cell walls of Listeria monocytogenes serotypes 3a, 4b, 4f, 6, and 7. The structure of the dephosphorylated repeating units, obtained by treatment with 46% hydrogen fluoride or alkaline hydrolysis, was examined by methylation analysis, acetolysis, and 1H-NMR spectroscopy. The results of Smith degradation of the teichoic acids and 13C-NMR spectroscopy led to the following most likely structures of the repeating units of the teichoic acids:----1-[N-acetylglucosaminyl(alpha 1----4)]ribitol-5-phosphate----for serotype 3a,----4-[galactosyl(alpha 1----6)][glucosyl(beta 1----3)]N -acetylglucosaminyl(beta 1----2)ribitol-5-phosphate----for serotype 4b,----4-[galactosyl(alpha 1----6)][N -acetylglucosaminyl(alpha 1----3)]N-acetylglucosaminyl(beta 1----2)ribitol -5-phosphate----for serotype 4f,----4-N-acetylglucosaminyl(beta 1----4)ribitol -5-phosphate----for serotype 6, and----1-ribitol-5-phosphate----for serotype 7. About 40% of the repeating units of the teichoic acid from serotype 4f were not substituted at C-3 of beta-N-acetylglucosaminyl residues.

The chemical compositions of the cell walls obtained from 10 strains (serotypes 1a, 3a, 4a, 4b, 4c, 4d, 4e, 4e, 4f, 6, and 7) of Listeria monocytogenes were analyzed. These cell walls were shown to be mainly composed of peptidoglycan and ribitol teichoic acids. Considerable variations in the composition of neutral sugars were observed among these cell walls. Chemical and NMR analyses indicated that the teichoic acids from L. monocytogenes serotypes 4a and 4d are composed of the following repeating units: Formula: See Text.

The cell-wall skeletons of Listeria monocytogenes strain EGD and Propionibacterium acnes strain C7, which have the ability to induce macrophage activation, were analyzed, and the structures of the peptidoglycans were investigated. The analytical data indicate that both peptidoglycans have glucosamine residues with free amino groups, which are responsible for the resistance to lysozyme. Possible structures of these peptidoglycans were deduced from the composition and the results of determination of N- and C-terminal amino acids, together with the characterization of fragments obtained by enzymatic treatment and partial acid hydrolysis of both peptidoglycans. The results suggested that the peptidoglycan of L. monocytogenes contains a cross-linkage region of peptide chains with meso-diaminopimelic acid and D-alanine, which belongs to the A1 gamma type (Schleifer, K.H. & Kandler, O. (1972) Bacteriol. Rev. 36, 407-477), whereas the peptidoglycan of P. acnes contains a cross-linkage region of peptide chains with L,L-diaminopimelic acid and D-alanine, in which two glycine residues combine with amino and carboxyl groups of two L,L-diaminopimelic acid residues. The latter type should be classified as a new type. These cell-wall skeletons and peptidoglycans were shown to have immunoadjuvant activity on the induction of delayed-type hypersensitivity and suppressive activity on the growth of 3-methylcholanthrene-induced fibrosarcoma in BALB/c mice, and the peptidoglycans were shown to be an immunological-active principle of these cell-wall skeletons.

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The lipoteichoic acids were isolated from phenol extracts of four Listeria strains representing serotypes 4a, 4b, 6a, and 6 to compare the differences in structure of amphiphilic polysaccharides from various serotypes of Listeria spp. The lipoteichoic acids from the four strains examined had the same structure in both hydrophilic chains and lipid portions. On the basis of the results of nuclear magnetic resonance spectroscopy and Smith degradation, the hydrophilic chains were shown to be 1,3-linked poly(glycerol phosphate) in which some of the glycerol residues had a-galactosyl substituents. The lipid portions were released by treatment with 46% hydrogen fluoride or 98% acetic acid. They were determined to be 3(1)-(2'-O-a-D-galactopyranosyl-a-Dglucopyranosyl)-1(3),2-diacylglycerol and 3(1)-[6'-phosphatidyl-2' -O-(a-D-galactopyranosyl)-a-Dglucopyranosyl]-1(3),2-diacylglycerol. The degrees of glycosyl substitution and proportions of the two lipids varied to some extent among these four strains.The antigenic scheme of Listeria spp., based on the somatic (0) and flagellar (H) antigens, includes 17 recognized serotypes, which can be divided into 13 groups by the differences in their somatic antigens (31). Ullmann and Cameron (35) reported the immunochemical properties of the cell wall carbohydrates of several serotypes of Listeria monocytogenes and described the monosaccharide constituents which should serve as the major antigenic determinant. Fiedler et al. (7) reported the composition of the cell walls from various serotypes of Listeria spp. and suggested that the glycosidic substituents of the cell wall teichoic acids determine the 0 antigenic specificities. Recently, the structures of cell wall teichoic acid from eight serotypes of Listeria spp. were reported (12,18,34), and some immunological properties of these teichoic acids were examined (18, 19) in our laboratory. In these studies, it seemed that the 0 serofactors of Listeria spp. cannot be entirely accounted for by cell wall teichoic acids; for instance, the structures of the cell wall teichoic acids from strain 5214 (serotype 4a) and strain 1383 (serotype 6) are identical (12,34). Therefore, other cell surface substances such as lipoteichoic acids also have to be taken into consideration in listerial 0 antigenicity.Lipoteichoic acids are membrane-associated amphiphilic molecules found in many gram-positive bacteria. They can act as surface antigens and often function as basis for serological classification of bacteria (20,39,40). Extracts obtained from L. monocytogenes contain apparent amphiphilic polysaccharides. The monocytosis-producing agent from serotype 1 cells is present in a high-molecularweight fraction containing phosphorus, lipids, and sugars (13). A phenol-water extract from serotype 1 cells contained a complex of peptide, polysaccharide, and lipid which had the biological activity of lipopolysaccharide (16,23 amphiphilic polysaccharide which was strikingly similar to gram-negative bacterial lipopolysaccharides in both chemical composition an...