Keijiro Kato

Isolation and Characterization of the Serotype g Carbohydrate Moiety from an Enzyme Lysate of Streptococcus mutans 6715 Cell Walls

The serotype‐specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell‐wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo‐N‐acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C‐25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G‐100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan—polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A‐25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re‐chromatographed on a Bio‐Gel P‐100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti‐serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4 : 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti‐serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an α‐linked galactose‐glucose terminal linkage.

Immunomodulating and Related Biological Activities of Bacterial Cell Walls and Their Components, Enzymatically Prepared or Synthesized

In 1959, we isolated the cell walls of tubercle bacilli (BCG) which have long been known to have a strong immunoadjuvant activity, especially in the stimulation of cell-mediated immune responses, and elucidated the chemical and immunological properties of this cell wall fraction (32, 31). The most remarkable finding on the chemical composition of this subcellular fraction was that various lipids, especially ethanol-ether insoluble but chloroform-soluble lipids and bound lipids, which characterized tubercle bacilli were almost exclusively localized in their cell walls. It was also demonstrated that the cell walls were the most active fraction of subcellular components isolated from sonicated BCG cells, in inducing tuberculin hypersensitivity in guinea pigs and that the cell walls were involved in the development of an acquired cell-mediated resistance of mice to tuberculous infection.

Chemical Composition of Streptococcus mutans Cell Walls and Their Susceptibility to Flavobacterium L-11 Enzyme

The susceptibility to a cell wall lytic L‐11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non‐cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS‐1 (serotype a), BHT (b), NCTC10449 (c), C67–1 (c), C67–25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS‐1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ 176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L‐11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L‐11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.