Shigeo Kawata

Mutanolysin, Bacteriolytic Agent for Cariogenic Streptococci: Partial Purification and Properties

Mutanolysin partially purified from the culture filtrate of Streptomyces globisporus 1829 consists of two main lytic enzymes with an isoelectric point near pH 8.5 and 10, respectively, and proteolytic enzyme is associated with the latter lytic enzyme. Mutanolysin exhibited maximal lytic activity at 60 C in the pH range 6.5 to 7.0 and was stable at 50 C in the acid range. N-bromosuccinimide caused complete inhibition of lytic activity at 1 mM, whereas calcium and magnesium ions at the same concentration caused activation. Mutanolysin had lytic or bactericidal activity against the living cells of Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis, Lactobacillus acidophilus, and Actinomyces viscosus, which are considered to be etiologic agents of dental caries, but had no activity against S. aureus and all gram-negative strains tested. The lytic activity was well retained in human saliva. Digestion of the cell walls of S. mutans BHT by mutanolysin was accompanied by the liberation of free amino groups and reducing sugars. Mutanolysin may be expected to be a useful agent for dental caries control. Images

Activation of the human complement cascade by bacterial cell walls, peptidoglycans, water-soluble peptidoglycan components, and synthetic muramylpeptides--studies on active components and structural requirements.

Cell walls isolated from 29 strains of 24 gram‐positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandlers classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement‐activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water‐soluble “polymer” of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross‐bridge degrading endopeptidase, retained most of the complement‐activating ability of the parent cell walls. A peptidoglycan “monomer,” SEPS‐M, which was obtained by hydrolysis of the glycan chain of SEPS with endo‐N‐acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N‐acetylmuramyl‐l‐alanyl‐d‐isoglutamine (MDP) nor MDP‐l‐Lys‐d‐Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6‐O‐(3‐hydroxy‐3‐docosylhexacosanoyl)‐MDP‐l‐Lys‐d‐Ala (BH48‐MDP‐l‐Lys‐d‐Ala) and 6‐O‐(2‐tetradecylhexadecanoyl)‐MDP (B30‐MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a d‐isoasparagine analog of B30‐MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30‐MDP is different from that caused by cell wall peptidoglycans and a water‐soluble “polymer” of peptidoglycan subunits.

Polyclonal B Cell Activation by Cell Wall Preparations of Gram‐Positive Bacteria

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram‐positive bacteria were studied using the anti‐sheep red blood cell (SRBC) or anti‐trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS‐responder), C3H/HeJ (LPS‐non‐responder), (CBA/N × Balb/c) F1 male with an X‐linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA‐ability of synthetic peptidoglycan, muramyl dipeptide (N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.

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.

Oral immunoadjuvant activity of lipophilic derivatives of N-acetylglucosaminyl-β(1 → 4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-(L)-2,6-meso-diaminopimeric acid-(D)-amide

Twenty-four kinds of the acylated, amidated or esterified derivatives of N-acetylglucosaminyl (GlcNAc)-beta(1-->4)-N-acetylmuramyl (MurNAc)-L-alanyl (Ala)-D-isoglutaminyl (isoGln)-(L)-meso-2,6-diaminopimeric acid (A2pm)-(D)-amide (GMP3-A) which were prepared by chemical modifications of an enzymatic hydrolysate of Lactobacillus plantarum cell-wall peptidoglycans were examined for oral adjuvant activity by gastric intubation with bovine serum albumin (BSA) in liposomes into BALB/c mice. The gastric intubation of GlcNAc-beta(1-->4)-MurNAc-L-Ala-D-isoGln-(L)- stearoyl-(D)-meso-A2pm-(D)-amide-(L)-O-octyl, -nonyl and -dodecyl esters exhibited the most marked oral adjuvant activity in terms of enhanced production of serum anti-BSA IgG antibody. Some derivatives showed a less marked adjuvanticity and others were totally inactive. Thus the oral adjuvanticity of test lipophilic derivatives of GMP3-A in liposomes was dependent upon their chemical structure.

T cell line specific for bacterial peptidoglycan subunit: Possible role of the COOH-terminal amino acid of the disaccharide tetrapeptide in binding to the T cell receptor

The T cell line specific for a bacterial cell wall peptidoglycan subunit, disaccharide tetrapeptide of diaminopimelic acid type, was examined for epitope specificity in elicitation of delayed-type hypersensitivity (DTH) in X-irradiated Lewis rats, using pairs of analogs different in optical configuration of the COOH-terminal amino acid. The test cell line induced DTH against analogs with the COOH-terminal D-amino acid but not against those with the L-amino acid at the COOH terminus. A close correlation was found between the T cell line-induced DTH reaction in vivo and the proliferative response in vitro, in terms of clear discrimination of the optical configuration of COOH-terminal amino acid of disaccharide tetrapeptide. The L-isomers (non-stimulatory analogs of T cell proliferation) competitively inhibited the proliferation of the T cell line by the corresponding D-isomers. Thus the L-isomers appear to interact with Ia molecules on antigen-presenting cells. We conclude that COOH-terminal D-amino acid of the disaccharide tetrapeptide could be involved in binding to the T cell receptor, induction of T cell proliferation, and elicitation of DTH.