Kazuhito Hisatsune

An immunochemical study of serological cross-reaction between lipopolysaccharides from Vibrio cholerae O22 and O139.

A comparative chemical and serological study of the LPS of Vibrio cholerae O139 and O22 was performed. Chemical analysis revealed that the sugar composition of the LPS of strain O22 was quite similar to that of O139 LPS. Each contained D-glucose, L-glycero-D-manno-heptose, colitose (3,6-dideoxy-L-galactose), D-fructose, D-glucosamine, D-quinovosamine and D-galacturonic acid. The O-antigenic relationship between the two strains was analysed by passive haemolysis (PH) and passive haemolysis inhibition (PHI) tests with the respective LPS being used as antigens to sensitize sheep red blood cells (SRBC) and, in the latter case, as inhibitors in a PH system that consisted of LPS-sensitized SRBC, guinea-pig complement and anti-O139 or anti-O22 antiserum, both unabsorbed and absorbed with the heterologous antigen. In the PH experiment, unabsorbed anti-O139 antiserum had haemolytic titres of 66,000 and 22,000 against O139 LPS- and O22 LPS-sensitized SRBC, respectively; unabsorbed anti-O22 antiserum had haemolytic titres of 900 and 13,000, respectively. Thus, the anti-O139 antiserum contained an antibody that reacted with a heterologous O22 antigen at a high titre (22,000) and this antibody was completely removed from anti-O139 antiserum with the O22 antigen. The anti-O22 antiserum contained an antibody that reacted with the heterologous O139 antigen at a low titre (900) and this antibody was completely removed from anti-O22 antiserum with the O139 antigen. In PHI tests O139 LPS and O22 LPS each strongly inhibited (the ID50 of LPS ranged from 0.03 to 0.14 microgram ml-1) the heterologous haemolytic systems of both O139 LPS-sensitized SRBC/anti-O22 antiserum and O22 LPS-sensitized SRBC/anti-O139 antiserm, which are substantially equivalent to the common antigen factor in the O139 LPS-sensitized SRBC/anti-O22 antiserum system and the common antigen factor in the O22 LPS-sensitized SRBC/anti-O139 antiserum system, respectively. The results indicated that the O antigen of O139 is closely related to that of O22 in an a,b-a,c type of relationship where a is common antigenic factor, b is an O139-specific antigenic factor and c is an O22-specific antigenic factor.

The O polysaccharide chain of the lipopolysaccharide from Vibrio cholerae O76 is a homopolymer of N-[(S)-(+)-2-hydroxypropionyl]-alpha-L-perosamine.

Chemical and serological studies of LPS from Vibrio cholerae O76 (O76) were performed. The LPS of O76 contained D-glucose, D-galactose, L-glycero-D-manno-heptose, D-fructose, D-glucosamine, D-quinovosamine (2-amino-2,6-dideoxy-D-glucose) and L-perosamine (4-amino-4,6-dideoxy-L-mannopyranose). The sugar composition of the LPS from O76 was quite similar to that of LPS from V. cholerae O1 with the exception of the presence of a small amount of D-galactose in the LPS of O76. However, perosamine, a major sugar component of the LPS from O76, was in the L configuration in contrast to the D configuration of the perosamine in the LPS of V. cholerae O1. The L-perosamine was N-acylated with an (S)-(+)-2-hydroxypropionyl group in the LPS from O76. Structural analysis by NMR spectroscopy, as well as GC/MS, revealed that the O polysaccharide chain of the LPS from O76 was an alpha(1-->2)-linked homopolymer of N-[(S)-(+)-2-hydroxypropionyl]-L-perosamine. The serological cross-reactivity between the LPS of O76 and the LPS from other strains, such as V. cholerae O1 (Ogawa and Inaba O forms), Vibrio bio-serogroup 1875 (Original and Variant strains), V. cholerae O140 (Hakata) and Yersinia enterocolitica O9, was examined in passive haemolysis tests with sheep red blood cells that had been sensitized with LPS and antisera raised against whole cells of these bacteria. The latter six strains have in common the O antigen that includes Inaba antigen factor C, in addition to their own O-antigenic factors. Thus, they crossreact serologically. The O polysaccharide chains of the LPS of these six trains are known to consist exclusively of alpha(1-->2)-linked D-perosamine homopolymers and differences are found only among the N-acyl substituents. In passive haemolysis tests, the LPS of O76 did not cross-react serologically with any of the other LPS examined. Thus, the results obtained in this study support the hypothesis that Inaba antigen factor C, associated with the O antigens of these six strains, which include V. cholerae O1, is related substantially and exclusively to their alpha(1-->2)-linked homopolymers of N-acylated D-perosamine, and not to such homopolymers of N-acylated L-perosamine.

Relationship between structure and antigenicity of O1 Vibrio cholerae lipopolysaccharides

The relationship between the release of fructose from O1 Vibrio cholerae lipopolysaccharides (LPS) by dilute acetic acid hydrolysis and the decrease in their antigenicity was examined. Decrease in the antigenicity of LPS was not parallel with the release of fructose, and occurred very much later than the latter. Periodate oxidation of LPS resulted in the total elimination of the fructose and glucose, and two-thirds of the heptose constituents, but no difference in the antigenicity of LPS was observed before and after oxidation. These findings indicate that the fructose present in O1 V. cholerae LPS is not substantially involved in their specific antigenicity. In the O1 V. cholerae LPS, the fructose is in the branch structure, most probably in the core region.

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238.

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.

Immunological Properties of Vibrio cholerae Lipopolysaccharides

Immunological effects of wall lipopolysaccharide (LPS) preparations obtained from Vibrio cholerae Inaba 569B, Ogawa NIH 41 and NAG 4715 strains by the hot phenol‐water procedure were examined in mice. Although these LPS lack KDO, which are basic components of the core region of most gram‐negative LPS, they still have potencies as B‐cell mitogens, adjuvants, immunosuppressants, polyclonal B‐cell activators and phagocytic stimulants for macrophages. The activities of these V. cholerae LPS on murine immune system seemed to be weaker than those of Salmonella typhimurium LT2‐LPS. Among these V. cholerae LPS, NAG 4715‐LPS showed the strongest mitogenic activity and phagocytic stimulation, while the potencies of this NAG 4715‐LPS for the induction of polyclonal B cell activation, adjuvant effects and immunosuppression did not seem to be greater to those of the other LPS.

Correlation between the capacity of bacterial lipopolysaccharide to suppress experimental allergic encephalomyelitis and its mitogenic activity for lymph node cells in guinea pigs

Protection of guinea pigs from experimental allergic encephalomyelitis (EAE) was attempted using bacterial lipopolysaccharide (LPS) from 4 sources. The ability of these LPS to induce DNA synthesis in guinea pig lymph node (LN) cells in vitro was also investigated. It was found that there existed a good correlation between the capacity of LPS to suppress EAE and their degree of mitogenic activities for LN cells. LPS from Escherichia coli 0111:B4 (Ec-LPS), which was most effective in suppressing EAE and also best inducer of DNA synthesis in LN cells, enhanced the proliferation of cells forming antibody to myelin basic protein (BP) in the regional LN. These results, in addition to the previous report, suggested that at the inductive phase the proliferation of B lymphocytes or their products, antibodies to BP, could inhibit formation of T lymphocytes sensitized to BP, resulting in suppression of EAE. Lipid A but not PS fraction of Ec-LPS showed a protective activity against EAE and a mitogenic activity for LN cells although less so than whole LPS. In addition, Lipid A appeared to exert its mitogenic effect mainly on B rather than on T lymphocytes.

Differences among Shigella spp. in Susceptibility to the Bactericidal Activity of Human Serum

Clinical isolates of Shigella spp. were examined for their susceptibility to human serum. The susceptibility of the strains to immune and nonimmune human serum was dependent upon the size of the bacterial inoculum and the concentration of serum. There were differences among Shigella spp. in susceptibility to human serum: S. sonnei strains were the least susceptible, strains of S. boydii and S. flexneri serotype 6 were intermediate, and those of S. flexneri other than serotype 6 and S. dysenteriae were the most susceptible. Experiments in which heat-treated (56 degrees C for 30 min, or 50 degrees C for 20 min) serum was used, and analysis of activation of complement by lipopolysaccharides (LPS) from each Shigella sp., suggested that LPS composition, especially the O antigen polysaccharide chains, contributes to the differences among Shigella spp. in susceptibility to human serum.