Setsuko Naito

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type‐specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type‐specific capsular polysaccharide was isolated from ultraviolet‐irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine‐requiring marker by selecting unagglutinable cells with the antiserum to the type‐specific capsular polysaccharide. Serological tests showed that the type‐specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: relationship between lattice formation and uniform salt forms.

Various uniform salt forms of an R‐form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN‐111 (O3‐:K1‐) were prepared and their ultra‐structure was examined. The LPS, which was extracted by the phenol‐water method, freed from contamination with RNA by treatment with RNase, and precipitated by addition of two volumes of 10 mM MgCl2‐ethanol, was used as the original preparation for uniform salt forms. The original LPS preparation formed a hexagonal lattice structure with a lattice constant of 14.9±0.2 nm. The LPS after electrodialysis retained the ability to form a hexagonal lattice structure, although its lattice constant was large (18.7±0.5 nm) and the lattice structure of the electro‐dialyzed LPS was labile at pH 8.0 in contrast to that of the original LPS preparation. The magnesium salt form of the LPS formed essentially the same ordered hexagonal lattice structure (lattice constant of 15.0±0.2 nm) as that of the original LPS preparation. The calcium and ammonium salt forms formed a hexagonal lattice structure, but the lattice constants of the calcium and ammonium salt forms were larger (18.6±0.6nm and 19.3±0.4nm, respectively) than that of the magnesium salt form. The sodium and potassium salt forms consisted of freely branching ribbon‐like structures with an average width of 13 nm and an average thickness of 9 nm. The triethylamine salt form consisted principally of short rods (10 nm × 9–13 nm).

Potent adjuvant action of lipopolysaccharides possessing the O-specific polysaccharide moieties consisting of mannans in antibody response against protein antigen

It was previously reported that Klebsiella O3 lipopolysaccharide (LPS) exhibits extraordinarily strong adjuvant activity in augmenting antibody response against protein antigens in mice compared with other kinds of LPS, for example, LPS from Escherichia coli O55, O111, and O127 and Salmonella enteritidis. The present study was undertaken to clarify the relationship between the strong adjuvant activity in augmenting antibody response against deaggregated bovine gammaglobulin and the chemical structure of LPS. Among LPS from Klebsiella O1, O4, O5, and O7, only O5 LPS exhibited nearly the same degree of the strong adjuvant activity as did O3 LPS. The adjuvant activity of the other LPS was very weak in a degree similar to that of LPS from E. coli O55 and O127. Even when the natural forms of Klebsiella O3 LPS and O1 LPS were converted to various defined uniform salt forms, their adjuvant activity did not significantly differ from that of the respective natural forms. It is therefore unlikely that the difference in strength of the adjuvant activity between Klebsiella O3 LPS and O1 LPS is due to the difference in their salt forms. The common feature in the structures of Klebsiella O3 LPS and O5 LPS is their O-specific polysaccharide chains consisting of the mannose homopolysaccharides (mannans). LPS from E. coli O8 and O9, the O-specific polysaccharide chains of which consist of the mannans, also exhibited much stronger adjuvant activity than do LPS from E. coli O55 and O127, and the strength of the adjuvant activities of the former two was comparable to that of LPS from Klebsiella O3 and O5. On the other hand, LPS from Klebsiella O3 and O5 and E. coli O8 and O9 showed the ability to activate B lymphocytes polyclonally in vivo in a degree similar to that of the other kinds of LPS. From the present results it can be concluded that LPS possessing the O-specific polysaccharide moieties consisting of the mannans exhibit extraordinarily strong adjuvant activity in augmenting antibody response against protein antigen.

Experimental Murine Model for Autoimmune Myocarditis Using Klebsiella Pneumoniae O3 Lipopolysaccharide as a Potent Immunological Adjuvant

Experimental autoimmune myocarditis could be produced in mice by repeated injection of syngeneic heart extract together with Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) as a powerful adjuvant. Histological changes in the cardiac lesions were characterized by infiltration with mononuclear cells in the myocardium, degeneration and loss of myocardial fibers, and replacement of granulation tissues. No such cardiac lesions were produced in mice receiving injections of heart extract alone or KO3 LPS alone. Development of the autoantibody and the delayed type-hypersensitivity (DTH) against syngeneic heart extract was found in mice immunized repeatedly with the mixture of heart extract and KO3 LPS. Moreover, definite cardiac lesions were produced in normal recipient mice by transfer of sensitized spleen cells from hyperimmunized mice. Therefore, it was suggested that those cardiac lesions were caused by the autoimmune mechanism. Our methodology provided a new experimental murine model for autoimmune myocarditis.

In Vitro Hexagonal Assembly of Lipopolysaccharide of Escherichia coli K-12

We examined Escherichia coli K‐12 lipopolysaccharide (LPS), which is known to be an R‐form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K‐12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mm MgCl2‐ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2±0.3 nm and that of the calcium salt form 18.5±0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.

Ultrastructure of Klebsiella O3 Lipopolysaccharide Isolated from Culture Supernatant: Structure of Various Uniform Salt Forms

Various uniform salt forms of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant were prepared as follows. Basic materials present in KO3 LPS were rigorously removed by electrodialysis and the electrodialyzed KO3 LPS was neutralized with NaOH, KOH, NH4OH, Ca(OH)2, tris(hydroxymethyl)aminomethane, or triethylamine. The ultrastructure of the uniform salt forms of KO3 LPS was examined using preparations stained with uranyl acetate. The sodium, potassium, ammonium, and trisaminomethane salt forms were structurally very similar to the natural form of KO3 LPS which consisted of a mixture of flat ribbon‐like structures (average width of 16 nm and average thickness of 7 nm) and spheres with various diameters, both covered with fine hairy structures. When KO3 LPS was converted to the triethylamine salt form, the ribbon‐like structures were distrupted into very small granules (7–9 nm × 9–15 nm). The calcium salt form consisted of particles and rods of various sizes and ribbon‐like structures which were markedly extended (maximum width of 50 nm) and presented irregular shapes. When converted to the calcium salt form, the ribbon‐like structures were extended and eventually divided into particles and rods. For reasons still unknown, the sodium salt of KO3 LPS was mostly stained positively with uranyl acetate in contrast to the natural form and the other uniform salt forms which were always negatively stained. In the positively stained preparation of the sodium salt form, it was clearly shown that the ribbon‐like structures consisted of a bilayer.

Further studies of the polysaccharide of Klebsiella pneumoniae possessing strong adjuvanticity. II. Serological relationship between the adjuvant polysaccharide and O3 antigen of Klebsiella.

The serological specificity of the neutral polysaccharide possessing extraordinarily strong adjuvanticity originally isolated from the culture supernatant of Klebsiella K1 strain Kasuya has been investigated. Among all of the reference strains (K1–K82) of Klebsiella obtained from the International Escherichia and Klebsiella Center, Statens Seruminstitut, Copenhagen, only 13 strains have been shown to produce the adjuvant polysaccharide by the passive hemagglutination inhibition test. All of these 13 strains belong to the 03 group, and the strains which belong to other O groups or O groups of which were not identifiable did not produce it. The gel precipitation test has demonstrated that the adjuvant polysaccharide is antigenically identical to O3 antigen isolated from the cells of the decapsulated mutant (strain LEN 1) of Klebsiella K1 strain Kasuya and to O9 antigen of Escherichia coli isolated from either the culture supernatant or the cells, which has already been shown to be antigenically and structurally identical to the O3 antigen of Klebsiella.

Rapid small-scale preparation method of cell surface polysaccharides.

A rapid small‐scale method of extraction of lipopolysaccharide (LPS) and capsular polysaccharides was developed for the purpose of identification of chemotypes of LPS and serotypes of capsular antigens. Cell surface polysaccharides were prepared within less than 2 hr from 1.5 ml of broth or suspension of colonies cultured overnight. The preparations were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for LPS, and by double diffusiori gel precipitation (Ouchterlony) test and blotting to nitrocellulose membrane for capsular polysaccharide. The analyses with the preparations obtained by the method could provide adequate results capable of identifying chemotypes of LPS and serotypes of capsular antigens.

Staining of the O-Specific Polysaccharide Chains of Lipopolysaccharides with Alkaline Bismuth

granules occurred on the LPS (3). We also showed that Klebsiella O3 LPS and Salmonella minnesota S-form LPS were positively stained with ruthenium red, which is known to stain polysaccharides in the slime layer or the capsule of bacterial cells (5), whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-), Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by the treatment (4). Alkaline bismuth has been used for electron microscopic observation of glycogen granules in the liver cells (6). As far as we are aware, there has been no report dealing with application of alkaline bismuth to staining of the polysaccharide moiety of LPS. In the present study, we tried to stain LPS with alkaline bismuth and found that the staining procedure gave sufficient results for distinguishing between Sand R-form LPS lacking the

Stability of the Hexagonal Lattice Structure Formed by an R-Form Lipopolysaccharide of Klebsiella: Decrease in the Stability by Electrodialysis and Recovery by Addition of the Magnesium

The R‐form lipopolysaccharide (LPS) from Klebsiella strain LEN‐111 (O3‐:K1‐) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2‐ethanol. When the LPS was suspended in various buffers (50 mM) at pH 2 to 12 for 24 hr at 4 C, at pH 2 and 3 pits of the hexagonal lattice structure markedly disappeared, at pH 4 to 8.5 the lattice structure was stable, and at pH 9 to 12 it tended to loosen somewhat. The LPS from which cations were removed by electrodialysis retained the ability of hexagonal assembly, although the lattice constant of the hexagonal lattice of the electrodialyzed LPS was large. The lattice structure of the electrodialyzed LPS was much more labile than that of the non‐electrodialyzed LPS at alkaline pH levels and the former was completely disintegrated into ribbon‐like structures when the LPS was suspended in 50 mM Tris buffer at pH 7.7 or higher. However, the electrodialyzed LPS formed a hexagonal lattice structure in Tris buffer at pH 8.5 containing 0.1 to 100 mM MgCl2. The lattice constants of the hexagonal lattice formed by the electrodialyzed LPS at 10 or 100 mM MgCl2 were very similar to that of the lattice of the non‐electrodialyzed LPS. From these results it is concluded that the lability of the hexagonal lattice structure of the electrodialyzed LPS at alkaline conditions is due to removal of Mg2+ by electrodialysis.