Masashi Mori

The bceT gene of Bacillus cereus encodes an enterotoxic protein.

A toxin gene (bceT) on a 2.9 kb DNA fragment of Bacillus cereus B-4ac was cloned and expressed in Escherichia coli, and its nucleotide sequence determined. The DNA fragment contained an open reading frame capable of encoding a polypeptide of 336 amino acids with a molecular mass of 41039 Da. The translated product in E. coli exhibited Vero cell cytotoxicity, and was positive in a vascular permeability assay. It also caused fluid accumulation in a ligated mouse ileal loop and was lethal to mice upon injection. These biological activities are considered characteristic of diarrhoeal enterotoxins. We therefore conclude that this gene, designated bceT, encodes one of the enterotoxic proteins of B. cereus which cause food-borne diarrhoea.

Chromosomal beta-lactamase of Klebsiella oxytoca, a new class A enzyme that hydrolyzes broad-spectrum beta-lactam antibiotics.

The chromosomally encoded beta-lactamase gene of Klebsiella oxytoca E23004, a strain resistant to cefoperazone and aztreonam, was cloned and expressed in Escherichia coli HB101. The molecular mass and pI of this enzyme were 28 kilodaltons and 7.4, respectively. Although the beta-lactamase of K. oxytoca hydrolyzed many cephalosporins, including broad-spectrum drugs, the nucleotide sequence and deduced amino acid sequence lacked homology with chromosomal class C beta-lactamase genes (ampC) of E. coli or Citrobacter freundii. Rather, about 45% nucleotide sequence homology and 40% deduced amino acid sequence homology were observed between the K. oxytoca beta-lactamase and TEM-1, a class A beta-lactamase which does not efficiently hydrolyze cephalosporins. Values of Km, relative Vmax, and relative Vmax/Km for the K. oxytoca beta-lactamase indicated that the enzyme is a penicillinase but that it can hydrolyze cefoperazone effectively and other broad-spectrum cephems weakly. Hence, the chromosomal beta-lactamase of K. oxytoca E23004 belongs to class A but differences in its amino acid sequence provide a broader spectrum of activity. Images

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.

Identification of Species and Capsular Types of Klebsiella Clinical Isolates, with Special Reference to Klebsiella planticola

In the 77 reference strains for Klebsiella K types, there are 17 strains (22.1%) of Klebsiella planticola, 6 strains (7.8%) of Klebsiella oxytoca, 1 strain (1.3%) of Klebsiella terrigena, and 53 strains (68.8%) of Klebsiella pneumoniae. The species K. planticola, which was originally isolated from botanical and aquatic environments and hence thus named, was also identified at high incidence (81 strains, 18.5%) among the 439 recent clinical isolates of Klebsiella species. Among these K. planticola strains of hospital origin, 52 (64%) were isolated from sputum, 17 (21%) from urine, and the remaining 12 (15%) from other sources. The capsular types of these isolates were determined by the gel precipitation reaction. Seventy of 81 K. planticola isolates (86.4%) were typable by antisera to Klebsiella reference strains for K types and the K types of the clinical isolates distributed to 35 kinds of K types. The proportion of typable strains among clinical isolates of K. planticola was very similar to those in K. pneumoniae (87.5%) and K. oxytoca (86.0%).

The Klebsiella O3 lipopolysaccharide isolated from culture fluid: structure of the polysaccharide moiety.

In a series of our earlier studies, the O3 antigen isolated from culture supernatant of Klebsiella pneumoniae strain Kasuya (O3:K1) (KO3) was shown to exhibit very strong adjuvant activity in mice. KO3 obtained was homogeneous in analyses by either gel filtration or ultracentrifugation. Its molecular weight determined by ultracentrifugal analysis was greater than 2 × 106. It contained 37.9% C, 6.20% H, 0.24% N, and less than 0.1% P. KO3 was degraded into the polysaccharide moiety and lipid moiety (about 20%) by hydrolysis with 1% acetic acid at 100 C for 1 hr. The molecular weight of the polysaccharide moiety obtained by the hydrolysis was 16,200 as determined by the Somogyi‐Nelson method. Chemical analyses using methylation analysis and Smith degradation as the principal methods indicated that the polysaccharide moiety consisted of a mannan which has a pentasaccharide repeating unit of α‐mannosyl‐1,3‐α‐mannosyl‐1,2‐α‐mannosyl‐1,2‐α‐mannosyl‐1,2‐α‐mannose joined through α‐1,3‐mannosyl linkages. The number of repetitions was less than 20. The fact that minor components such as 2‐keto‐3‐deoxyoctonate and glucose were detected suggests the presence of a core oligosaccharide, but its precise structure is unknown.

Structure of cereulide, a cyclic dodecadepsipeptide toxin from Bacillus cereus and studies on NMR characteristics of its alkali metal complexes including a conformational structure of the K+ complex

The structure and stereochemistry of cereulide 1 or 1a, an emetic toxin produced by Bacillus cereus, have been determined through spectral analysis and chemical methods. It is a 36-membered cyclic depsipeptide with the sequence of cyclo(D-O-Leu-D-Ala-L-O-Val-L-Val)3. The outstanding property of cereulide is a Rb+-and K+-ion-selective ionophore, the same as in valinomycin 2. Cereulide forms a 1:1 complex with alkali metal ions. Owing to its structural similarity to valinomycin 2 and our well established conformational knowledge about compound 2, the NMR data of cereulide–alkali metal complexes in chloroform gave us a partial view of the conformational structure in solution and a calculated modelling of the complex with K+ is proposed. The main chain shows a hexagonal cyclinder-like framework which is similar to the known bracelet-like structure of the valinomycin–K+ complex. All amide carbonyls arrange along the cylindrical side-wall planes so that they can form β-turn hydrogen bonds with NH protons. Every alpha carbon is located at a corner in both the top and the bottom planes, enabling alpha protons of oxy acids to turn inwards towards the cavity and those of the amino acids turn outwards. The K+ is centrally situated in the cavity of the host cereulide by the ion–dipole interactions with three carbonyl oxygens of L-Val in the top plane and the three carbonyls of D-Ala in the bottom plane. The experimental coupling constant and NOE data of the K+ complex had conformity with the calculated dihedral angle and distance measured from the proposed conformational structure. Partial views of the conformations of cereulide complexed with Li+ and Na+ are analysed as follows: The frameworks of the Li+ complex main chain lose their compactness, becoming more linearly extended, which results in a bigger cavity diameter, have fewer hydrogen bonds, and all the residual side-chains can freely rotate whereas the conformation(s) of the Na+ complex takes the intermediate conformation(s) between those of the highly stable (the K+ and Rb+) and that of the less stable (the Li+) complexes.

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.

Adjuvant action of capsular polysaccharide of Klebsiella pneumoniae on antibody response. VII. Further purification of the active substance.

Previous studies from this laboratory have demonstrated that the capsular polysaccharide of Klebsiella pneumoniae (CPS-K), prepared from the culture fluid of type 1 Kasuya strain, exhibits a strong adjuvant effect on antibody responses in mice to various antigens (9). Furthermore, CPS-K acts as a polyclonal B-cell activator (11), has interferon-inducing (7) and infection-promoting effects (6) and is pyrogenic for rabbits and lethal for mice (8). CPS-K is fractionated into acidic and neutral polysaccharide fractions (acidic CPS-K and neutral CPS-K) by the addition of cetyl-pyridinium chloride (4, 10). Previous studies have revealed that neutral CPS-K is chiefly responsible for these immunological and biological activities of CPS-K (6, 8, 10,11). Neutral CPS-K is antigenically distinct from acidic CPS-K which is the type-specific capsular antigen. The properties of the active substance have been characterized (10). However, the preparation of neutral CPS-K used in our previous study contained 6.8% hexuronic acids, indicating that a significant amount of acidic CPS-K still contaminated the preparation. In the course of investigation thereafter, we found that complete removal of acidic CPS-K from the preparation of neutral CPS-K was not necessarily easy. Then, we tried to purify the preparation further with neutral detergent Tween 20 and succeeded in obtaining a purified preparation of neutral CPS-K (PN CPS-K) which was free from contamination of acidic CPS-K. K. pneumoniae type 1 Kasuya strain isolated in our laboratory was used. CPSK was prepared from the culture fluid of Kasuya strain by the procedures described by Batshon et al ( 1) . The culture fluid (15 liters) was concentrated approximately 10-fold by evaporation at 40 C. After bacterial cells were removed by centrifugation, three volumes of 95% ethanol were added to the culture fluid. The precipitate was dissolved and dialyzed in water. The solution was deproteinized by treating 15 times with half volumes of a chloroform-butanol (5 : 1) mixture and 600-700 mg of CPS-K were obtained. The neutral fraction of CPS-K was obtained from CPS-K by using cetyl-pyridinium chloride as described previously (10). Acidic CPS-K was precipitated as a complex with cetyl-pyridinium chloride, while neutral CPS-K remained in the supernatant. Neutral CPS-K was precipitated from the supernatant by the addition of 95% ethanol and 60-70 mg of neutral CPS-K were obtained.

Serological response to P-fimbriae ofEscherichia coli in patients with urinary tract infections

The antibody response to P-fimbriae ofEscherichia coli in patients with upper urinary tract infections was investigated. In the sera of patients with pyelonephritis obtained at the initial visit to hospital (3 to 7 days after the onset of symptoms), a high incidence of antibodies to P-fimbriae was detected (12 out of 14 patients). P-fimbriatedEscherichia coli strains were isolated from urine samples in all of these antibody-positive patients. Antibodies detected by ELISA using purified antigen were essentially IgG and specifically recognized P-fimbriae. These antibodies inhibited completely, or in some cases partially, mannose-resistant hemagglutination with P-fimbriatedEscherichia coli.