Kazue Fukushi

Biochemical and immunological comparison of lipopolysaccharides from Bordetella species.

Lipopolysaccharides (LPS) isolated from Bordetella pertussis, B. parapertussis and B. bronchiseptica were analysed for their chemical composition, molecular heterogeneity and immunological properties. All the LPS preparations contained heptose, 3-deoxy-D-manno-2-octulosonic acid, glucosamine, uronic acid, phosphate and fatty acids. The fatty acids C14:0, C16:0 and beta OHC14:0 were common to all the LPS preparations. LPS from B. pertussis strains additionally contained isoC16:0, those from B. parapertussis contained isoC14:0 and isoC16:0, and those from B. bronchiseptica contained C16:1. By SDS-PAGE, LPS from B. pertussis had two bands of low molecular mass, and the LPS from B. parapertussis and B. bronchiseptica showed low molecular mass bands together with a ladder arrangement of high molecular mass bands. Immunodiffusion, quantitative agglutination and ELISA demonstrated that the LPS from B. pertussis strains reacted with antisera prepared against whole cells of B. pertussis and B. bronchiseptica; LPS from B. parapertussis reacted with antisera to B. parapertussis and B. bronchiseptica, and LPS from B. bronchiseptica reacted with anti-whole cell serum raised against any of the three species. From these results, it is concluded that LPS from B. bronchiseptica has structures in common with LPS from B. pertussis and B. parapertussis, while the LPS from B. pertussis and B. parapertussis are serologically entirely different from each other.

Chemical Characterization of Lipopolysaccharides from Proteus Strains Used in Weil‐Felix Test

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil‐Felix test, were characterized by chemical analysis and SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). To separate the O‐polysaccharide, core‐oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G‐50 column. The core‐oligosaccharides contained L‐glycero‐D‐mannoheptose, D‐glycero‐D‐man‐noheptose, glucose (Glc), galactose, 3‐deoxy‐D‐mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN‐phosphate, and three fatty acids (myristic, palmitic, and β‐hydroxymyristic acids). However, the O‐polysaccharides of OX2‐ and OXK‐LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19‐LPS seemed to lack O‐polysaccharide.

Immunological Characterization of Lipopolysaccharides from Proteus Strains Used in Weil-Felix Test and Reactivity with Patient Sera of Tsutsugamushi Diseases

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil‐Felix (WF) test, were performed by quantitative agglutination, enzyme‐linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS. and the reaction was inhibited by the O‐polysaccharide fraction isolated from the homologous LPS except OX19‐LPS. which lacked O‐polysaccharide moiety. The immunological data support the findings that the O‐polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121 133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK‐WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK‐WC and OXK‐LPS independently of the titers of WF or IP tests.

Chemical and Ultrastructural Differences in Endotoxic Glycolipids from Salmonella Minnesota Re Mutant Extracted with Various Solvent Systems

Endotoxic glycolipids (ReGl) extracted from the whole cells (WC) and cell walls of heptose‐less Re mutant of Salmonella minnesota with hot phenol‐water (PW), phenol‐chloroform‐petroleum ether (PCP), and chloroform‐methanol (CM) were analyzed chemically and examined with an electron microscope. ReGl‐PW‐(WC) contained mannose and proteins as contaminants, ReGl‐PCP(WC) consisted of an excess amount of amino compound (cadaverine), and ReGl‐CM(WC) consisted of proteins and cadaverine, in addition to the ReGl constituents.

Chemical and Ultrastructural Comparison of Endotoxins Extracted from Salmonella minnesota Wild Type and R Mutants

Endotoxic lipopolysaccharide and glycolipids (RGl) extracted from Salmonella minnesota wild type and R mutant cells (chemotypes Ra, Rb, Re, Rd1, and Rd2), respectively, with hot phenol‐water (PW) and phenol‐chloroform‐petroleum ether (PCP) were analyzed chemically and electron microscopically. All RGl extracted with PW (RGl‐PW) contained excess amounts of phosphate, O‐ester linked fatty acids and neutral sugars, while all RGl extracted with PCP (RGl‐PCP) contained excess amounts of free amino groups and fatty acids, in addition to the RGl constituents. Polyamine (cadaverine), phosphoethanolamine, and an unidentified amino compound were contained in RGl‐PCP as free amino groups.

Electron Microscopic Studies of Lipopolysaccharides from Phase I and Phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.

Electron Microscopic Studies of Endotoxins Treated with Alkaline and Acid Reagents

Endotoxins extracted from Salmonella minnesota wild strain and R mutants (Ra to Re) were treated with two different alkaline reagents. Treatment with diluted sodium hydroxide, which caused partial removal of O‐ester linked fatty acids, changed the ultrastructures of endotoxins from an onion‐like structure to monolayer particles (approximately 100 Å in diameter) except for endotoxic glycolipids from Rd2 and Re mutants which showed mixed ultrastructures of untreated and treated endotoxins. Treatment with alkaline hydroxylamine, which caused liberation of all O‐ester linked fatty acids, changed the ultrastructures of all endotoxins to monolayer particles. The results suggested that the ultrastructures of alkaline‐treated endotoxins were dependent on the degree of their hydrophobicity. On the other hand, the micrographs of acid treated endotoxins did not show a constant structure because of the high hydrophobicity.

Effect of pH on Turbidity and Ultrastructures of Endotoxins Extracted from Salmonella Minnesota Wild Type and Re Mutant

It has been reported that the physical state of endotoxic lipopolysaccharide (LPS) is influenced by inorganic cations (Nat, K+, Mg2t, and Fe2+) and low molecular basic amines (putrescine, spermidine, spermine, and ethanolamine) (6, 8). On the other hand, Schramm et al (9) showed the ultrastructural differences between Escherichia coli LPS in pH 7 and 10 solutions. However, they did not observe these structures at acidic pH. In this paper, we observed the effect of pH on the turbidity and the ultrastructures of endotoxic LPS and glycolipid (ReG1) from Re mutant, and suggested that the ultrastructure and the turbidity of the endotoxins were under the influence of pH. S. minnesota 1114 (wild type) and R595 (Re mutant) were cultivated as described previously (1-3). The LPS and ReG1 were extracted with hot phenol-water (PW) according to the method of Westphal and Jann (10). The ReG1 was also extracted with phenol-chloroform-petroleum ether (PCP) according to the method of Galanos et al (7) and chloroform-methanol (CM) according to the method of Chen et al (4). The extracts (crude endotoxins) were purified by ultra-centrifugation (100,000 x g for 2 hr, repeated twice). Endotoxins and phosphatidylethanolamine (PEA, Sigma Chemical Co., St. Louis, MO, U.S.A.) (1 mg) were solubilized using an ultrasonic bath (Branson Cleaning Equipment, Shelton, CT, U.S.A.) in 2.2 ml each of the following 20 mm buffers; sodium citrate-Na2HPO4 (pH 3.0, 5.0, and 6.7), sodium citrate-NaOH (pH 3.0, 4.5, and 6.0), tris(hydroxymethyl)aminomethane-HC1 (pH 7.0 and 9.0), and glycine-NaOH (pH 9.0 and 11.0), and incubated at 60 C for 20 min. After standing for 30 min at room temperature, the turbidity of these samples was measured with the absorbance at 500 nm. Electron microscopy was performed as described previously (1-3). We measured the turbidity in different pH solutions of the LPS and ReG1 to determine the effect of pH on the bacterial endotoxins in aqueous solution. As shown in Fig. 1, the wild-type LPS in the acidic buffers (pH 3.0) represented relatively high turbidity (0.044-0.058 absorbance at 500 nm (Abs)/mg/2.2 ml), while the LPS in the buffers between pH 4.5 and 11.0 showed low turbidity (0.008-0.016