Endotoxic Properties Research Articles

The chemical structure of bacterial endotoxin in relation to bioactivity

Lipopolysaccharides (LPS) constitute the O-antigens and endotoxins of Gram-negative bacteria. Whereas both the polysaccharide and lipid portion of LPS contribute to the pathogenic potential of this class of bacteria, it is the lipid component (lipid A) which determines the endotoxic properties of LPS. The primary structure of lipid A of various bacterial origin has been elucidated and Escherichia coli lipid A has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique structural arrangement and conformation. Such analyses have furthermore provided insight into the determinants required for lipid A binding to and activation of human target cells. Present research efforts aim at the molecular characterization of the specificity, modulation and biomedical consequences of the interaction of lipid A with host cells.

Synthesis, characterization, and some immunological properties of conjugates composed of the detoxified lipopolysaccharide of Vibrio cholerae O1 serotype Inaba bound to cholera toxin.

Protection against cholera has been correlated with the level of serum vibriocidal antibodies. The specificity of these vibriocidal antibodies was mostly to the lipopolysaccharide (LPS). We synthesized conjugates of detoxified LPS with cholera toxin (CT) and other proteins in order to elicit serum LPS antibodies with vibriocidal activity. Treatment with hydrazine (deacylated LPS) reduced the endotoxic properties of the LPS to clinically acceptable levels and resulted in a molecule larger and more antigenic than the saccharide produced by acid hydrolysis. More immunogenic conjugates resulted from multipoint compared with single-point attachment of the deacylated LPS to the protein. The conjugates containing CT had low levels of pyrogen and no toxic activity upon Chinese hamster ovary cells and elicited booster responses of vibriocidal and CT antibodies when injected subcutaneously as saline solutions into mice; the vibriocidal titers were similar to those elicited by comparable doses of cellular vaccines. We suggest how serum vibriocidal antibodies might prevent cholera.

Interaction of melittin with endotoxic lipid A

Several amphipathic and cationic substances are known to bind lipid A, the toxic component of bacterial lipopolysaccharides. In this report, we have characterized, by fluorescence methods, the interaction of melittin, an amphipathic and basic 26-residue polypeptide isolated from bee venom, with lipid A. The stoichiometry of the complex appears to be two molecules of melittin to one of lipid A with a dissociation constant of 2.5 × 10 −6 M . The binding of melittin not only modifies the endotoxic properties of lipid A in a number of biological assays, but also results in abrogation of the hemolytic activity of melittin. A model of the complex is proposed based on the known structures of lipid A and melittin, and the observed stoichiometry of binding.

Lipid a mutants of [formula omitted]: Isolation and partial characterization

Vibrio cholerae mutants resistant to common antibiotics and neutral and anionic detergents were isolated. Analysis of isolated outer membranes revealed a significant deficiency in the acylation of lipid A in the resistant strains. The content of amide-linked and ester-bound fatty acids in the lipid A of the mutant strains compared to that of the wild type was about 50–56% and 29–37% respectively. This defect was specific for lipid A as there was no change in the acylation of phospholipids. The reduction in fatty acid content of lipid A was reflected in the altered endotoxic properties in the mutant strains.

Endotoxic properties of chemically synthesized lipid A analogs. Studies on six inflammatory reactions in vivo, and one reaction in vitro.

Biological activities of two groups of synthesized lipid A analogs, the counterpart of biosynthetic precursor, Lehmann's Ia type, 406, and E. coli lipid A type, 506, as well as their non-phosphorylated, and mono-phosphorylated analogs were investigated. The activities employed included four bone marrow cell reactions in mice, mice skin reaction, leukocytes migration in rabbits' cornea, and hemagglutination. Compound 406 and 506 elicited bone marrow reactions in mice and hemagglutination of mouse RBC, although 406 failed to elicit hemorrhage and necrosis also in mice skin. Compound 406 did not elicit corneal reaction in rabbits. The results suggest that for elicitation of this reaction and mice skin reaction, acyloxyacyl structure is required. Cytotoxicity and thromboplastin production of four bone marrow reactions had been reported by us to be endotoxic reactions, since these had not been elicited by peptidoglycan of Lactobacillus and Staphylococcus (1981) and 300 series synthesized analogs (1984) which did not have endotoxic structures. From these results, it seems that these two marrow reactions and hemagglutination require, as does the limulus test, the lipid A part structure as is present in 406.

Chemical and biological properties of a phenol-water extract from leptospira interrogans. Evidence for the absence of lipopolysaccharide

Leptospira interrogans, serovar copenhageni was extracted by the phenol-water method and the resulting preparation examined for chemical composition and endotoxic activity. Chemical analysis revealed that a number of sugars were present, however, the amount of lipid content was very low. Further, the preparation was devoid of characteristic endotoxic properties, like lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction. The extract, however, was active in the limulus lysate gelation test and in the induction of monocyte activation. It is concluded that the leptospira preparation is devoid of endotoxin properties, both from the chemical and from the biological point of view.

The Enzymatic Synthesis of Lipid A: Molecular Structure and Biologic Function of Monosaccharide Precursors

Certain Escherichia coli mutants, deficient in phosphatidylglycerol, accumulate novel, glucosamine-derived phospholipids that appear to be very early precursors of lipid A. The simplest of these, lipid X, is a derivative of glucosamine-1-phosphate substituted with beta-hydroxymyristoyl moieties at positions 2 and 3. The discovery of lipid X makes it possible to predict a biosynthetic pathway and a unified structure for lipid A. In addition, the minimal molecular requirements for the mitogenic and endotoxic properties of lipopolysaccharides can be elucidated with compounds of this kind.

The genus-specific antigen of Chlamydia: resemblance to the lipopolysaccharide of enteric bacteria.

A strong immunological cross-reaction between a major glycolipid antigen of Chlamydia and the innermost (Re) core of the lipopolysaccharide of enteric bacteria was demonstrated with the aid of mutants in which the Re structure is exposed. The chlamydial glycolipid resembled the Re lipopolysaccharide in molecular size, solubility, and endotoxic properties and may thus be functionally equivalent to lipopolysaccharide, an essential and characteristic component of the outer membrane of Gram-negative bacteria.

Characterization of the lipopolysaccharide from Vibrio cholerae 395 (Ogawa).

The chemical structure and biological properties of the lipopolysaccharide (LPS) from Vibrio cholerae 395 (Ogawa), isolated by the phenol-water procedure, were studied. Upon acid hydrolysis, the LPS was split into its polysaccharide and lipid A moieties. The polysaccharide contained both neutral (glucose, heptose, fructose) and amino (glucosamine, quinovosamine) sugars. The LPS contained the acid-labile amino sugar, 4-amino-arabinose, which was absent in the Inaba serotype of V. cholerae. The LPS differed from the LPSs of Enterobacteriaceae by the absence of 2-keto-3-deoxyoctonate and the presence of fructose. Analysis of the methylated polysaccharide by gas-liquid chromatography and mass spectrometry showed that it had a branched structure with glucose and heptose residues primarily appearing at the nonreducing-end groups. Interactions with lectins, concanavalin A. and wheat germ agglutinin suggested that terminal glucose residues were alpha linked, whereas terminal glucosamine residues were connected by alpha-1,3 linkages. The major fatty acids of the LPS were C14:0, C16:0, C12h:0, and C14h:0 compounds, of which only the C14h:0 were amide linked, the remainder being ester linked to the backbone. Biological studies showed that the LPS possessed endotoxic properties such as lethality, pyrogenicity, limulus lysate gelation, and ability to induce non-specific resistance to infection. Thus, the LPS from V. cholerae 395 (Ogawa) possessed both common and distinct features as compared with the LPSs from the Enterobacteriaceae.

Biological activities of fragments derived from Bordetella pertussis endotoxin: isolation of a nontoxic, Shwartzman-negative lipid A possessing high adjuvant properties.

Endotoxin from fresly sedimented Bordetella pertussis cells, isolated by the phenol/water procedure when submitted to kinetically controlled, mild acidic hydrolysis released a polysaccharide (polysaccharide 1), a complex lipid (lipid X), and a glycolipid. When treated with somewhat stronger acid, the glycolipid yielded a second polysaccharide (polysaccharide 2) and another complex lipid (lipid A). The intact pertussis endotoxin had all the usual properties of endotoxins extracted from enteric bacteria. Lipid X and the intermediary glycolipid retained all the endotoxic properties of the unfractionated endotoxin. In lipid A, pyrogenicity was reduced to a very low level and toxicity and Shwartzman reactivity were absent; however, this fraction retained most of the endotoxin's antiviral activity, and its adjuvant power was considerably higher than that of the intact endotoxin. Lipid A elicited nonspecific resistance against challenge with certain bacteria, but not against others.

The modification of biophysical and endotoxic properties of bacterial lipopolysaccharides by serum.

Normal rabbit serum reduces the buoyant density of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (d = 1.44 g/cm3) and Salmonella minnesota R595 (d = 1.38 g/cm3) to a value less than g/cm3. This density shift is associated with the inhibition of a number of endotoxic activities of the LPS; namely, the pyrogenic activity, the ability to produce an immediate neutropenia in rabbits, lethality in adrenalectomized mice, and anticomplementary activity. A qualitatively similar change in buoyant density was observed to occur after intravenous injection of the LPS into rabbits. Preliminary evidence suggests that the density shift does not occur as a result of the degradation of the glycolipid backbone of the LPS. These data suggest that the interactions of LPS with plasma (or serum) components leading to reduction in buoyant density may account for a major pathway of LPS detoxification.

Studies on the Chemical Composition of Lipopolysaccharide from Neisseria meningitidis Group B

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.

Relationship of structure to function in bacterial endotoxins. IX. Differences in the lipid moiety of endotoxic glycolipids

Chemical, immunochemical, chromatographic, and endotoxic properties of five chromatographically pure glycolipids were compared. The preparations were extracted by chloroform-methanol from three Escherichia coli, one Salmonella minnesota, and one S. typhimurium Re heptoseless mutant strains. The local Shwartzman skin assay, the nonspecific resistance-enhancing effect, and the Limulus assays could not distinguish among the five glycolipids, all five being active in all three assays. Significant differences could be seen when the tumor resistance-enhancing effect of the glycolipids in mice was compared with the nonspecific TA3-Ha murine mammary adenocarcinoma growing in ascites form. Even greater variation was observed in the capacity of the preparations to enhance the nonspecific resistance of mice to virulent S. typhi 0901 infections. The data show that the five glycolipids are quite dissimilar in their biological effects. Similarly, thin-layer chromatography and molecular ratio determinations showed that differences exist in the chemical structure of the glycolipids. Accordingly, we claim that not only the polysaccharide but the lipid moiety as well may vary in various gram-negative endotoxin preparations.

Effects of a bacterial vaccine on the marginal zone of the spleen.

AbstractThe effect of systemic administration of a typhoid vaccine (T.A.B.) on the marginal zone of rat spleen has been investigated by light and electron microscopy. This bacterial preparation damages granulocytes in the circulating and marginated pools and the damaged cells are sequestered in the spleen, particularly in the marginal zone. The activities of the vaccine are attributed to its endotoxic properties. Terminations of arterial vessels in the marginal zone are also described.

Isolation and Biological Characterization of Pasteurella pestis Endotoxin

Endotoxin containing 2.1% nitrogen, 1.6% phosphorus, 22.5% neutral hexose, 15% hexosamine, 25% esterified and amide-linked fatty acids, and 1.4% protein was isolated from Pasteurella pestis strain Alexander by slight modification of a method adapted by Tauber and Russell. The lipopolysaccharide exhibited classical endotoxic biological properties including: (i) toxicity in mice, guinea pigs, and rabbits; (ii) antigenicity in rabbits; (iii) capacity to evoke a biphasic pyrogenic response in rabbits; (iv) capacity to induce tolerance in mice to the lethal effect of endotoxin; (v) capacity to stimulate rapidly acquired resistance in mice to bacterial infection, and (vi) the capacity to produce the localized and generalized Shwartzman phenomena in rabbits. Findings obtained during the study concerning the occurrence, isolation, toxicity, and other biological properties of P. pestis endotoxin provide new evidence that endotoxin could contribute to death in plague.

Isolation, Purification and Physico‐Chemical Characteristic of Shigella sonnei Phase I Free Endotoxin

The specific extracellular material with endotoxic properties (free endotoxin) was isolated from liquid culture of Shigella sonnei phase I.Bio‐Gel P‐300 filtration and subsequent ultracentrifugation at 10500μg was used to purify and separate free endotoxin into two fractions: the sediment and the supernatant.The sediment fraction was identified as lipopolysaccharide‐protein complex of the molecular weight of several millions, containing the same constituents as cell lipopolysaccharide of Shigella sonnei phase I, prepared by phenol–water extraction. This substance was a better endotoxin than cell lipopolysaccharide; it showed strong toxicity (L.D.50= 15 μg), pyrogenicity and elicited local and general Shwartzman reaction.The supernatant fraction represents phase I specific substance practically free of heptose, 2‐keto‐3‐deoxyoctonic acid and phosphorus. It contained glucose, galactose and glucosamine as does phase I lipopolysaccharide, and also galactosamine and small amounts of mannose. Phase I specific substance was very active serologically and revealed very weak endotoxic properties (L.D.50= 1800 μg). The homogeneity of the supernatant fraction was proved in sedimentation experiments, free electrophoresis and rechromatography on Bio‐Gel P‐300 and Sephadex G‐200 columns. The examination of hydrodynamic properties (sedimentation coefficient, limiting viscosity number, diffusion constant and partial specific volume) of the supernatant fraction showed that this substance has a linear structure with molecular weight 90800, and diameter and length of the molecule of about 15 Å and 800 Å, respectively.

Relationship of chemical structure to hemodynamic properties of endotoxins.

SummaryThe present study was carried out to gain insight into the relationship between chemical structure and hemodynamic properties of endotoxins. Preparations of crude endotoxins, obtained by the trichloroacetic acid extraction of two strains of S. marcescens, were fractionated by hot aqueous phenol treatment and sequential acetone precipitation and the carbohydrate, uronic acid, aminonitrogen, and fatty acid content of each fraction were determined. The effect of fractionation on the endotoxic properties was followed by measuring changes in systemic arterial pressure, portal venous pressure, heart rate, blood pH, and hematocrit produced by intravenous administration of the fractions in dogs. Results indicate that the crude endotoxins of mixture of macromoliecular compounds and that sequential removal of nucleic acids, acidic polysaccharides, and peptidolipids had little effect on the hemodynamic properties of remaining lipopolysaccharides. It is suggested that a macromolecular fatty acid—carbohydrate c...

Endotoxic properties of gramnegative bacteria and their susceptibility to the lethal effect of normal serum.

effect of normal serum (Rowley, 1956), (b) an array of normal serum components including specific antibody, complement and lysozyme are required for conversion of Gram-negative bacteria to spheroplasts (Michael and Braun, 1959; Carey et al, 1960), and (c) in the course of the bactericidal action of serum there occurs a striking reduction in the endotoxic and antigenic properties of the bacteria (Landy et al, 1960). In the light of these developments implicating the somatic complexes as at least the primary target for the bactericidal effects of serum, an attempt was made to determine whether marked differences in the bactericidal effectiveness of serum on members of antigenically related bacterial strains are related to the content of endotoxin in these bacteria.

Endotoxic properties associated with cell walls of group A streptococci.

Journal Article Endotoxic Properties Associated with Cell Walls of Group A Streptococci Get access Bob S. Roberson, Bob S. Roberson Search for other works by this author on: Oxford Academic PubMed Google Scholar John H. Schwab John H. Schwab Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 108, Issue 1, January 1961, Pages 25–34, https://doi.org/10.1093/infdis/108.1.25 Published: 01 January 1961 Article history Received: 31 May 1960 Published: 01 January 1961

Absence of endotoxic activity in materials derived without bacterial contamination from mammalian tissue.

SummaryIn the attempted isolation from mammalian tissues of polysaccharides with endotoxic properties, only materials devoid of such activity were obtained under conditions in which exclusion of bacteria was demonstrated.