Endotoxins stimulate generation of superoxide radicals and lipid peroxidation in blood platelets

Lipopolysaccharide (LPS, endotoxin) is an important structural constituent of the membrane of gram-negative bacteria with a wide range of biological effects. It can activate blood platelets. The purpose of present study was to determine the direct effect of endotoxins from Proteus mirabilis, differing significantly in their composition, on the generation of superoxide radicals and thiobarbituric acid reactive substances (TBARS) in blood platelets. Superoxide radicals were measured by means of superoxide dismutase-inhibitable reduction of cytochrome C. The TBARS determination (malonyldialdehyde) was used as a marker of endogenous arachidonate metabolism and thromboxane A2 synthesis. Results demonstrate that three endotoxins (LPS S1959, LPS R110, LPS R45) after 2 min of action, even at the lowest concentration (0.03 microg/10(8) platelets) stimulated the generation of TBARS and release of superoxide radicals. All LPS contain lipid A as a component but differ in their chemical composition in the polysaccharide part. It is suggested that the observed effects of LPS on blood platelets are attributable to their lipid A portion.     

Composition of the fractions separated by polyacrylamide gel electrophoresis of the lipopolysaccharide of a marine bacterium.

The sugar composition of lipopolysaccharide (LPS) isolated from whole cells of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonas B-16, ATCC 19855), variant 3, of the lipid A, core, and side-chain fractions derived from it, and of the LPS fractions (LPS I, II, and III) obtained by subjecting it to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been determined. Conditions optimum for the release of constituent monosaccharides by hydrolysis were established. Sugars were quantitated by gas-liquid chromatography of their alditol acetate derivatives. Lipid A was detected by gel electrophoresis and by the spectral shift obtained with a carbocyanin dye. A comparison of the molar ratios of the various fractions suggest that LPS III is an LPS molecule lacking an O-antigenic side chain, whereas LPS I and II are LPS molecules differing in side-chain composition. LPS I may be a mixture of two LPS species. In double immunodiffusion experiments using anti-whole-cell serum, LPS I and II showed a homologous cross-reaction with isolated whole-cell LPS. LPS III as well as lipid A, core, and side-chain fractions failed to give rise to precipitin lines.     

A rapid method for determining the fatty acid composition of lipid A

Lipid A is the most conservative part of LPS. Its fatty acids composition can serve as an important taxonomic marker of bacteria. The isolation of LPS and studying their chemical composition are difficult and protracted procedure. We propose the rapid method of determining the prevailed fatty acids of lipid A without isolation of LPS from the cell. The essence of the method is in the release of cell from the lipids which are not components of LPS. These lipids, in contrast to the lipid A, are more easily extractable from the cell structures. The fatty acids, which prevailed in the lipid-free cells, are the structural components of lipid A.     

Core region in Proteus mirabilis lipopolysaccharide.

Four R mutants of P. mirabilis were isolated. The composition of their degraded polysaccharides (PS) obtained from the respective lipopolysaccharides (LPS) as well as the composition and properties of the PS-fractions separated by column chromatography were examined. The results were compared with those obtained with PS of the wild type. One of the mutants could be classified as an Ra-type mutant, presenting a complete LPS core. This polysaccharide core contains: galacturonic acid, glucosamine, glucose, D-glycero-D-mannoheptose, L-glycero-D-mannoheptose in a molar ratio of 1 : 1 : 1 : 1 : 2 and 2-keto-3-deoxyoctonate. Taking into consideration the common sugars described previously in the LPS chemotypes of P. hauseri, the composition of the complete core region mentioned above represents the LPS core part of all the chemotypes, containing two different heptoses.     

Characteristics of the antigenic complexes that determine cross reactions between brucellae and Yersinia enterocolitica 09

In the study of antigens with different chemical composition, isolated from different Brucella species and from Y. enterocolitica 09, the common character and antigenic relationship of Brucella and Y. enterocolitica 09 in respect to their lipopolysaccharide (LPS) components and protein antigens have been established. The occurrence of serological cross reactions between the above microorganisms is due to their common LPS determinants.     

Immunochemical Studies on Lipopolysaccharide from Agglutinable and Non-Agglutinable Vibrios

Lipopolysaccharide (LPS) prepared from Vibrio cholerae and a non-agglutinable (NAG; not agglutinable with O-group I serum according to Gardner and Venkatraman [13]) vibrio strain, isolated from a patient with cholera-like clinical symptoms, have been compared with respect to their chemical composition and immunological behavior. In addition to a significant difference in the chemical composition between the two lipopolysaccharides, the LPS from V. cholerae, unlike that from the NAG vibrio, requires prior treatment with alkali for it to be an effective antigen in the indirect hemagglutination test with sheep cells. It has been suggested that the alkali acts by removing excess O-acetyl group from LPS of agglutinable vibrios. LPS from the NAG vibrio consistently showed a lower antibody response in rabbits in terms of agglutinin and vibriocidal titer. Also, the class of agglutinin antibody elicited by LPS of the NAG vibrio was predominantly immunoglobin M, and that from V. cholerae was immunoglobulin G under comparable conditions.     

Composition of the lipopolysaccharide from different capsular serotype strains of Haemophilus influenzae

Lipopolysaccharide (LPS) from all six serotype strains of Haemophilus influenzae was similar in composition. The oligosaccharide, of each LPS, was composed of glucose, galactose, heptose and 2-keto-3-deoxyoctonic acid. The lipid A was composed of glucosamine, phosphate and the fatty acids 14:0 and 3-OH 14:0. Each LPS also contained ethanolamine and ethanolamine phosphate, and the oligosaccharides from two strains additionally contained small amounts of glucosamine. Although the LPS was similar in composition, different serotypes had quantitative differences, especially in the galactose content, which correlated with the antigenic specificity of their homologous antisera and with their mobility on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A survey by SDS-PAGE showed that LPS from strains of the serotypes a, c and d was characteristically of lower Mr than the LPS from most (80%) serotype b strains.     

Aspects of Vibrio cholerae lipopolysaccharide

In this review information on the chemical structure, biosynthesis, antigenic and biological properties of V. cholerae lipopolysaccharide (LPS) is presented. The specific structural feature of this LPS is a small size of the polysaccharide chain of O-antigen. In vibrios of serogroup O 139 it is oligosaccharide. The modification of the O-chain (methylation of individual sugars, shortened chain, etc.) plays an essential role in the antigenic specificity of V. cholerae LPS. All these factors affect of endotoxin function, the microbial resistance to external influences. V. cholerae LPS takes part in the formation of microcapsules and biofilms. The evolutional development of V. cholerae in this direction determines, to some extent, their increased resistance in the environment. In human body the heterogeneity of the LPS composition permits the preservation of vibrios and ensures, together with cholerogen, their pathogenetic action.     

Parameters involved in antimicrobial and endotoxin detoxification activities of antimicrobial peptides

Endotoxin [lipopolysaccharide (LPS)] covers more than 90% of the outer monolayer of the outer membrane of Gram-negative bacteria, and it plays a dual role in its pathogenesis: as a protective barrier against antibiotics and as an effector molecule, which is recognized by and activates the innate immune system. The ability of host-defense antimicrobial peptides to bind LPS on intact bacteria and in suspension has been implicated in their antimicrobial and LPS detoxification activities. However, the mechanisms involved and the properties of the peptides that enable them to traverse the LPS barrier or to neutralize LPS endotoxic activity are not yet fully understood. Here we investigated a series of antimicrobial peptides and their analogues with drastically altered sequences and structures, all of which share the same amino acid composition (K 6L 9). The list includes both all- l-amino acid peptides and their diastereomers (composed of both l- and d-amino acids). The peptides were investigated functionally for their antibacterial activity and their ability to block LPS-dependent TNF-alpha secretion by macrophages. Fluorescence spectroscopy and transmission electron microscopy were used to detect their ability to bind LPS and to affect its oligomeric state. Their secondary structure was characterized in solution, in LPS suspension, and in LPS multibilayers by using CD and FTIR spectroscopy. Our data reveal specific biophysical properties of the peptides that are required to kill bacteria and/or to detoxify LPS. Besides shedding light on the mechanisms of these two important functions, the information gathered should assist in the development of AMPs with potent antimicrobial and LPS detoxification activities.     

Characterization of LPS mutants of peanut specific Bradyrhizobium (GN17)

Two mutants of Bradyrhizobium sp. (Arachis) strain GN17 having altered lipopolysaccharide (LPS) composition were isolated upon random Tn5 mutagenesis to study their binding with peanut root lectin (PRA II). These mutant strains designated as GN17M1 and GN17M2 produced rough colonies and showed autoagglutination. Flow cytometric analyses indicated that strain GN17M1 bind to PRA II with highest efficiency. Both the mutants synthesized only high molecular weight lipopolysaccharides as observed by silver staining of polyacrylamide gel. The LPSs from both the mutants cross-reacted with anti-GN17 LPS, however, GN17M1 LPS showed 3 times higher cross-reactivity as detected by ELISA. Carbohydrate analysis by high performance anion exchange chromatography (HPAEC) showed that glucose was the major constituent of the purified LPS from the parent strain whereas mannose appeared as major component in the GN17M2 LPS. Equivalent amount of glucose and galactosamine with significant amount of mannose and galactose was the characteristics of the GN17M1 LPS. Purified LPS from GN17M1 and GN17M2 were respectively 17 and 10 times more potent inhibitors of PRA II activity than that of parent strain GN17. Similar binding efficiencies of the mutant LPS towards PRA II was also observed by ELISA. The results of this study indicate that the composition and the arrangement of the LPS are crucial for lectin binding.     

Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action

Binding of lipopolysaccharide (LPS) to macrophages results in proinflammatory cytokine secretion. In extreme cases it leads to endotoxic shock. A few innate immunity antimicrobial peptides (AMPs) neutralize LPS activity. However, the underlying mechanism and properties of the peptides are not yet clear. Toward meeting this goal we investigated four AMPs and their fluorescently labeled analogs. These AMPs varied in composition, length, structure, and selectivity toward cells. The list included human LL-37 (37-mer), magainin (24-mer), a 15-mer amphipathic alpha-helix, and its D,L-amino acid structurally altered analog. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 and bone marrow-derived primary macrophages, to bind LPS in solution, and when LPS is already bound to macrophages (fluorescence spectroscopy and confocal microscopy), to compete with LPS for its binding site on the CD14 receptor (flow cytometry) and affect LPS oligomerization. We conclude that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein lipopolysaccharide-binding protein, or alternatively to its receptor, and hence inhibits cytokine secretion.     

The role of lipopolysaccharide in complement-killing of Aeromonas hydrophila strains of serotype O:34

The role of lipopolysaccharide (LPS) in the susceptibility of Aeromonas hydrophila strains of serotype O:34 to non-immune human serum was investigated using isogenic mutants (serum-sensitive), previously obtained on the basis of phage resistance, and characterized for their surface components. The classical complement pathway was found to be principally involved in the serum-killing of these sensitive strains. LPS preparations from serum-resistant or serum-sensitive strains, or purified core oligosaccharides (low-molecular-mass LPS) inactivated both bactericidal and complement activity of whole serum, while the O-antigen molecules (high-molecular-mass LPS) did not. The results indicate that LPS core oligosaccharide composition contributes to complement resistance of A. hydrophila strains from serotype O:34 with moderate virulence.     

Sugar composition of the polysaccharide portion of lipopolysaccharides of Vibrio fluvialis, Vibrio vulnificus, and Vibrio mimicus

A chemotaxonomic study was carried out on Vibrio fluvialis and V. vulnificus on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharides (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. Nineteen O-serogroups of V. fluvialis were divided into 14 chemotypes while seven O-serogroups of V. vulnifucus were divided also into seven chemotypes since the polysaccharide portion of LPS of each serogroup has a different sugar composition from that of the other serogroups. Close similarities in the sugar composition of the same portion were demonstrated between serologically cross-reacting non-O1 group V. cholerae and V. fluvialis, and non-O1 V. cholerae and V. mimicus.     

Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant.

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-deficient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS.     

Lipopolysaccharide Variability inPseudomonas aeruginosa

The lipopolysaccharide (LPS) ofPseudomonas aeruginosa (ATCC 9027) exhibited gross changes in both the amount of LPS produced per cell and the composition of the LPS in response to changes in magnesium ion concentrations. Compositional variation in the LPS was detected under both batch and continuous culture conditions, and was particularly apparent as changes in the levels of heptose in the molecule. Compositional changes in the molecule were also reflected as functional alterations in the LPS. However, functionally, these alterations were counterbalanced by increased production of the lipopolysaccharide. We propose that LPS composition changed in accordance with environmental factors, and that cells responded to these changes by increasing or decreasing the amount of LPS produced.     

Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.     

Evaluation of polysaccharide antigens of Vibrio cholerae O139 of different origin by monoclonal antibodies

The epitope composition of O-polysaccharides in the lipopolysaccharide (LPS) of V. cholerae, serogroup O139, isolated from clinical material and water of surface reservoirs was analyzed with the use of monoclonal antibodies. The analysis demonstrated that these O-polysaccharides were similar in their structure and chemical composition. In LPS of V. cholerae O139 clinical strains O-polysaccharide determinants occurred more often. Among V. cholerae isolated from water strains on whose surface individual epitopes of O-polysaccharide occurred less frequently or were absent appeared to be more numerous. A decrease in the concentration of microbial cells in the process of their testing by immunological methods led to increased percent of negative reactions with specific antibodies. Some V. cholerae O139 strains isolated from water were similar in the epitope composition of their O-polysaccharide and binding activity to cultures isolated from humans. As indicated by the results of these studies, cholera vibrios Bengal and vibrios isolated from river water on the territory of Russia had quantitative differences due to a higher level of the production of O-polysaccharide determinants and their occurrence in V. cholerae of serogroup O139.     

Immunochemical characteristics and serologic properties of lipopolysaccharides isolated from various Brucella species using various extraction methods

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.     

Monosaccharide composition of the lipopolysaccharides of bacteria of the genus Citrobacter

The authors studied antigens obtained by Grasset's method from 13 strains of Citrobacter of the International collection. The strains possessed O- and H-antigens whose behaviur in the electric field differed. All the strains under study were divided into two groups (by the number of serologically-active components of their O-antigens); representatives of the second group had no cathode O-antigen component. Chemical composition of specific lipopolysaccharides (LPS) obtained by Westphal's method was determined. Fourteen different sugars were revealed. The strains under study were referred to the known chemotypes. Strain 16/52 (8a, 8c) was for the first time studied in respect to the monosaccharide composition of specific LPS, and was referred to chemotype designated as CC-L.     

Occurrence of lipopolysaccharide alterations among Tn5 mutants of Rhizobium leguminosarum bv. trifolii strain 24.1 with altered colony morphology.

Transposon mutants of Rhizobium leguminosarum bv. trifolii 24.1 showing less glossy or smaller colonies were screened for properties usually associated with lipopolysaccharide (LPS) defects in R. leguminosarum, i.e. motility, growth rate, tendency to agglutination in liquid media and symbiotic efficiency. Neither any of the above mutants nor the earlier isolated 24.12 strain, defective in LPS, showed all these properties changed simultaneously. According to PAGE/sodium deoxycholate analysis the mutant 24.12 was the only one producing defective lipopolysaccharide. GC-MS analysis revealed in this mutant qualitative changes in composition of its LPS in comparison with LPS isolated from the parent strain. Other Tn5 mutants produced LPSs similar in composition, however the proportion between LPS I and LPS II differed from that in the parent strain.