Intact Lipopolysaccharide Research Articles

Structural studies utilizing 13C-enrichment of the O-antigen polysaccharide from the enterotoxigenic Escherichia coli O159 cross-reacting with Shigella dysenteriae type 4.

The structure of the O-antigen polysaccharide from Escherichia coli O159 has been determined using primarily NMR spectroscopy of the 13C-enriched polysaccharide. The sequence of the sugar residues could be determined by heteronuclear multiple bond connectivity NMR experiments. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [sequence: see text] Matrix assisted laser desorption ionization mass spectrometry was performed on intact lipopolysaccharide and from the resulting molecular mass the O-antigen part was estimated to contain approximately 23 repeating units. Cross-reactivity of this O-antigen to that of Shigella dysenteriae type 4 was confirmed using enzyme-linked immunoabsorbant assay.

Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae strain RM.118‐26

The structure of the lipopolysaccharide of Haemophilus influenzae mutant strain, RM.118-26, was investigated. Electrospray ionization-mass spectrometry on intact lipopolysaccharide, O-deacylated lipopolysaccharide and core oligosaccharides obtained from lipopolysaccharide after mild acid hydrolysis provided information on the composition and relative abundance of the glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. The structure of the major glycoform containing phosphocholine is identical to the Hex2 glycoform described for H. influenzae RM.118-28 [Risberg, A., Schweda, E.K.H. & Jansson, P.-E. (1997) Eur. J. Biochem. 243, 701-707]. A second major glycoform, containing three hexose residues (Hex3), in which a lactose unit, beta-D-Galp-(1-->4)-beta-D-Glcp, is attached at the O-2 position of the terminal heptose of the inner core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-( 1-->4)-]- L-alpha-D-Hepp-(1-->5)-alpha-Kdo, carries no phosphocholine. Instead this lipopolysaccharide glycoform is partly (40%) substituted by an O-acetyl group linked to the 6-position of the glucose residue in the lactose unit and has the following structure:

Identification of a plasmid-borne locus in Rhizobium etli KIM5s involved in lipopolysaccharide O-chain biosynthesis and nodulation of Phaseolus vulgaris.

Screening of derivatives of Rhizobium etli KIM5s randomly mutagenized with mTn5SSgusA30 resulted in the identification of strain KIM-G1. Its rough colony appearance, flocculation in liquid culture, and Ndv(-) Fix(-) phenotype were indicative of a lipopolysaccharide (LPS) defect. Electrophoretic analysis of cell-associated polysaccharides showed that KIM-G1 produces only rough LPS. Composition analysis of purified LPS oligosaccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (alpha-D-GalA-1-->4[alpha-D-GalA-1-->5]-Kdo) and tetrasaccharide (alpha-D-Gal-1-->6[alpha-D-GalA-1-->4]-alpha-D-Man-1-->5Kdo), strongly suggesting that the transposon insertion disrupted a locus involved in O-antigen biosynthesis. Five monosaccharides (Glc, Man, GalA, 3-O-Me-6-deoxytalose, and Kdo) were identified as the components of the repeating O unit of the smooth parent strain, KIM5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5s that, when provided in trans on a broad-host-range vector, restored the smooth LPS and the full capacity of nodulation and fixation on its host Phaseolus vulgaris. The mTn5 insertion in KIM-G1 was located at the N terminus of a putative alpha-glycosyltransferase, which most likely had a polar effect on a putative beta-glycosyltransferase located downstream. A third open reading frame with strong homology to sugar epimerases and dehydratases was located upstream of the insertion site. The two glycosyltransferases are strain specific, as suggested by Southern hybridization analysis, and are involved in the synthesis of the variable portion of the LPS, i.e., the O antigen. This newly identified LPS locus was mapped to a 680-kb plasmid and is linked to the lpsbeta2 gene recently reported for R. etli CFN42.

The structure of the core part of Proteus vulgaris OX2 lipopolysaccharide

The identity of a novel structural component, an open-chain acetalic linkage, in the core part of the lipopolysaccharide (LPS) from Proteus vulgaris serotype OX2 has been determined by extensive NMR spectroscopic analysis of fragments isolated after mild acid hydrolysis of the intact LPS. The open-chain N-acetylgalactosamine fragment is substituted in the 4-position by non-stoichiometric amounts of a β-galactopyranose residue and the overall structure of the core is as follows: All sugars except the N-acetylgalactosamine are in the pyranose form, α-Hep refers to l- glycero-α- d- manno-heptopyranose and α- ddHep to d- glycero-α- d- manno-heptopyranose. Bold italics indicate non-stoichiometric substituents.

The structure of the core part of Proteus mirabilis O27 lipopolysaccharide with a new type of glycosidic linkage

The structural assignment of the intact lipopolysaccharide core from Proteus mirabilis O27 has been completed based on a combination of chemical degradation studies, NMR spectroscopy and ES-MS spectroscopy. The overall core structure is as follows: where all sugars are in the pyranose form except the N-acetylglycosamine residue, Hep refers to l- glycero-α- d- manno-heptopyranose and α- ddHep to d- glycero-α- d- manno-heptopyranose. Bold italics indicate non-stoichiometric substituents. A new type of glycosidic linkage has been discovered wherein a GalNAc residue is linked as an open form acetal to the 4- and 6-positions of a 2-amino-2-deoxygalactopyranose residue. This structural element is abbreviated Gal oNAc-4,6-, where the ‘ o’ indicates the open form of the sugar residue.

Antigenic components of Actinobacillus actinomycetemcomitans lipopolysaccharide recognized by sera from patients with localized juvenile periodontitis.

The dominant antigen of Actinobacillus actinomycetemcomitans recognized by high-titer sera from patients with localized juvenile periodontitis is the serotype antigen located in the O-side chains of lipopolysaccharide. Whether such sera contain antibodies reactive with other epitopes in lipopolysaccharide, as is the case for patients with rapidly progressive periodontitis, remains unknown. We prepared and characterized by gas liquid chromatography lipopolysaccharide, lipid A, core carbohydrate with no or few O-side chains (core) and high-molecular-mass carbohydrate-rich in O-side chains (oligosaccharide) from A. actinomyce-temcomitans ATCC 43718 (serotype b, Y4). Using enzyme-linked immunosorbent assay (ELISA), sera from 36 patients with localized juvenile periodontitis were surveyed using whole-cell sonicate as plate antigen. The seven highest titer sera were selected for further study. Specific IgG antibody binding was observed to intact lipopolysaccharide and to all the lipopolysaccharide fractions. The mean titers were highest for intact lipopolysaccharide (138.8 ELISA units), and lipid A (122 ELISA units), followed by the core fraction (81 ELISA units) and the oligosaccharide fraction (69.5 ELISA units). ELISA inhibition revealed that the core fraction at a concentration of 10 micrograms/test well inhibited antibody binding to A. actinomycetemcomitans lipopolysaccharide by a mean value of 56.7%. To further characterize antibody binding to the core fraction, ELISA inhibition was performed using as inhibitor the core carbohydrate fraction of the Re mutant of Salmonella minnesota, which is known to contain only alpha-keto-3-deoxyoctonate residues and phosphate. This fraction at 10 micrograms/test well inhibited binding of antibodies from 6 of 7 test sera with a mean value of 49.2%. Thus, sera from patients with localized juvenile periodontitis contain antibodies that bind to the O-side chains of lipopolysaccharide, as has been previously reported, but they also contain antibodies that bind to lipid A and to lipopolysaccharide core polysaccharide epitopes, specifically to alpha-keto-3-deoxyoctonate moieties. The humoral immune response to A. actinomycetemcomitans in patients with localized juvenile periodontitis is more complex than previously reported and is very similar to that of patients with rapidly progressive periodontitis.

Lipopolysaccharide O-antigen expression and the effect of its absence on virulence in rfb mutants of Vibrio cholerae O1

Using defined rfb mutants, defective in the biosynthesis of the O-antigen of the lipopolysaccharide (LPS), and monoclonal antibodies (MAbs) to the A, B and C LPS antigens, we have examined the distribution of the antigens and the effects of their loss. By immunogold electron microscopy, it has been possible to determine the relative amounts of the A, B and C antigens on Inaba and Ogawa cells, confirming previous studies based upon bacterial agglutination and hemagglutination inhibitions. These antigens are absent from rfb::Tn mutants selected as resistant to phages which have been shown to use the O-antigen as their receptor. These mutants were severely attenuated as measured by both LD 50 and their ability to compete with the wild-type parents when analyzed in the infant mouse cholera model. These mutants were unchanged in the export of cholera toxin or other secreted proteins but revealed an altered outer membrane protein profile. The competition defect suggested an effect on TCP (toxin-coregulated pilus). An analysis of the rfb::Tn mutants revealed that they were unable to assemble TCP on their surface, but the major subunit, TcpA, could be found as an intracellular pool. These mutants could be complemented back to wild-type using the cloned rfb region, implying that functional TCP assembly is dependent upon an intact LPS.

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359: THE COMPLETE STRUCTURE OF THE CORE REGION OF R. ETLILIPOPOLYSACCHARIDES

The structural arrangement of oligosaccharides comprising the core region of Rhizobium etli CE3 lipopolysaccharide (LPS) has been elucidated through the characterization of the LPSs from two R. etli mutants. One mutant, CE358, completely lacks the O-chain polysaccharide, while the second mutant, CE359, contains a truncated portion of this polysaccharide. This structural arrangement of the core oligosaccharides in these LPSs was determined using electrospray ionization mass spectrometry, tandem mass spectrometry, and methylation analysis. Mild acid hydrolysis of the CE359 LPS produces two major core oligosaccharides: a tetrasaccharide (1) with the structure alpha-D-Galp-(1-->6)-[alpha-D-GalpA-(1-->4)]-alpha-D-Manp-(1 -->5)-Kdo p (where Kdo represents 3-deoxy-D-manno-2-octulosonic acid) and a trisaccharide (2) having the structure alpha-D-GalpA-(1-->4)-[alpha-D-GalpA-(1-->5)]-Kdop. Structure 1 in CE358 LPS lacks the galacturonosyl residue. Glycosyl linkage and tandem mass spectrometry analyses show that the intact LPS core region consists of trisaccharide (2) attached to O-4 of the Kdo residue in tetrasaccharide 1, and that an additional Kdo residue is attached to O-6 of the galactosyl residue of 1. [structure: see text] The additional terminally linked Kdo residue is not in close proximity to the lipid A moiety, a unique location for a core Kdo residue. The mutant LPS preparations also contain minor LPS species, one of which lacks the Kdo linked to O-6 of the galactosyl residue, another that lacks the galacturonic acid attached to O-5 of Kdo, and a third that lacks two galacturonosyl residues and one Kdo residue. Thus, in addition to lacking both heptose and phosphate, the R. etli LPS core region differs substantially from the typical enterobacterial cores. The abundance of galacturonosyl residues in the R. etli core might serve as a suitable functional replacement for phosphate, such as would be predicted for Ca2+ binding.

Hydrophobic characterization of thermophilicCampylobacterspecies and adhesion to INT 407 cell membranes and fibronectin

Cell surface hydrophobicity ofCampylobacter jejuni,C. coli,C. lariandC. upsaliensiswas tested by hydrophobic interaction chromatography on octylsepharose CL-4B. The hydrophobicity was influenced by cultivation mode, presence or absence of intact lipopolysaccharide (LPS) and outer membrane protein structures. Species-specific differences of hydrophobic characteristics were not detected. Bacteria grown in fluid medium exhibited a high degree of hydrophobicity. Agar-grown bacteria showed hydrophobic interaction to a significant lower extent. By oxidation of LPS with sodiummeta-periodate the hydrophobicity of agar-grown bacteria was slightly increased. Bacteria pretreated with proteinase K exhibited a marked decrease of hydrophobic interaction, whereas pretreatment with trypsin did not influence the hydrophobic interaction. Live bacteria were allowed to adhere to INT 407 cell membranes. With exception of one aflagellate strain, bacteria grown in fluid medium adhered better to the cellular substrate than agar-grown bacteria. This difference was not found when adhesion to fibronectin was tested. LPS-oxidized bacteria adhered significantly better to both cell membranes and fibronectin, whereas proteinase K treated bacteria exhibited a significant loss of adhesion capacity for both substrates.

Epitopes of Bordetella pertussis lipopolysaccharides as potential markers for typing of isolates with monoclonal antibodies.

Three hybridomas (P1P3, D7 and 60.5) producing monoclonal antibodies (mAbs) against Bordetella pertussis lipopolysaccharide (LPS) were established. All reacted with the LPS from a typical, vaccine strain of B. pertussis (1414), but not with that of a variant strain (A100). Two of these mAbs (P1P3 and 60.5) cross-reacted with a B. bronchiseptica LPS; only one (P1P3) reacted with a B. parapertussis LPS. ELISA reactivities with intact LPSs, and defined partial structures covalently linked to bovine serum albumin, were compared. mAb 60.5 bound to the terminal region of a distal trisaccharide consisting of N-acetylated amino sugars. D7 reacted with a substructure which can be modified in the B. parapertussis and B. bronchiseptica LPSs by addition of a polymeric O-chain. P1P3 bound to a nonacetylated glucosamine substituted with L-glycero-D-manno-heptose, present in the 'core' of the B. pertussis LPS. These mAbs may be useful for rapid typing of Bordetella in clinical isolates.

Separation and Characterization of O-Deacylated Lipooligosaccharides and Glycans Derived fromMoraxella catarrhalisUsing Capillary Electrophoresis–Electrospray Mass Spectrometry and Tandem Mass Spectrometry

Electrophoretic methods have been developed for the analysis of complex carbohydrates derived from lipooligosaccharides (LOS) ofMoraxella catarrhalisusing capillary electrophoresis coupled to electrospray mass spectrometry (CE–ESMS). Separation of lipooligosaccharides (LOS) arising from mild hydrazinolysis of the intact lipopolysaccharides (LPS) was achieved using aqueous ammonium formate, and enabled identification of sites of heterogeneity (phosphates, phosphoethanolamine, and pendant acyl groups) on either the lipid A or the core oligosaccharide. More complex mixtures of carbohydrates obtained from the complete deacylation and dephosphorylation of LOS were amenable to electrophoretic conditions using both anionic and cationic separation. In particular, electrophoretic conditions were developed which permitted resolution of closely related oligosaccharides according to the number of carbohydrate residues appended to the core structure. Structural characterization of carbohydrates and LOS released from the hydrazinolysis and acid hydrolysis treatment of the intact LPS was achieved using tandem mass spectrometry (MS-MS) for samples introduced by direct flow injection. Taken together, the combination of CE–ESI-MS and MS-MS analyses provided valuable information on the heterogeneity of the LOS population in which a significant level of variability was found mostly in the lipid A portion.

Molecular characterization of a virulence-associated epitope on the lipopolysaccharide ofLegionella pneumophilaserogroup 1

For identification of lipopolysaccharide (LPS)-associated epitopes of Legionella pneumophila serogroup 1, LPS of strain Philadelphia 1 was investigated using monoclonal antibodies (MAbs). The O-specific chain of LPS is a homopolymer of 5-acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-D-glycero- L-galacto- nonulosonic acid. At least four immunoaccessible epitopes were recognized by different MAbs on the intact LPS. After O-deacetylation of LPS, the reactivity of one of the MAbs (MAb 3/1) was lost, indicating thus that the corresponding epitope is associated with the 8-O-acetyl group. Since the reactivity pattern of the MAb 3/1 is identical with those of the MAb 2 which was considered as a virulence marker for serogroup 1, this epitope may be involved in mediating virulence in L. pneumophila. Four MAbs specific to strains of serogroup 1 other than the monoclonal subtype Philadelphia recognized epitopes on the O-deacetylated LPS of strain Philadelphia 1 and, therefore, the virulence-associated epitope blocks recognition of the immunodeterminants that are accessible on the intact LPS of the strains lacking this epitope.

Immunochemistry of group A and Inaba C antigen factors constituting the O antigen of O1 Vibrio cholerae.

Serological cross-reactivity among intact lipopolysaccharides (LPS) from O1 Vibrio cholerae Inaba O-form (Inaba), Yersinia enterocolitica O9 (O9), non-O1 V. cholerae serogroup Hakata (Hakata) and Vibrio bio-serogroup 1875 Variant (1875 Variant) (all of which share Inaba antigen factor C), as well as a total of six kinds of chemically modified LPS (three from O9 and three from Inaba) was demonstrated by passive hemolysis and passive hemolysis inhibition by using these LPS as antigen for sensitizing sheep red blood cells and as inhibitor. These intact as well as chemically modified LPS contained, in their O polysaccharide chain, alpha(1-->2)-linked linear perosamine (4-amino-4,6-dideoxy-D-manno-pyranose) homopolymers with different N-acyl groups: their acyl groups comprise 3-deoxy-L-glycero-tetronyl (Inaba LPS), formyl (O9 LPS), 3-hydroxypropionyl (1875 Variant LPS), acetyl (Hakata LPS and artificially introduced into Inaba and O9 LPS), propionyl and butyryl (both artificially introduced into Inaba and O9 LPS) groups. N-Deacylation of the alpha(1-->2)-linked N-(3-deoxy-L-glycero-tetronyl)perosamine homopolymer of Inaba and the N-formyl one of O9 LPS resulted in virtual elimination of their serological reactivity with both homologous and heterologous antisera. Furthermore, when the resultant NH2 groups of the N-deacylated perosamine homopolymers of both LPS were N-acylated with acetyl, propionyl or butyryl groups, they markedly recovered both of their serological reactivities. These results are compatible with the interpretation that the Inaba antigen factor C possessed by the four bacteria is substantially related to the common presence of N-acyl groups, regardless of their identity, residing in the perosamine residues constituting the O polysaccharide chain of their LPS. It was also indicated that the group antigen factor A of O1 V. cholerae is substantially related to the 3-deoxy-L-glycero-tetronyl groups residing in the perosamine homopolymer of Inaba LPS.

Structural studies of the O-polysaccharide from the lipopolysaccharide of Moraxella ( Branhamella) catarrhalis serotype A (strain ATCC 25238)

The polysaccharide of the Moraxella ( Branhamella) catarrhalis serotype A lipopolysaccharide was prepared by mild acid hydrolysis followed by gel permeation chromatography. The structure was established by methylation analysis, mass spectrometry, and NMR spectroscopy. It is concluded that the O-antigenic polysaccharide has the following structure. ▪ Methylation analysis of the intact lipopolysaccharide showed that the lipid A portion consisted of 6-substituted glucosamine residues. Methylation followed by methanolysis showed that two Kdo residues were present, one terminal and one 4,5-substituted residue. A terminal Kdo thus substitutes the branch-point Kdo in the 4-position.

Alkali-treated LPS of Yersinia enterocolitica does not induce expression of E-selectin, ICAM-1 or VCAM-1 on endothelial cells but may mediate antibody- and complement-dependent cell injury.

Lipopolysaccharide (LPS) prepared from a rough mutant of Salmonella typhimurium and deacylated enzymatically (dLPS) does not promote neutrophil adherence to human umbilical vein endothelial cells (HUVECs). This paper reports that similarly, a smooth form of LPS prepared from Yersinia enterocolitica O:3, a serotype known to trigger reactive arthritis in humans, and treated with alkali (yersinia LPS-OH) failed to augment neutrophil adherence to HUVECs. Studies of the mechanism underlying the poor augmentation revealed that neither enzymatically deacylated LPS from Escherichia coli J5 (J5 dLPS) nor yersinia LPS-OH stimulated expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1, whereas both intact J5 LPS and yersinia LPS were stimulatory. Impaired up-regulation could not be explained by decreased binding of yersinia LPS-OH to HUVECs. Furthermore, 51Cr-labelled HUVECs treated with different concentrations of yersinia LPS-OH released 51Cr in the presence of anti-yersinia anti-O antibody and complement. J5 dLPS and yersinia LPS-OH inhibited up-regulation of the adhesion molecules induced by J5 LPS and yersinia LPS but not that induced by tumour necrosis factor alpha. Taken together, the results suggest that although yersinia LPS-OH can depress development of acute inflammation by inhibiting up-regulation of endothelial-cell adhesion molecules, sufficient LPS-OH is bound to induce cell injury and thereby inflammation in the presence of specific antibody and complement. The findings may have pathogenetic implications in yersinia-triggered reactive arthritis characterized by dissemination of yersinia LPS throughout the body.

Chemical structure of the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide.

The complete structure for the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide (LPS) was assigned through studies on derivatives of the liberated oligosaccharide (OS 2) and the intact LPS. Structure determinations were performed using 1H-NMR spectroscopy, methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analysis by gas chromatography/mass spectrometry, Smith degradation, and oxidation with chromium trioxide. It was concluded that complete oligosaccharide chains had the following structure: [formula: see text]

Chemical structures of the core regions of Campylobacter jejuni serotypes O:1, O:4, O:23, and O:36 lipopolysaccharides.

Complete structures, including the location of N-acetylneuraminic acid (Neu5Ac) residues, were assigned for the core regions of Campylobacter jejuni serotypes O:1, O:4, and O:23 and O:36 lipopolysaccharides (LPS). In continuation of earlier studies, structure determinations of liberated oligosaccharides and, where necessary, of intact LPS, were by 1H-NMR spectroscopy, Smith degradation, chromium trioxide and enzymic degradations, in conjunction with methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analyses by gas chromatography/mass spectrometry. It was concluded on the basis of the following structures, in which each was linked 1-->5 to a terminal 3-deoxy-D-manno-octulosonic acid residue, that the core regions with qualitatively similar sugar compositions showed serotypic differences in one or more of their sequences, linkage types, and anomeric configurations: [formula: see text] [corrected]. The outer regions of each structure carry Neu5Ac residues linked 2-->3 to available beta-D-Galp residues and show striking similarities with various glycosphingolipids of the ganglioside family. However, Neu5Ac epitopes are not apparently involved in determining serospecificity.

4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-3-deoxy-D-manno-2-octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus.

A disaccharide containing GlcN and 3-deoxy-D-manno-2-octulosonic acid (Kdo) was detected after acidic methanolysis and peracetylation of hydrofluoric-acid-treated smooth-type and rough-type lipopolysaccharide of Pectinatus cerevisiiphilus and Pectinatus frisingensis, which are strictly anaerobic bacteria capable of growing in packaged beer. The disaccharide was also found in alkali-treated lipopolysaccharide, but was not directly detectable from intact lipopolysaccharide. This suggested that the disaccharide carried a phosphate residue. The position of this phosphate was shown, by GLC/MS of appropriately degraded and derivatized samples, to be O6 of the GlcN. Methylation analysis of the purified disaccharide revealed that GlcN was linked to position 4 of Kdo. The acetylated derivative of the disaccharide was isolated in pure form, and, by 1H-NMR and 13C-NMR spectroscopy, it was confirmed to possess the structure alpha-D-GlcpN-(1'-->4)-Kdo. In the lipopolysaccharide the amino group of GlcN is free.

Location of fructose in lipopolysaccharide isolated from 01 Vibrio cholerae NIH 41R.

Fructose, a rarely occurring sugar constituent of Gram-negative bacterial lipopolysaccharides (LPS), is distributed ubiquitously in LPS of 01 Vibrio cholerae so far examined, but its location in LPS has not hitherto been elucidated. It was found that hydrazinolysis of LPS successfully affords a derivative retaining virtually all the fructose of intact LPS, but no ester-bound phosphate. Structural analysis carried out on the LPS derivative prepared by the hydrazinolysis of R-type LPS isolated from a rough mutant strain (NIH 41R) of 01 V. cholerae NIH 41 (Ogawa) revealed that the fructose is present as a non-reducing terminal residue bound to position C-6 of a glucose residue in the core region. This finding is considered to exclude the possibility that, in the LPS of 01 V. cholerae, the fructose is present in the region of the inner core in place of 2-keto-3-deoxyoctonate.

Succinylated lipid A is a potent and specific inhibitor of endotoxin mitogenicity.

Chemically modified lipopolysaccharides of Salmonella abortus-equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide (LPS). All the lipopolysaccharide preparations deacylated by different alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost when succinic residues were introduced on hydroxyl groups. Partially deacylated alkaline-treated preparations (but not completely deacylated preparations) inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the mitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Succinylated lipid A, on the other hand, was not toxic to the cells and inhibited the B-cell mitogenicity of lipopolysaccharide (but not the T-cell mitogenicity of concanavalin A). Chemical analysis revealed that about 4.6 mol of succinic acid had been introduced into lipid A by succinylation, and that the fatty acid and phosphate composition was unchanged by this treatment. Macrophages do not seem to participate in this inhibition. Inhibition was observed when succinylated lipid A was added either at the same time or after lipid A mitogen, but optimal inhibition was expressed when it was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the succinylated lipid A competes with intact LPS.