Isolation of monoclonal antibodies reacting with the core component of lipopolysaccharide from Rhizobium leguminosarum strain 3841 and mutant derivatives.

Monoclonal antibodies reacting with the core oligosaccharide or lipid A component of Rhizobium lipopolysaccharide (LPS) could be useful for the elucidation of the structure and biosynthesis of this group of macromolecules. Mutant derivatives of Rhizobium leguminosarum 3841 with LPS structures lacking the major O-antigen moiety were used as immunogens, and eight antibodies were selected for further study. All the antibodies reacted with the fast-migrating species known as LPS-2 following gel electrophoresis of Rhizobium cell extracts. For four of these antibodies, reactivity with affinity-purified LPS was lost after mild acid hydrolysis, indicating that they probably recognized the core oligosaccharide component. The four other antibodies still reacted with acid-treated LPS and may recognize the lipid A moiety, which is stable to mild acid hydrolysis. The pattern of antibody staining after gel electrophoresis revealed differences in LPS-2 epitope structure between each of the mutants and the wild type. Furthermore, for each of the mutants the antibodies crossreacted with a minor band that migrated more slowly than LPS-2; we have termed this more slowly migrating form LPS-3. The majority of the antibodies also reacted with LPS from strain CE109, a derivative of Rhizobium etli CE3, confirming that the LPS core antigens can be relatively conserved between strains of different Rhizobium species. One of the antibodies isolated in this study (JIM 32) was unusual because it appeared to react with all forms of LPS from strain 3841 (namely, LPS-1, LPS-2, and LPS-3). Furthermore, JIM 32 reacted positively with the LPS from many strains of Rhizobium tested (excluding the Rhizobium meliloti subgroup). JIM 32 did not react with representative strains from Bradyrhizobium, Azorhizobium or other related bacterial species.     

Phosphorylation of the lipid A region of meningococcal lipopolysaccharide: identification of a family of transferases that add phosphoethanolamine to lipopolysaccharide

A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions, N. meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A."     

Studies on the antigenic S-type lipopolysaccharides of Brucella abortus strains 7 and Mustapha

Antigenic phenol phase S-type lipopolysaccharides (LPS) isolated from Brucella abortus (B. abortus) strains 7 and Mustapha were observed to have 13C n.m.r. spectra which were almost identical to the one reported for the Brucella abortus 1119-3. The glycosyl content of the lipid A obtained from the LPS of strain 7 was found to be 2-acetamido-2-deoxyglucose only while strain Mustapha was found to contain both 2-acetamido-2-deoxyglucose and 2-acetamido-2-deoxygalactose. The fatty acid present in the lipid A of both strains was mainly n-hexadecanoic acid. Octadecanoic acid, 3-hydroxytetradecanoic acid as well as small quantities of 3-hydroxydodecanoic acid were also identified. This contrasts with the earlier reports of the absence of 3-OH-14:0 in the LPS of Brucella abortus.     

Blocking of bacteriophages phi W and phi 5 with lipopolysaccharides from Escherichia coli K-12 mutants.

In the preceding paper we presented a formula for the composition of lipopolysaccharides (LPS) from Escherichia coli K-12. This formula contains four regions defined from analyses of LPS from four key strains, the parent and mutants which had lost one, two, or three regions of their carbohydrates. Support for the formula was derived from the susceptibility of the key mutants to several bacteriophages. One of these, phage phi W, was found specific for strains which had lost region 4. In this paper we described inactivation in vitro of phage phi W and its host-range mutant phi 5, using LPS devoid of regions 2 to 4. The blocking of phi W was found to require about 0.15 M concentrations of monovalent cations and to be inhibited by low concentrations of calcium and magnesium ions. One particle of phage phi W required 2 times 10-16 g of LPS devoid of region 4 for stoichiometric inactivation. Phage phi 5 was blocked by both heptose-less LPS (devoid of regions 2 to 4) and glucose-less LPS (devoid of regions 3 to 4) but was unaffected by LPS devoid of region 4. LPS from a heptose-less mutant of Salmonella minnesota showed the same inactivation ability as did LPS from heptose-less strains of E. coli K-12. Lipid A was prepared from LPS containing all four regions. Such lipid A was found to inactivate phi 5, whereas both the polysaccharide moiety as well as the intact LPS were without effect. It is suggested that lipid A is part of the receptor site for phage phi 5.     

Lipopolysaccharides of Thiocystis violacea, Thiocapsa pfennigii, and Chromatium tepidum, species of the family Chromatiaceae.

The lipopolysaccharides (LPS) of three species of purple sulfur bacteria (Chromatiaceae), Thiocystis violacea, Thiocapsa pfennigii, and the moderately thermophilic bacterium Chromatium tepidum, were isolated. The LPS of Thiocystis violacea and Chromatium tepidum contained typical O-specific sugars, indicating O-chains. Long O-chains were confirmed for these species by sodium deoxycholate gel electrophoresis of their LPS. Thiocapsa pfennigii, however, had short or no O-chains. The core region of the LPS of all three species comprised D-glycero-D-mannoheptose as the only heptose and 2-keto-3-deoxyoctonate. The lipid A, obtained from the LPS by mild acid hydrolysis, contained glucosamine as the main amino sugar. Amide-bound 3-hydroxymyristic acid was the only hydroxy fatty acid. The main ester-bound fatty acid in all lipid A fractions was 12:0. Mannose and small amounts of 2,3-diamino-2,3-dideoxy-D-glucose were common constituents of the lipid A of the three Chromatiaceae species investigated. All lipid A fractions were essentially free of phosphate.     

Chemical, immunobiological and antigenic characterizations of lipopolysaccharides from Bacteroides gingivalis strains.

Lipopolysaccharides (LPS) were extracted from whole cells of seven strains of Bacteroides gingivalis--381, ATCC 33277, BH18/10, OMZ314, OMZ406, 6/26 and HW24D-1--by the phenol/water procedure, and purified by treatment with nuclease and by repeated ultracentrifugation. These LPS were composed of hexoses, hexosamines, fatty acids, phosphorus and phosphorylated 2-keto-3-deoxyoctonate (KDO). The major components of the lipid portion of these LPS were hexadecanoic, 3-hydroxyhexadecanoic, branched 3-hydroxypentadecanoic and branched 3-hydroxyheptadecanoic acids. All the LPS preparations induced marked mitogenic and in vitro polyclonal B cell activation responses in spleen cells from both C3H/HeN and C3H/HeJ mice, exhibited no definitive preparatory activity in the local Shwartzman reaction in rabbits, but were active in the chromogenic Limulus amoebocyte lysate test. A monoclonal antibody (mAb) raised against the LPS from B. gingivalis strain 6/26 reacted with LPS from all other B. gingivalis strains tested. Other mAbs raised against LPS from B. gingivalis strains 381 and 6/26 reacted with the LPS from strains 381, ATCC 33277, BH18/10 and 6/26 (these strains were termed LPS serogroup I), as revealed by ELISA and immunodiffusion. The LPS from these strains except for 6/26 showed almost identical patterns in SDS-PAGE stained with ammoniacal silver. A mAb raised against the LPS from B. gingivalis HW24D-1 reacted with the LPS from strains OMZ314, HW24D-1 and OMZ409 (LPS serogroup II). These LPS, except OMZ409, exhibited very similar profiles in SDS-PAGE. These results indicate that there are at least two different antigenic groups present among LPS from B. gingivalis strains, as well as a common, species-specific antigen.     

IL-1 induction-capacity of defined lipopolysaccharide partial structures

Natural and synthetic lipid A as well as natural and synthetic oligosaccharide partial structures of LPS were examined in dose-response experiments to define the minimal structure necessary for IL-1 induction and release in cultures of human mononuclear cells. Wild type LPS (S. abortus equi) and rough mutant LPS was active in minimal-doses of 1 to 100 pg/ml, whereas synthetic heptaacyl and hexaacyl lipid A (Salmonella minnesota and Escherichia coli lipid A, respectively) induced IL-1 in minimal-doses of 100 to 1,000 pg/ml and 10 to 1,000 pg/ml, respectively. Nanogram amounts (0.1 to 10 ng/ml) of synthetic monodephospho partial structures of E. coli lipid A were necessary for IL-1 induction. Synthetic pentaacyl partial structures induced IL-1 very weakly. Synthetic tetraacyl and bisacyl partial structures lacking non-hydroxylated fatty acids were not active. Compared to LPS million-fold higher doses of natural and synthetic 3-deoxy-D-manno-octulosonic acid containing core oligosaccharides were necessary for IL-1 induction. Dose-response investigations with LPS and natural or synthetic partial structures established the following hierarchy in IL-1 induction-capacity: LPS greater than lipid A much greater than lipid A partial structures greater than core oligosaccharides greater than oligoacyl lipid A. Lipid A was shown here to be the portion of LPS mainly responsible for induction of IL-1 activity. The high potency of lipid A in inducing IL-1 release and the failure of the precursor Ia of lipid A to induce IL-1 production and release was also observed measuring intracellular IL-1 activity after freeze-thawing the cells. Levels of IL-1 beta mRNA in extracts of mononuclear cells correlated with biologic activity. In co-incubation experiments, precursor Ia of lipid A produced dose-dependent inhibition of production and release of IL-1 activity induced by lipid A or LPS, but not by Staphylococcus epidermidis or PHA. Incubation of cells with precursor Ia for 1h, followed by a medium change and further incubation of stimulus without precursor Ia of lipid A also resulted in inhibition. We conclude that lipid A is the main portion of LPS responsible for induction of IL-1, and that specific activation- and/or binding-mechanisms are involved in stimulation of cells with LPS and/or lipid A.     

Characterization of lipopolysaccharides from Ralstonia solanacearum

Lipopolysaccharides (LPSs) from four strains of Ralstonia solanacearum belonging to biovar I (ICMP 6524, 8115, 5712, and 8169) were isolated and investigated. The structural components of the LPS molecule, such as lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS), were obtained after mild acid hydrolysis of the LPS preparations. In lipid A from all the LPS samples studied, 3-hydroxyhexadecanoic, 2-hydroxyhexadecanoic, tetradecanoic, and hexadecanoic fatty acids prevailed. The dominant monosaccharides of the core oligosaccharides of all of the strains studied were rhamnose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and heptose. However, individual strains varied in the content of galactose, ribose, xylose, and arabinose. Three types of the O-PS structure were established, which differed in their configuration (alpha or beta), as well as in the type of the bond between glucosamine and rhamnose residues (1-->2 or 1-->3).     

Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells

The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-alpha induction and NF-kappaB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-alpha release and activated NF-kappaB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-alpha and NF-kappaB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-alpha induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-alpha release and NF-kappaB activation in THP-1 cells.     

Lipophilic O-antigens containing D-glycero-D-mannoheptose as the sole neutral sugar in Rhodopseudomonas gelatinosa.

Lipopolysaccharides (LPS, O-antigens) of 12 strains of the photosynthetic bacterium Rhodopseudomonas gelatinosa were obtained by the phenol/chloroform/petroleum ether method, recommended for extracting lipophilic glycolipids of enterobacterial R-mutants. All R. gelatinosa LPS have essentially the same chemical composition. Similar to LPS of Salmonella R-mutants of chemotypes Rd1 and Rd2, the sole neutral sugar constituent is an aldoheptose. The heptose of R. gelatinosa LPS has the D-glycero-D-manno- configuration, in contrast to the L-glycero-D-mannoheptose of enterobacterial LPS. 2-Keto-3-deoxyoctonate forms the acid-labile linkage between the lipid moiety (lipid A) and the oligosaccharide moiety of R. gelatinosa LPS. Like enterobacterial lipid A, lipid A of this species contains phosphate and D-glucosamine as the sole amino sugar. The fatty acid spectrum conprises beta-hydroxycapric, lauric, and myristic acids. Beta-Hydroxymyristic acid, the typical fatty acid of enterobacterial LPS, is lacking. The R. gelatinosa LPS show O-antigenic acitivity; passive hemagglutinations with untreated or heat-treated (not well alkali-treated) LPS and antisera prepared against heat-killed cells yield high titers. According to the serological cross-reactions observed, the LPS of the 12 strains could be arranged into two different serotypes: serotype I comprising strains 29/1, 29/2, 25/2, and serotype II comprising strains 44/K/6, 3/1, IS/10, 39/2, Dr2, 2150, P8P9, K32, P18f3.1. No serological cross-reactions were observed between LPS of these two different serotypes in passive hemagglutinations.     

Compositional analysis of Helicobacter pylori rough-form lipopolysaccharides.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the macromolecular heterogeneity of lipopolysaccharides (LPS) from seven fresh clinical isolates and three culture collection strains of the human pathogen Helicobacter pylori. All the clinical isolates produced smooth-form LPS with O side chains of relatively homogeneous chain length, whereas the culture collection strains yielded rough-form LPS. A better yield of the latter LPS was obtained when combined protease pretreatment and hot phenol-water extraction were used than when the conventional phenol-water technique alone was used for extraction. The LPS of the three culture collection strains (S-24, C-5437, and NCTC 11637) were chemically characterized. Constituents common to all the LPS were fucose, D-mannose, D-glucose, D-galactose, D-glycero-D-manno-heptose, L-glycero-D-manno-heptose, and 3-deoxy-D-manno-2-octulosonic acid. The molar ratios of the hexoses differed between different strains, thereby reflecting structural differences. Phosphate, phosphorylethanolamine, and pyrophosphorylethanolamine were present also. Free lipid A contained D-glucosamine and fatty acids, with phosphate and a minor amount of ethanolamine. The major fatty acids were ester- and amide-bound 3-hydroxyoctadecanoic acid and ester-bound octadecanioc and 3-hydroxyhexadecanoic acids, with minor amounts of ester-bound tetradecanoic and hexadecanoic acids. In addition to the uncommonly long 3-hydroxy fatty acids, an unusual phosphorylation pattern was deduced to be present in the lipid A.     

Serological properties and immunobiological activities of lipopolysaccharides from black-pigmented and related oral Bacteroides species

Lipopolysaccharides (LPS) from five species of oral Bacteroides, B. gingivalis strains 381 and ATCC 33277, B. oralis ATCC 33269, B. loescheii ATCC 15930, B. intermedius ATCC 25611 and B. corporis ATCC 33547, were extracted from whole cells by the phenol/water procedure, and subsequently purified by treatment with nuclease and ultracentrifugation. The LPS were composed of hexoses, glucosamine, fatty acids and phosphorus. Heptose and 2-keto-3-deoxyoctonate were not detected. The LPS preparations from B. gingivalis strains 381 and ATCC 33277 presented very similar SDS-polyacrylamide gel electrophoresis patterns when stained with ammoniacal silver. They produced a fused precipitin band against an antiserum to B. gingivalis 381 LPS in immunodiffusion tests. Antisera raised against the LPS from B. loescheii and B. intermedius reacted with the LPS prepared from all the oral Bacteroides strains except those of B. gingivalis. All the LPS preparations were mitogenic for spleen cells of BALB/c (nu/nu) mice, but not for thymus cells from C3H/HeN mice. The LPS induced marked mitogenic responses and polyclonal B cell activation for spleen cells of not only C3H/HeN (LPS responder) mice, but also C3H/HeJ (LPS nonresponder) mice. The mitogenic responses were not suppressed significantly upon addition of polymyxin B to the reaction mixture. These LPS also enhanced interleukin-1 production by murine peritoneal macrophages and mouse cell line J744. 1 macrophages. Hydrolysis of B. gingivalis ATCC 33277 LPS in 1 m-HCl at 100 degrees C for 1 h yielded lipid and polysaccharide. The lipid portion was largely composed of fatty acids and glucosamine, and was mitogenic for spleen cells from C3H/HeJ as well as C3H/HeN mice, while the polysaccharide portion induced no significant mitogenic responses under similar experimental conditions.     

Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.     

Lipid A, the active part of bacterial endotoxins in inducing serum colony stimulating activity and proliferation of splenic granulocyte/macrophage progenitor cells.

An analysis of which component of lipopolysaccharides (LPS), the lipid or the polysaccharide (PS), is active in stimulating the murine granulopoietic system has been performed. LPS with different structures, isolated from different mutant strains of Salmonella and chemical degradation products of lipopolysaccharides have been used. Lipid A obtained by acid hydrolysys of the LPS and complexed to bovine serum albumin (BSA) (lipid A-BSA) was shown to be active in generating serum colony stimulating factor (CSF) and in increasing the splenic colony forming cells (CFC) levels, although it was less active than the parent LPS. The polysaccharide (PS) showed no significant activity at the concentrations used. LPS (glycolipids) from R mutants of Salmonella minnesota were active to the same extent as the LPS. The fact that even the most defective LPS from the R mutant R595 which contains lipid A and KDO only is a potent endotoxin, points unequivocally, to lipid A, as the active principle in stimulating the granulopoietic system.     

Strong interaction of lipopolysaccharides possessing the mannose homopolysaccharides with complement and its relation to adjuvant action

LPS from Klebsiella pneumoniae O3 (KO3 LPS) exhibited an extremely high anticomplementary activity by the hemolysis assay using human sera. The free lipid A isolated from KO3 LPS by acid hydrolysis and R form LPS from a mutant lacking the O-specific polysaccharide portion possessed lower anticomplementary activity, and the O-specific polysaccharide fraction isolated from KO3 LPS alone did not activate the C system. It was suggested that the O-specific polysaccharide moiety enhanced the C activation by the lipid A portion. This was also supported by the finding that modification of the O-specific polysaccharide moiety with Con A or tyramine decreased anticomplementary activity of KO3 LPS, and that the other LPS preparations possessing the mannose homopolysaccharides as the O-specific polysaccharide portions such as KO3 LPS, such as LPS from Klebsiella O5, Escherichia coli O8 and O9, exhibited a high anticomplementary activity. KO3 LPS could activate the C system in either the classical or the alternative pathway, whereas the lipid A or R form LPS activated the classical pathway alone. The intensity of anticomplementary activity of LPS was parallel to that of their adjuvant action on antibody response to deaggregated BSA. The role of the anticomplementary activity in the expression of the adjuvant action of LPS is discussed.     

Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity

A major problem in the development of vaccines against Gram-negative bacteria is the endotoxic -activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the O-linked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-alpha. Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O-linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.     

Glycolipoprotein cytotoxin from Leptospira interrogans serovar copenhageni

Lipopolysaccharide (LPS), glycolipoprotein (GLP) and lipid extract were prepared from Leptospira interrogans serovar copenhageni. GLP, lipid extract or purified fatty acids from lipid extract produced cytotoxic effects seen as cell enzyme leakage followed by cytotoxic death when tested in mouse fibroblast L929 cells in tissue culture. All extracts also agglutinated mouse erythrocytes but purified LPS was not cytotoxic. Neither GLP nor LPS were pyrogenic but both gelled Limulus amoebocyte lysate. Specific anti-GLP IgG neutralized the cytotoxic and haemagglutinating effect of GLP; however, at higher concentrations it enhanced the cytotoxicity of GLP and mediated lysis of the erythrocytes. A high dose of leptospires (i.e. 10(10) organisms) killed weanling mice causing pathological changes similar to those seen in acute leptospirosis. Similar results were obtained with live, dead, pathogenic and saprophytic leptospires. The results suggest that toxicity is involved in leptospiral infection and that lipid components either of whole leptospires or of a leptospiral GLP may contribute to the pathogenesis of acute leptospirosis.     

Structural heterogeneity regarding local Shwartzman activity of lipid A

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin-layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515--Ec-A2 and Ec-A3--exhibited strong activity, similar to that of previous synthetic E. coli-type lipid A (compound 506 or LA-15-PP). The Ec-A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec-A2 fraction was structurally similar to compound 506 except that it carried a 3-hydroxytetradecanoyl group at the C-3' position of the backbone in place of a 3-tetradecanoyloxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec-A2 (12 C) and Ec-A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec-A2 or Ec-A3. In these preparations, a large part of the 3-dodecanoyloxytetradecanoyl group might be replaced by 3-hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella-type lipid A (compound 516 or LA-16-PP) exhibited low activity. A lipid A fraction, Cv-A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C-2 and C-2' positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester, and these lipid A (Pd-A3 and Pv-A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.     

In vitro stimulation of C3H/HeJ spleen cells and macrophages by a lipid A precursor molecule derived from Salmonella typhimurium

C3H/HeJ mice possess a genetic lesion that renders them significantly less responsive to the biologic effects of protein-free lipopolysaccharide (LPS) preparations, and more specifically, to the lipid A region of the LPS molecule. The in vivo manifestations of this mutation are also reflected in vitro in that cells derived from this mouse strain fail to respond to LPS when compared with cells derived from fully endotoxin-responsive mouse strains. The precise nature of this gene defect has not yet been established. In this study, we have examined in vitro the biologic activities of a structurally less complex "lipid A precursor" molecule, produced by a conditionally lethal, temperature-sensitive mutant of Salmonella typhimurium. In contrast to the intact LPS or wild-type lipid A extracted from the parental strain of Salmonella typhimurium, the lipid A precursor induced a highly significant, polymyxin B-inhibitable mitogenic response in splenic cultures derived from LPS-hyporesponsive C3H/HeJ and C57BL/10ScN (nu/nu) mice. In addition, the lipid A precursor was found to stimulate cultures of C3H/HeJ macrophages to produce significant levels of both interleukin 1 (IL 1, previously referred to as "lymphocyte activating factor" or "LAF") and prostaglandins of the E series (PGE). These findings suggest the possibility that the defect in endotoxin responsiveness exhibited by C3H/HeJ mice may be related to a defect in the processing of wild-type lipid A or LPS to a suitably stimulatory form that is structurally related to the lipid A precursor molecule.