Core Region Of Lipopolysaccharide Research Articles

Outer membrane polysaccharide deficiency in two nongliding mutants of Cytophaga johnsonae

Phenol-extractable polysaccharides firmly associated with the outer membrane of the gliding bacterium Cytophaga johnsonae could be resolved by gel filtration in sodium dodecyl sulfate (SDS) or by SDS-polyacrylamide gel electrophoresis into a high-molecular-weight (H) fraction (excluded by Sephadex G-200) and a low-molecular-weight (L) fraction. Fraction L was rich in components typical of lipid A and the core region of lipopolysaccharide (P, 3-hydroxy fatty acids, and 2-keto-3-deoxyoctonate) and evidently was a lipopolysaccharide with a limited number of distal, repeating polysaccharide units, as judged by SDS-polyacrylamide gel electrophoresis. In relation to total carbohydrate, the H fraction was rich in amino sugar but poor in (possibly devoid of) the lipid A and core components. Two nongliding mutants were highly deficient in the H fraction; one of these was deficient in sulfonolipid but could be cured by provision of a specific sulfonolipid precursor, a process that also resulted in the return of both the H fraction and gliding, as well as the ability to move polystyrene latex spheres over the cell surface. Hence, the polysaccharide may be the component that is directly involved in motility, and the presence of sulfonolipids in the outer membrane is necessary for the synthesis or accumulation of the polysaccharide. This conclusion was reinforced by the fact that the second nongliding, polysaccharide-deficient mutant had a normal sulfonolipid content.

Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) are potent endotoxins that are thought to be involved in the pathogenesis of Gram-negative septicemia. The liver is known to be the primary organ responsible for the clearance of LPS from the systemic circulation in mammals. In this work, 125I-labeled LPS have been used in a filtration assay for the specific binding of LPS to intact rat hepatocytes. Eight S-form (smooth) LPS with complete O-specific polysaccharide chains isolated from different O-serotypes of Salmonella and Escherichia coli as well as nine R-form (rough) LPS isolated from Salmonella mutants deficient in synthesis of their core oligosaccharides were used in this study. All 125I-labeled S-form LPS and R-form LPS, except Re, show specific binding to isolated hepatocytes. The binding is saturable, is inhibited with excess unlabeled homologous or heterologous LPS but not lipid A, and is trypsin sensitive. L-Glycero-D-mannoheptose (heptose), a constituent of the inner core region of almost all LPS, is a potent inhibitor of the specific binding of 125I-labeled Rb2 LPS, whereas other monosaccharides, including 3-deoxy-D-manno-2-octulosonic acid (KDO), have weak or negligible inhibitor activity. These results strongly suggest the presence of a lectin-like receptor for the LPS inner core region (heptose-KDO region) on the plasma membrane of rat hepatocytes.

Use of antibodies to the lipopolysaccharide core region in the treatment of gram‐negative sepsis and shock

AbstractIn this paper we review evidence in favor of a protective role for antibodies directed at the lipopolysaccharide (LPS) core region of Gram‐negative bacilli. It will be shown that, given the right animal model, polyclonal antisera raised against O‐antigen lacking, rough mutant strains, can be shown to protect animals against lethal sepsis due to a variety a bacterial species. Evidence for a protective role obtained from clinical studies will also be discussed. It will be stressed how difficult it is to prove that the antibodies directed against the LPS‐core, and not other proteins present in the polyclonal anti‐rough strain antisera, were responsible for the observed protective effects. The solution to some of these problems is the production of monoclonal antibodies (Mabs). We will discuss the findings that purified anti‐core Mabs may give false‐positive outcomes of protection studies, due to the induction of tolerance by small amounts of contaminating endotoxin, present in the antibody preparation. The need to administer the Mab therapeutically instead of prophylactically to the test animals, will be stressed. It will be shown how we succeeded in determining the epitope‐specificities of several anti‐core antibodies. It will be shown also that use of Elisa alone for characterization of anti‐core Mabs may lead to false conclusions concerning epitope specificities. We will prove that synthetic KDO‐(=ketodeoxycctanate) and lipid A‐containing antigens are indispensable tools for thorough characterization of Mabs. Using this methodology we were able to discriminate between specific antigen‐antibody interactions, and the ability of some Mabs to bind “non‐specifically” to hydrophobic surfaces. The relationship between epitope specificity and protective effects of anti‐core Mabs will be described. Finally, we will demonstrate that a KDO‐specific Mabs is capable of protecting mice against lethal sepsis; in other words, it will be shown conclusively that Mabs to defined epitopes in LPS core region are cross‐protective.

Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We utilized the recently described tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) system to study the lipooligosaccharides (LOS) and lipopolysaccharides (LPS) of gram negative bacteria. TSDS-PAGE resulted in a high degree of resolution of LOS and LPS in the ‘mini-gel’ format. TSDS-PAGE resulted in the LOS and LPS migrating as a function of their M r during electrophoresis and allowed estimation of M r from a protein standard. Several species of LOS were analyzed. The newly described procedure allowed a more rapid and accurate analysis of LOS and the core region of LPS.

Structure and serology of the lipopolysaccharides: Cell components of citrobacter

AbstractThe genus Citrobacter consists of enteric rods that utilize citrate as a sole carbon source. Citrobacter serotypes are normal intestinal inhabitants, but some of them have been reported in gastroenteritis and in extraintestinal infections.Our immumochemical studies on Citrobacter lipopolysaccharides of chemotype G comprised serotypes: 04, 027 and 036. As we found the serotypes mentioned above are distinguished in having O‐specific polysaccharides that are homopolymers of β [1–2] linked 4‐deoxy‐D‐arabinohexopyranose. In accordance with the structural indentity, the Citrobacter lipopolysaccharides showed strong serological cross‐reactivity. Nevertheless, serotypes 04, 027, and 036 form separate entities, as observed in early studies of serologists. To find the reason for the immunological diversity the structural studies on lipopolysaccharide core regions of these Citrobacter serotypes were carried out. Three novel enterobacterial cores were identified. The complete cores 04 and 036 are decasaccharides differing in one structural element only: side‐chain glucose is present in 04 oligosaccharide in the place of galactose in the 036. The core 027 is octasaccharide.In conclusion, our structural studies have shown hitherto that in Citrobacter chemotype G group only one O‐specific polysaccharide, but three core regions, exist.

ChemInform Abstract: Building Units of Oligosaccharides. Part 92. Synthesis of Trisaccharide Units of the Inner Core Region of Lipopolysaccharides.

ChemInform Abstract: Building Units of Oligosaccharides. Part 92. Synthesis of Trisaccharide Units of the Inner Core Region of Lipopolysaccharides.

Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy- d- manno-octulosonate-containing synthetic antigens

Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy- d- manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivatives of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective allyl glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, IgM) recognizing a terminal Kdo p group was shown to require for its binding the α-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG 3), which recognizes a (2→4)-linked Kdo disaccharide, was shown to require for its binding the α-anomeric configuration of both residues. The isomer having a reducing β-Kdo residue was significantly less active, and that with a terminal β-Kdo group was completely inactive. The OH-5 group of the reducing residue was shown to be not important for the specificity of this antibody, since it could be replaced by a hydrogen atom without loss of serological reactivity. The α-(2→8)-linked Kdo disaccharide was strongly cross-reactive with its (2→4)-linked isomer. The antibody recognized also parts of the 2-amino-2-deoxy- d-glucose backbone of lipid A.

ChemInform Abstract: Building Units of Oligosaccharides. Part 90. Synthesis of Structures of the Inner Core Region of Lipopolysaccharides.

AbstractThe heptopyranosyl chloride (I) reacts with the KDO‐glycosyl acceptor (II) (KDO: 3‐deoxy‐D‐manno‐2‐octulosonic acid) stereoselectively to give the α(1 → 5)‐linked disaccharide (III), which undergoes a deprotection sequence to give the title compound (IV).

Immunochemical Specificity of Human Antibodies to Lipopolysaccharide from the J5 Rough Mutant of Escherichia coli O111:B4

Antibody to the core region of lipopolysaccharide (LPS) of Escherichia coli O111:B4 (J5) is reported to cross-react with LPS from other gram-negative bacteria. We used an enzyme-linked immunosorbent assay to test various LPSs and their constituents for their ability to inhibit binding of human IgG antibody to J5 LPS to J5 LPS. Results were expressed as the concentration of inhibitor required to cause 50% inhibition of binding (I50). I50 values of antibody to J5 LPS for J5 LPS ranged from 5 x 10(-7) M to 8 x 10(-9) M. I50 values for Salmonella minnesota (Rc) were higher (2 x 10(-4) M to 2 x 10(-7) M). Other rough LPS failed to inhibit to 50% (up to 2 x 10(-4) M). J5 lipid A and core oligosaccharide inhibited, with I50 values ranging from 2 x 10(-5) M to 3 x 10(-8) M. Fifty percent inhibition was not achieved by smooth LPS (8 x 10(-5) M), heterologous lipid A (3 x 10(-4) M), or core LPS constituents (5 x 10(-3) M). These data suggest that IgG J5 LPS antibodies are specific for the Rc chemotype of the core of LPS.

Comparison of monoclonal antibodies to the deeper core region of Gram-negative lipopolysaccharide by means of polymyxin B inhibition studies

Previously we have described a panel of 12 monoclonal antibodies (Mabs) directed to lipopolysaccharide (LPS) of the Salmonella minnesota Re mutant R595. Six of them had been found to decrease mortality of LPS for actinomycin D-sensitized mice. The other six clones were not effective. It is known and we have confirmed that polymyxin B (PMB) also neutralizes LPS endotoxicity. We now tested the hypothesis that protective clones bound near or at the PMB binding site, by an in vitro assay where PMB and Mab competed for binding to R595 LPS. Our results show that this hypothesis must be rejected and that the LPS epitopes recognized by protective clones are interspersed by those recognized by non-protective ones. We could, however, demonstrate that this sort of inhibition assays are of value in estimating the localization on the core of the binding sites of various Mabs.

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa.

It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysaccharide also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.

Production and characterisation of mouse monoclonal antibodies reacting with the lipopolysaccharide core region of gram-negative bacilli

Monoclonal antibodies to the lipopolysaccharide (LPS) core region were produced by immunising mice with Escherichia coli strain J5 (chemotype Rc). One of these bound to the deepest part of the core, i.e., Lipid A, and reacted with other heat-killed but not live gram-negative bacilli, including E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Eight other monoclonal antibodies, binding to the terminal glucose residue of Rc LPS, reacted with live cells of E. coli strains only. Thus, the O antigen does not necessarily render the core inaccessible to antibody. However, despite binding to live bacteria, these monoclonal antibodies neither enhanced phagocytic killing, nor protected mice from dying from gram-negative infection or endotoxaemia. It is concluded that antibodies reacting with the most immunodominant parts of the J5 core are not protective.

Unmasking of bacteriophage Mu lipopolysaccharide receptors in Salmonella enteritidis confers sensitivity to Mu and permits Mu mutagenesis

The human pathogen Salmonella enteritidis 3b was found to be highly resistant to phage P22 and Mu derivatives. The Mu sensitivity (musA1) allele from Salmonella typhimurium could be transferred to S. enteritidis 3b at low frequency by cotransduction with hisG::Tn10. Sensitivity to Mu resulted in a large reduction in the number of lipopolysaccharide core-region oligosaccharides that were substituted with O-antigen polysaccharide. The residual high-molecular-weight lipopolysaccharide appeared to be a hybrid displaying O antigens which were immunologically related to those of S. typhimurium and not to those of S. enteritidis. Consequently, Mu d1(Ap lac) could then be transduced into Mus strains forming stable lysogens. On temperature induction, Mu transposition could easily be used to generate mutations in genes coding for cell surface antigens including fimbriae, lipopolysaccharide, and flagella.

Synthese von O-(3-desoxy-α- d- manno-2-octulopyranosylonsäure)-(2→4)- O-(3-desoxy-α- d- manno-2-octulopyranosylonsäure)- (2→6)- O-(2-amino-2-desoxy-β- d-glucopyranosyl)-(1→6)-2-amino-2-desoxy- d-glucopyranose

Benzyl-3- O-benzyl-2-benzyloxycarbonylamino-6- O-[2-benzyloxycarbonyl-amino-2-deoxy-3,4- O-(tetraisopropyldisiloxane-1,3-diyl)- β- d-glucopyranosyl]-2-deoxy-α- d-glucopyranoside was coupled with methyl (4,5,7,8-tetra- O-acetyl-3-deoxy-α- d- manno-2-octulopyranosyl bromide)onate ( 13) to yield the α-glycosidically linked trisaccharide. After deacetylation and selective introduction of a second 7′,8′- O-tetraisopropyldisiloxane group, a further glycosidation reaction with 13 led regioselectively to the tetrasaccharide benzyl O-[methyl (4,5,7,8-tetra- O-acetyl-3-deoxy-α- d- manno-2-octulopyranosyl)onate]-(2→4)- O-{methyl [3-deoxy-7,8- O-(tetraisopropyldisiloxane-1,3-diyl)-α- d- manno-2-octulopyranosyl]-onate}-(2→6)- O- [2-benzyloxycarbonylamino-2-deoxy-3,4- O-(tetraisopropyldisiloxane-1,3-diyl)-β- d-glucopyranosyl]- (1→6)-3- O-benzyl-2-benzyloxycarbonyl-amino-2-deoxy-α- d-glucopyranoside. A series of deblocking steps gave O-(3-deoxy-α- d- manno-2-octulopyranosylonic acid)-(2→4)- O-(3-deoxy-α- d- manno-2-octulopyranosylonic acid)- (2→6)- O-(2-amino-2-deoxy-β- d-glucopyranosyl)-(1→6)-2-amino-2-deoxy- d-glucopyranose which was identical with a tetrasaccharide that had been isolated by hydrazinolysis of the lipopolysaccharide from Salmonella minnesota R 595. Hence, synthetic proof is provided for the linkages in this part of the inner core region of lipopolysaccharides.

Novel Disaccharides Containing 3-Deoxy-D-Manno-2-Octulosonic Acid (KDO) and Lipid a Subunit Analogs

Abstract 3-Deoxy-D-manno-2-octulosonic acid (KDO)1 is a prominent constituent of bacterial lipopolysaccharides (LPS),2 and recently it has been demonstrated 3,4 that the KDO part in the core region of LPS is α-ketosidically linked to 0-6′ of the o-glucosamine-disaccharide backbone of lipid A. Although the portion, including KDO, has been suggested5,6 to be necessary for expression of antitumor and interleukin 1 (IL 1)-induction activities of LPS, the disticnt biological roles of KDO in LPS are still obscure.

Conservation and diversity of Campylobacter pyloridis major antigens.

Infection with Campylobacter pyloridis has been strongly associated with gastritis in humans although its etiologic significance is currently undefined. We examined the structure and antigenicity of whole-cell, outer-membrane, acid-extractable surface protein, and proteinase K-treated whole cell lysate preparations from eight C. pyloridis strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with homologous and heterologous immune rabbit serum. Whole-cell and outer-membrane profiles observed in all strains of C. pyloridis were nearly identical; none were similar to those of C. jejuni and C. fetus. Major whole-cell bands migrated at 26,000, 29,000, 56,000, and 62,000 molecular weights. The acid-extracted protein profiles of all C. pyloridis strains also were similar to one another and showed similarities with acid-extracted proteins from C. jejuni, with major bands migrating at 29,000, 48,000 to 53,000, and 62,000. All proteinase K-treated lysates showed different lipopolysaccharide (LPS) profiles, ranging from rough to smooth with multiple repeating side chains. Immunoblots of whole-cell and proteinase K-treated preparations of the C. pyloridis strains showed that there was antigenic cross-reactivity of proteins migrating at 62,000 and 56,000, but not in other regions, and cross-reactivity between LPS core regions but not side chains. These results suggest that C. pyloridis has both protein and core LPS group antigens and strain-specific protein and LPS side chain antigens.

ChemInform Abstract: Branched and Chain‐extended Sugars. Part 30. Diastereoselective Synthesis of L‐glycero‐D‐manno‐Heptose, a Constituent of the Inner Core Region of Lipopolysaccharides.

AbstractMit der Mannofuranose (I) als Ausgangsverbindung läßt sich die Titelverbindung (VIIb) (keine Ausb.‐Angaben) sowohl über die Zwischenstufe (IV) als auch über das Gemisch (V)/(VI) synthetisieren.

Use of mucin and hemoglobin in experimental murine gram-negative bacteremia enhances the immunoprotective action of antibodies reactive with the lipopolysaccharide core region.

An antiserum with a high content of antibodies, binding to the Gram-negative lipopolysaccharide core region, was prepared by immunizing rabbits with the rough Escherichia coli mutant J5. This antiserum was capable of protecting mice against lethal challenge doses of E. coli 0 111:B4 in a mouse model where the animals were compromised by means of mucin plus hemoglobin (LD 50 = 10(3) bacteria). However, no protection was observed in a non-compromised mouse model (LD 50 = 10(7) bacteria). This observation might explain why in the past so many discrepant results have been obtained in mouse protection studies with cross-reactive antisera.

Lipopolysaccharide structures in Enterobacteriaceae, Pseudomonas aeruginosa, and Vibrio cholerae are immunologically related to Campylobacter spp

To determine whether lipopolysaccharide (LPS) structures of Campylobacter species are immunologically related to those of 11 other gram-negative organisms, we immunoblotted from polyacrylamide gels the LPS of these strains with immune rabbit serum raised against six Campylobacter jejuni strains and two Campylobacter fetus strains. The LPS studied were from Salmonella minnesota wild type and Ra to Re mutants, Salmonella typhi, Escherichia coli, Yersinia enterocolitica, Vibrio cholerae, and Pseudomonas aeruginosa. None of the 11 LPS preparations was recognized by the eight antisera, but antisera to each of the Campylobacter strains recognized core determinants of some LPS preparations. Antiserum directed against the most serum-sensitive C. jejuni strain, 79-193, was the only antiserum sample that recognized core regions of the rough Salmonella mutants. In converse experiments, when LPS preparations from five Campylobacter strains were blotted with antiserum to Salmonella lipid A, recognition of core structures of each was shown; data from an enzyme-linked immunosorbent assay confirmed this result. In contrast, antiserum to Salmonella typhimurium Re LPS showed no reactivity. We conclude that LPS of Campylobacter strains share lipid A antigenic determinants with the core region of LPS of several other gram-negative organisms.

A 28,000-dalton protein of normal mouse serum binds specifically to the inner core region of bacterial lipopolysaccharide.

Normal mouse serum was found to contain a protein, referred to here as factor, which binds to the inner core region of lipopolysaccharides (LPSs) of various bacterial families. Since factor-LPS interactions resulted in activation of guinea pig complement, factor activity could be assayed by a passive hemolysis test with sheep erythrocytes coated with LPS or lipid A from Acinetobacter calcoaceticus (which was found earlier to bind particularly well to factor). Factor was purified by G-50 and hydroxyapatite chromatography whereby the specific hemolytic activity was enriched 1,675-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed the presence of a 28,000-dalton protein as the main band. The identity of this band was determined by absorption experiments with LPS-coated sheep erythrocytes or latex beads, whereby the 28,000-dalton band disappeared after specific absorption and could be recovered from the absorbent. The binding specificity of factor was determined in a passive hemolysis inhibition assay with defined oligosaccharides representative for the inner core region of LPS. Thus, the di- and trisaccharides alpha-D-mannoheptopyranosyl-(1----5)-2-keto-3-deoxy-D-mannoocto nic acid and alpha-D-mannoheptopyranosyl-(1----3)-alpha-D-mannoheptopyranosy l-(1----5)-2- keto-3-deoxy-D-mannooctonic acid, respectively, were able to inhibit binding of factor to LPS. The results are in accordance with our earlier observation that the heptose-2-keto-3-deoxy-D-mannooctonic acid region represents a common antigen of bacterial LPS. Rabbit hyperimmune serum directed against this common antigen and purified factor was found to exhibit the same specificity for LPS. Factor activity was followed in mice in vivo after injection of LPS; it disappeared completely 15 min after the injection of LPS and reappeared within 1 h.