Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo.

The survival of Salmonella montevideo during serum treatment depends on the presence of an O antigen (O-Ag) associated with the lipopolysaccharide molecule. In this organism, the O antigen is a polysaccharide composed of 0 to more than 55 subunits, each containing 4 mannose residues together with glucose and n-acetylglucosamine. We used a mutant strain of S. montevideo that requires exogenous mannose for the synthesis of O-Ag. Lipopolysaccharide (LPS) was prepared from these cells grown under three different conditions where the availability of exogenous mannose was regulated such that the average number of O-Ag units per LPS molecule, the percentage of LPS molecules bearing long O-Ag side chains, and the percentage of lipid A cores bearing O-Ag were all varied. These changes in LPS profiles were monitored on sodium dodecyl sulfate-polyacrylamide gels, and cells with different LPS profiles were tested for their ability to survive treatment with pooled normal human serum. Survival in serum was associated with LPS that contained an average of 4 to 5 O-Ag units per LPS molecule, and 20 to 23% of the LPS molecules had more than 14 O-Ag units per LPS molecule. Serum survival was less clearly associated with the percentage of lipid A cores covered with O-Ag. We propose, based on these data and on previous work, that the O-Ag polysaccharide provides the cell protection from serum killing by sterically hindering access of the C5b-9 complex to the outer membrane and that a critical density of long O-Ag polysaccharide is necessary to provide protection.     

Subunit glycosylation of Lupinus angustifolius seed globulins

Reduced polypeptide subunits of α-, β- and γ-conglutins from Lupinus angustifolius seeds were resolved by preparative SDS gel electrophoresis of the fluorescent labelled proteins, into four, six and two major components, respectively. All subunits were glycosylated, to varying degrees, containing mannose, galactose and glucosamine. The major glycopeptides released by pronase digestion of each conglutin had similar galactose/mannose ratios; the MW of the glycopeptide released from α- and β-conglutin was ca 5000. Although on average, each molecule of α-conglutin contains one main oligosaccharide chain, and β-conglutin two, the presence of carbohydrate in all polypeptide subunits suggests that some subunits may arise by proteolytic cleavage of a larger polypeptide after glycosylation. The presence of minor glycopeptide components indicates that modification of carbohydrate chains during seed development may also occur.     

Cell wall lipopolysaccharide response to the ColIb plasmid mutants.

Mutants of ColIb plasmid affected the synthesis of O-side chains of lipopolysaccharides (LPS) in Salmonella. The plasmid srd 25 (defective in colicin synthesis) caused a significant decline of rhamnose and mannose content and lack of abequose in LPS of S. typhimurium. The number of repeating units in O-side chains was decreased after the indroduction of srd 25. Cultures of S. typhimurium and S. enteritidis harboring drd2 (derepressed in colicin production) polymerised dideoxyhexose-defective O-side chains i.e. deprived of abequose and tyvelose, respectively. In dideoxyhexoseless S. meleagridis the content of rhamnose and mannose were reduced. The information for the alterations of Salmonella LPS was contained in the plasmid genome. In the wild-type plasmids the genes controlling the O-antigen changes were not expressed.     

1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.     

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.     

Composition of O-antigenic lipopolysaccharides from Enterobacter cloacae

Analyses have been carried out on lipopolysaccharides (LPS) from 14 strains of Enterobacter cloacae representing different O serotypes. All of the products appeared to have a composition and architecture typical of enterobacterial LPS, but points of interest include the absence of phosphate residues from the core oligosaccharide, the presence of both L-glycero-D-mannoheptose and D-glycero-D-mannoheptose (ratio usually about 4:1), and the presence in lipid A of small amounts of fatty acids with odd numbers of carbon atoms (mainly C13) in addition to tetradecanoic acid and 3-hydroxytetradecanoic acid. Monosaccharides identified as components of polymeric fractions from the LPS were glucose, galactose, mannose, rhamnose, glucosamine, galactosamine, fucosamine, and galacturonic acid. Most polymeric fractions also probably contained an O-acetyl substituent. Closely similar chemotypes found for the polymeric fractions from the LPS of cross-reacting serotypes support the view that these fractions contain the O-antigenic determinants and represent the side chains of the LPS.     

Investigation of lipopolysaccharides from Sinorhizobium meliloti SKHM1-188 and two of its mutants with decreased nodulation competitiveness

A comparative study of the lipopolysaccharides (LPS) isolated from Sinorhizobium meliloti SKHM 1-188 and two its LPS-mutants (Th29 and Ts22) with sharply decreased nodulation competitiveness was conducted. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all the three strains: a higher molecular-weight LPS1, containing O-polysaccharide (O-PS), and a and lower molecular-weight LPS2 without O-PS. However, the LPS1 content in mutants was significantly smaller than in the parent strain. The LPS of the strains studied contained glucose, galactose, mannose, xylose, three nonidentified sugars--X1 (TGlc 0.53), X2 (TGlc 0.47), and X3 (TGlc 0.43), glucosamine, and ethanolamine, while the LPS of S. meliloti SKHM1-188 additionally contained galactosamine, glucuronic and galacturonic acids, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as fatty acids, such as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T3-OH C14:0 1.33), C18:0, and unsaturated C18:1 fatty acids. The LPS of both mutants were similar in the component composition but differed from the LPS of the parent strain by a lower X2, X3, and 3-OH C 14:0 content and a higher KDO, C18:0, and hydroxy X content. The LPS of all the strains were subjected to mild hydrolysis with 1% acetic acid and fractionated on a column with Sephadex G-25. The higher molecular weight fractions (2500-4000 Da) contained a set of sugars typical of intact LPS and, supposedly, corresponded to the LPS polysaccharide portion (PS1). In the lower molecular weight fractions (600-770 Da, PS2), glucose and uronic acids were the major components; galactose, mannose, and X1 were present in smaller amounts. The PS1/PS2 ratio for the two mutants was significantly lower than for strain SKHM1-188. The data obtained show that the amount of O-PS-containing molecules (LPS1) in the heterogeneous lipopolysaccharide complex of the mutants was smaller than in the SKHM1-188 LPS; this increases the hydrophobicity of the cell surface of the mutant bacteria. This supposedly contributes to their nonspecific adhesion on the roots of the host plant, thus decreasing their nodulation competitiveness.     

Identification of three oligo-/polysaccharide-specific ligases in Yersinia enterocolitica

In lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria the lipid A-core oligosaccharide (LA-core) and O-polysaccharide (O-PS) biosynthesis pathways proceed separately and converge in periplasmic space where the waaL-encoded ligase joins O-PS onto LA-core. Enterobacterial common antigen (ECA) biosynthesis follows that of O-PS except that ECA is usually ligated to phosphatidylglycerol (PG) and only rarely to LA-core. In Yersinia enterocolitica serotype O:3 LPS is composed of LA-inner core (IC) onto which a homopolymeric O-PS, a hexasaccharide called outer core (OC), and/or ECA are ligated. We found that an individual O:3 LPS molecule carries either OC or O-PS substitution but not both. Related to this, we identified three genes in Y. enterocolitica O:3 that all expressed O-PS ligase activity in the Escherichia coliΔwaaL mutant. The LPS phenotypes of Y. enterocolitica O:3 single, double and triple ligase mutants indicated that two of ligases, named as WaaL(os) and WaaL(ps) , had a preferred substrate specificity for OC and O-PS, respectively, although with some promiscuity between the ligases; the third ligase named as WaaL(xs) was not involved in LPS or ECA biosynthesis. In Y. enterocolitica O:8 the WaaL(os) homologue (Ye1727) ligated a single pentasaccharide O-unit to LA-IC suggesting that in both Y. enterocolitica O:3 and O:8 WaaL(os) is an oligosaccharide (OS)-specific ligase. Finally, Yersinia pestis and Y. pseudotuberculosis carry only the waaL(ps) gene, while either waaL(os) or waaL(xs) or both are additionally present in other Yersinia species. This is the first report on the presence of three different oligo-/polysaccharide-specific ligases in a single bacterium.     

Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length

We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain-length determinant (CLD) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli 0111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states:‘E’facilitating extension and T facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cld gene.     

Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance

This study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa , rfb , and rol , and in a plasmid-encoded O-antigen chain-length regulator, cld _pHS-2. LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O-antigen side chains or produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX , affected the LPS core region and resulted in reduced amounts of O-antigen. Mutants defective in cld _pHS-2 or rol had different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Serény test, and serum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cld _pHS-2 is required for resistance to serum killing and for full inflammation in the Serény test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque formation, but not for serum resistance. Thus, O-antigen synthesis and chain-length regulation genes encoded on both the chromosome and the small plasmid pHS-2 play important roles in S. flexneri invasion and virulence.     

Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli.

Lipopolysaccharide (LPS), a glycolipid molecule found on the outer leaflet of outer membranes of gram-negative bacteria, consists of three moieties: lipid A, core oligosaccharide, and the O-specific polysaccharide chain. The O-specific side chain, which extends to the extracellular milieu, plays an important role in pathogenicity, especially during the initial stages of infection, because of its ability to interact with serum complement. In recent years, several laboratories have used recombinant DNA tools to determine, at the molecular level, the organization, expression, and regulation of genes involved in LPS biosynthesis in Salmonella and Escherichia coli. An increased understanding of the molecular aspects of the O-specific side-chain genes will shed light on the intimate details related with the formation of the O-specific side chain, its assembly onto the lipid A--core, and the translocation and insertion of the complete LPS molecule into the outer membrane. It will also contribute to the understanding of the evolution of these genes and the correlation of chemical diversity of O-specific side chains with the genetic diversity of O-specific side-chain genes. In addition, since the O-specific side chains are involved in the pathogenicity of medically important gram-negative bacteria, a basic understanding of the regulation and expression of O-specific side chain LPS genes will contribute to the field of molecular pathogenesis. This article provides an overview of the role of O-specific side chains in septicemic infections and also discusses the current status of molecular genetic studies on O-specific side-chain genes from E. coli.     

Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine

Many studies have shown that the human blood fluke Schistosoma mansoni contains glycoproteins whose oligosaccharide side chains are antigenic in infected hosts. We report here that adult male schistosomes synthesize glycoproteins containing complex-type N-linked chains that have structural features not commonly found in mammalian glycoproteins. Adult male worms were incubated in media containing either [3H]mannose, [3H]glucosamine, or [3H]galactose, and the metabolically radiolabeled oligosaccharides on newly synthesized glycoproteins were analyzed. Schistosomes synthesize triantennary- and biantennary-like complex-type asparagine-linked chains that contain mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Interestingly, none of the complex-type chains contain sialic acid, and few of the chains contain galactose. Since N-acetylgalactosamine is not a common constituent of mammalian-derived N-linked chains, we investigated the position and linkage of this residue in the schistosome-derived glycopeptides. Virtually all of the N-acetylgalactosamine was beta-linked and in a terminal position. The unusual features of the S. mansoni glycoprotein oligosaccharides support the possibility that they may be involved in the host immune response to infection.     

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.     

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).     

Serological diversity and chemical structures of Campylobacter jejuni low-molecular-weight lipopolysaccharides.

Low-Mr lipopolysaccharides (LPS) of Campylobacter jejuni reference strains for serotypes O:1, O:4, O:23, and O:36 were examined through the liberation of core oligosaccharides by mild acid cleavage of the ketosidic linkage of 3-deoxy-D-manno-2-octulosonic acid residues to the lipid A moiety. The liberated oligosaccharides were examined for chemical structure by compositional analysis and methylated linkage analysis in conjunction with fast atom bombardment-mass spectrometry of permethylated oligosaccharide derivatives. The results showed (i) that the LPS contained short oligosaccharide chains of branched nonrepetitive structure, to many of which N-acetylneuraminic acid residues remained attached by 2----3 linkages to 4-linked D-galactose residues in the core structure; (ii) that serotypical differences, which are not readily defined through qualitatively similar compositions, are clearly reflected in variations in linkage types and sequences of sugar residues in the outer core attached to an inner region of invariable structure; but (iii) that the presence or absence of NeuAc residues does not appear to be a basis for serotypical differences. The results also showed that oligosaccharide chains from LPS of serotypes O:1 and O:4 are distinctly different and are distinct again from those of the cross-reacting serotypes O:23 and O:36, between whose core oligosaccharide chains no differences were found. It is concluded that the structurally variable low-Mr LPS from C. jejuni show greater similarities to the lipooligosaccharides from Neisseria spp. than to the highly conserved core regions of Salmonella species. Those strains (serotypes O:23 and O:36) which also furnish high-Mr LPS are unique among gram-negative bacteria in possessing both low-Mr molecules of the Neisseria lipooligosaccharide type and high-Mr LPS of the Salmonella smooth type.     

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.     

Identification of an ATP-binding cassette transporter for export of the O-antigen across the inner membrane in Rhizobium etli based on the genetic, functional, and structural analysis of an lps mutant deficient in O-antigen

For O-antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenyl pyrophosphate-bound O-antigen oligosaccharide subunits or polysaccharide occurs before ligation to the core region of the LPS molecule. In this study, we identified by mutagenesis an ATP-binding cassette transporter in Rhizobium etli CE3 that is likely responsible for the translocation of the O-antigen across the inner plasma membrane. Mutant FAJ1200 LPS lacks largely the O-antigen, as shown by SDS-polyacrylamide gel electrophoresis and confirmed by immunoblot analysis. Furthermore, LPS isolated from FAJ1200 is totally devoid of any O-chain glycosyl residues and contains only those glycosyl residues that can be expected for the inner core region. The membrane component and the cytoplasmic ATP-binding component of the ATP-binding cassette transporter are encoded by wzm and wzt, respectively. The Tn5 transposon in mutant FAJ1200 is inserted in the wzm gene. This mutation resulted in an Inf- phenotype in bean plants.     

Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium

Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45 degrees C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30 degrees C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45 degrees C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45 degrees C-grown cells of SA134 (rfa13020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45 degrees C; following a switch to 30 degrees C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complement activation via the alternative pathway by purified Salmonella lipopolysaccharide is affected by its structure but not its O-antigen length

Salmonellae, the lipopolysaccharide of which differ in the chemical structure of their O-antigenic side chains, were previously shown to activate C3 at differential rates via the alternative pathway. We wanted to test whether lipopolysaccharide isolated from these strains yields identical results, and also the effect of the polysaccharide chain length, which varies from 0 to 40 or more repeating units in a single strain. Lipopolysaccharide was purified from the above strains, hydrolyzed (0.1 N NaOH, 56 degrees C, 30 min), and used to coat sheep erythrocytes to different densities, and C3 activation in C4-deficient guinea pig serum was measured. C3 activation was proportional to lipopolysaccharide density and time, and the relative rates and extents of activation by this bacteria-free system were the same as for the original bacteria. Activation was reduced 10 to 15% when the serum was preabsorbed with strains either containing or lacking O-antigen side chain, suggesting augmentation by antibody; however, even after multiple absorptions, activation varied with O-antigen structure as expected. This differential activation was not due to differences in the average length of the O-antigenic polysaccharide chains, because the size was similar for all three lipopolysaccharides. Moreover, the extent of activation by lipopolysaccharide that had been fractionated on a column of Sephadex G-200 was independent of the polysaccharide chain length for lengths greater than 3 repeating units. The results prove that C3 activation by lipopolysaccharide via the alternative pathway is sensitive to slight variations in the chemical structure, but not to large variations in length of the O-antigen polysaccharide side chain of lipopolysaccharide.