S-type Lipopolysaccharides Research Articles

Structural characterization of bacterial lipopolysaccharides with mass spectrometry and on‐ and off‐line separation techniques

The focus of this review is the application of mass spectrometry to the structural characterization of bacterial lipopolysaccharides (LPSs), also referred to as "endotoxins," because they elicit the strong immune response in infected organisms. Recently, a wide variety of MS-based applications have been implemented to the structure elucidation of LPS. Methodological improvements, as well as on- and off-line separation procedures, proved the versatility of mass spectrometry to study complex LPS mixtures. Special attention is given in the review to the tandem mass spectrometric methods and protocols for the analyses of lipid A, the endotoxic principle of LPS. We compare and evaluate the different ionization techniques (MALDI, ESI) in view of their use in intact R- and S-type LPS and lipid A studies. Methods for sample preparation of LPS prior to mass spectrometric analysis are also described. The direct identification of intrinsic heterogeneities of most intact LPS and lipid A preparations is a particular challenge, for which separation techniques (e.g., TLC, slab-PAGE, CE, GC, HPLC) combined with mass spectrometry are often necessary. A brief summary of these combined methodologies to profile LPS molecular species is provided.

Contents: Electrophoresis 22'12

A novel, fast, and sensitive ME method was developed to analyze and differentiate the smooth (S) and rough (R) type bacterial endotoxin components labeled covalently with a fluorescent dye. The quantitative analysis of purified lipopolysaccharides, or partially purified samples from whole-cell lysates becomes possible with this method. Two groups with three sub-groups in the first group of S-type lipopolysaccharides can be classified based on the electrophoretic profiles. The LOD of the endotoxins from S- and R-type Gram-negative bacteria was found to be 2.6 ng and 6.9 ng, respectively. This method is capable to replace the commonly used SDS-PAGE combined with silver staining.

Antilipopolysaccharide II: An Antibody Protective Against Fatal Melioidosis

This was a study of IgG antibody responses to two S-type lipopolysaccharides (LPS I and LPS II) and flagellin of Burkholderia pseudomallei in patients with melioidosis. The specificity of these antibodies was 91.7%, 90.3%, and 93.8%, respectively, when compared to responses in a population where the organism is not endemic. Only the level of antibody to LPS II (anti-LPS II) was significantly higher in patients who survived than in those who died, as well as in patients with nonsepticemic vs. septicemic melioidosis. Results of logistic regression analysis, controlled for confounding factors such as duration of illness before treatment and bacteremic status, confirmed that a high level of anti-LPS II was a significant factor protective against fatal melioidosis. Thus, LPS II of B. pseudomallei would be a potentially useful component of a vaccine developed against fatal melioidosis. Further studies are in progress to determine the level of this antibody among those with asymptomatic infection in areas where melioidosis is endemic.

Role of lectins in the innate immunity of horseshoe crab

We have purified five types of lectins, named tachylectins, from circulating hemocytes and hemolymph plasma of the Japanese horseshoe crab, Tachypleus tridentatus. Tachylectin-1 interacts with Gram-negative bacteria probably through 2-keto-3-deoxyoctonate, one of the constituents of lipopolysaccharides (LPS). Tachylectin-1 also binds to polysaccharides such as agarose and dextran with broad specificity. Tachylectin-2 binds to d-GlcNAc or d-GalNAc and recognizes staphylococcal lipoteichoic acids and LPS from several Gram-negative bacteria. In contrast, tachylectins-3 and -4 specifically bind to S-type LPS from several Gram-negative bacteria through a certain sugar moiety on the O-specific polysaccharides (O-antigens). Tachylectin-5 identified in hemolymph plasma has the strongest bacterial agglutinating activity in the five types of tachylectins, and exhibits broad specificity against acetyl group-containing substances. Thus, the innate immune system of horseshoe crab may recognize invading pathogens through a combinatorial method using lectins with different specificities against carbohydrates exposed on pathogens. An encounter of these lectins derived from hemocytes and hemolymph plasma at injured sites, in response to the stimulation of LPS, suggests that they serve synergistically to accomplish an effective host defense against invading microbes and foreign substances.

A Newly Identified Horseshoe Crab Lectin with Specificity for Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial Lipopolysaccharides

A 14-kDa lectin, named tachylectin-3, was newly identified from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. This lectin exhibited hemagglutinating activity against human A-type erythrocytes, but not against the B- and O-types of erythrocytes and animal erythrocytes, including those of sheep, rabbit, horse, and bovine. The hemagglutinating activity of tachylectin-3 was equivalent to that of a previously identified lectin, named tachylectin-2, with affinity for N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. However, the activity of tachylectin-3 was not inhibited by these two N-acetylhexosamines at 100 mM but was inhibited by a blood group A-pentasaccharide at a minimum inhibitory concentration of 0.16 mM. Furthermore, the hemagglutinating activity was strongly inhibited by bacterial S-type lipopolysaccharides (LPSs) from Gram-negative bacteria but not by R-type LPSs lacking O-antigens. One of the most effective S-type LPSs was from Escherichia coli O111:B4, with a minimum inhibitory concentration of 6 ng/ml. These data suggest that tachylectin-3 specifically recognizes Gram-negative bacteria through the unique structural units of O-antigens. Ultracentrifugation analysis revealed that tachylectin-3 is present in dimer in solution. A cDNA coding for tachylectin-3 was isolated from a hemocyte cDNA library. Tachylectin-3 consisted of two repeating sequences, each with a partial sequence similarity to rinderpest virus neuraminidase. Tachylectin-3 and three previously isolated types of tachylectins were all predominantly expressed in hemocytes and released from hemocytes in response to external stimuli. These lectins present at injured sites suggest that they probably serve synergistically to accomplish an effective host defense against invading microbes.

A Newly Identified Horseshoe Crab Lectin with Binding Specificity to O-antigen of Bacterial Lipopolysaccharides

We identified a novel horseshoe crab hemocyte-derived lectin, which we named tachylectin-4. It has more potent hemagglutinating activity against human A-type erythrocytes than a previously identified hemocyte lectin with an affinity to N-acetylglucosamine, tachylectin-2. The purified tachylectin-4 is an oligomeric glycoprotein of 470 kDa, composed of subunits of 30 and 31.5 kDa. Ca2+ at 10 mM enhanced the hemagglutinating activity 4-fold, and the activity was inhibited by EDTA and o-phenanthroline. L-Fucose and N-acetylneuraminic acid at 100 mM completely inhibited the activity of tachylectin-4. The activity was also inhibited more strongly by bacterial S-type lipopolysaccharides (LPS) but not by R-type LPS lacking O-antigen. The most effective S-type LPS was from Escherichia coli O111:B4, and the minimum concentration required for inhibiting agglutination against human A-type erythrocytes (0.1 microg/ml) was 160-fold lower than those of S-type LPS from Salmonella minnesota. Therefore, colitose (3-deoxy-L-fucose), a unique sugar present in the O-antigen of E. coli O111:B4 with structural similarity to L-fucose, is the most probable candidate for a specific ligand of tachylectin-4. A cDNA coding for tachylectin-4 was isolated from a hemocyte cDNA library. The open reading frame of the 1344-base pair cDNA coded for the mature protein with 232 amino acids. There is no significant sequence similarity to any other known LPS-binding lectins, whereas tachylectin-4 is homologous to the NH2-terminal domain with unknown functions of Xenopus laevis pentraxin 1.

Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei.

A serologically typical strain of Burkholderia pseudomallei (strain 304b) was found to produce two S-type lipopolysaccharides (LPS) differing in the chemical structures of their O-polysaccharide (O-PS) components. Structural analysis revealed that one O-antigenic polysaccharide (O-PS I) is an unbranched high-molecular-weight polymer of 1,3-linked 2-O-acetyl-6-deoxy-beta-D-manno-heptopyranose residues. The other LPS O antigen (O-PS II) is an unbranched polymer of repeating disaccharide units having the structure -3)-beta-D-glucopyranose-(1-3)-6-deoxy-alpha-L-talopyranose-(1- in which ca. 33% of the L-6dTalp residues bear 2-O-methyl and 4-O-acetyl substituents while the other L-6dTalp residues carry only 2-O-acetyl substituents. Analysis of a serologically atypical strain of B. pseudomallei (strain 824a) produced a single LPS O-PS which was chemically identical to the 6-deoxy-D-manno-hepan O-PS I. The production of two distinct LPS raises the interesting question of their relative immunogenicities and consequently their relative importance for diagnostic serology and for the possible development of conjugate vaccines.

The sialic acid-containing lipopolysaccharides of Salmonella djakarta and Salmonella isaszeg (serogroup O: 48): chemical characterization and reactivity with a sialic acid-binding lectin from Cepaea hortensis.

Lipopolysaccharides (LPS) of Salmonella djakarta and Salmonella isaszeg, as well as of a spontaneous R-mutant of S. djakarta were investigated as to their content in neuraminic acid (Neu) and its individual linkage. The two Salmonella serovars both belong to the O:48 serogroup of Salmonella, but to two different subgroups. LPS of both S-forms contained high amounts of Neu, although in different quantities, whereas the R-form was completely devoid of it. Methylation analysis indicated that Neu is exclusively terminally linked in S. djakarta whereas both terminal and 4-linked Neu were recognized in S. isaszeg. Although terminally linked, a sialidase from Arthrobacter ureafaciens was unable to split Neu even after prolonged incubation from both S-type LPSs. When LPS was first treated by mild alkali, however, the total amount of Neu from S. djakarta LPS and about 50% from that of LPS of S.isaszeg could be removed. In contrast, alkali-treated LPS, but also the non-treated one, proved to be effective inhibitors for a sialic acid-binding lectin from Cepaea hortensis. The resistance of terminal Neu towards sialidase may be due to the presence of an O-acetyl group which would be removed during the methylation analysis but would, especially when linked to C-4, not interfere with the reactivity of the lectin.

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata, M. segnis, M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid ADAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid AGlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component “X”. In addition to “X”, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid ADAG), or in 3-OH-14:0 (in Lipid AGlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.

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.

Lipopolysaccharides ofVibrio fluvialis 181-86 Kobe possessing an antigenic factor in common with O1Vibrio cholerae

The chemical and serological characteristics of lipopolysaccharides (LPS) isolated fromVibrio fluvialis 181-86 Kobe were compared with those of LPS isolated from O1Vibrio cholerae and Vibrio bio-serogroup 1875 (Original and Variant), a marine vibrio possessing an antigenic factor in common with O1Vibrio cholerae. Two kinds of LPS (S-type LPS and R-type LPS) were extracted fromV. fluvialis 181-86 Kobe. The S-type LPS is different from LPS of the other serotypes (O1–O18) ofV. fluvialis in that the former was found to contain the pair perosamine and quinovosamine, a characteristic component of O1V. cholerae LPS. Furthermore, the sugar composition of the S-type LPS is close to that of Vibrio bio-serogroup 1875 LPS because both LPS contained mannose and the same two unidentified neutral sugars. Definite serological cross-reactivity in the passive hemolysis and the passive hemolysis inhibition tests were demonstrated between LPS fromV. fluvialis 181-86 Kobe and that from Vibrio bioserogroup 1875 Variant.