The R-type lipopolysaccharides of Neisseria meningitidis

The lipopolysaccharides of all the different serogroups of Neisseria meningitidis are of the "R" type despite the morphologically smooth appearance and the demonstrated virulence of the organisms from which they were derived. This was confirmed when each of the lipopolysaccharides was found to be devoid of detectable O-antigen side chains, giving only a low "molecular" weight core oligosaccharide when subjected to mild acid hydrolysis. The cores were modified by dephosphorylation and subjected to sugar and methylation analysis by gas-liquid chromatography. Although all the different cores contained identical components (glucose, galactose, glucosamine, heptose, and 2-keto-3-deoxyoctonate) they could be separated into three distinct categories according to their galactose:glucose ratios. These categories are typified by the cores obtained from groups A, C, and 29-e which have galactose:glucose ratios of 1:2, 2:2, and 2:1, respectively. The modified cores were methylated and analyzed by gas chromatography--mass spectrometry and on the basis of differences in the derived methylated sugars the cores could again be divided into the same three categories as above. This structural diversity also results in some serological specificity as demonstrated by the complete serogroup specificity of the group A lipopolysaccharide.     

Rapid purification of extracted bacterial lipopolysaccharides by continuous free-flow electrophoresis

The use of continuous free-flow electrophoresis for the purification of extracted lipopolysaccharides ( LPSs ) was investigated. Commercial (nucleic acid contaminated) LPS preparations, isolated by the hot phenol-water method of Westphal from Salmonella typhimurium and Escherichia coli 0111: B4, were analyzed. Continuous free-flow electrophoresis for purification of crude LPSs proved to be a rapid and useful means for the continuous purification of large amounts of LPS (more than 45 mg crude LPS per hr) and it showed good reproducibility and pure LPS. The electrophoretic profile of both crude LPSs obtained by continuous free-flow electrophoresis showed two distinct, sharp peaks; one representing the nucleic acid fraction and the other the LPS fraction. Under the continuous free-flow electrophoresis conditions employed, nucleic acid in the crude LPSs possessed low electrophoretic mobility, whereas LPS migration was negligible. Thus for both preparations pure LPS (no detectable nucleic acid) was obtained. Electrophoretic profiles of these purified LPSs on sodium dodecylsulfate-polyacrylamide gel electrophoresis were similar in both cases to those of crude LPS and of LPS purified by repeated ultracentrifugation. By immunological analysis using double immunodiffusion and immunoelectrophoresis, it was found that two components of crude E. coli 0111: B4 LPS were eliminated by continuous free-flow electrophoresis, but each component of purified E. coli 0111: B4 LPS was immunologically identical to the corresponding component in its crude LPS. In S. typhimurium LPS, none of its components were influenced by continuous free-flow electrophoresis but not by ultracentrifugation. In spite of these results, both purified LPSs possessed stronger mitogenic activity than each crude LPS. These results indicated that continuous free-flow electrophoresis is a useful means of purifying extracted crude LPS.     

Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry.

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.     

Characteristics of bacterial lipopolysaccharides depending on extraction method

Carbohydrate-containing biopolymers have been isolated from Erwinia carotovora subsp. atroseptica 549 by two methods--the aqueous-phenol and with using physiological solution--with addition and without addition of ethylenediaminetetraacetate (EDTA). The biopolymers yield from the cells of bacteria are shown to depend on the extraction method. Lipopolysaccharide-protein complex have been isolated by the sparing method. The purest lipopolysaccharide have been isolated by the aqueousphenol method. The preliminary treatment of cells by EDTA increased the biopolymers output. Carbohydrate containing biopolymers isolated from the cells of bacteria by different methods possess the similar qualitative composition of monosaccharides but they differ in the quantitative content of monosaccharides, spectrum of fatty acids of lipid components as well as in the protein content.     

Structure of bacterial lipopolysaccharides

Bacterial lipopolysaccharides are the major components of the outer surface of Gram-negative bacteria They are often of interest in medicine for their immunomodulatory properties. In small amounts they can be beneficial, but in larger amounts they may cause endotoxic shock. Although they share a common architecture, their structural details exert a strong influence on their activity. These molecules comprise: a lipid moiety, called lipid A, which is considered to be the endotoxic component, a glycosidic part consisting of a core of approximately 10 monosaccharides and, in "smooth-type" lipopolysaccharides, a third region, named O-chain, consisting of repetitive subunits of one to eight monosaccharides responsible for much of the immunospecificity of the bacterial cell.     

A method for the purification of bacterial flagellin that allows simple upscaling

There is a growing interest in enterobacterial flagellins that may result in a demand to produce flagellin on an industrial scale for possible applications as an adjuvant, immunomodulatory agent or vaccine antigen. Traditionally, small-scale production of flagellin has occurred in the laboratory by flagellar shearing of bacterial surfaces and subsequent ultracentrifugation. The main drawback of this method is the need to use low-agitation cultures to avoid the loss of flagella due to shearing during culture. In the present work, we describe a scalable protocol for the production of flagellin with higher yields than traditional laboratory-scale protocols. The use of cross-flow filtration to concentrate bacterial cultures combines extensive shearing of flagella with a reduction in volume, greatly simplifying downstream processing. This technique also allows the use of highly-agitated culture conditions because any sheared flagella are retained in the bacterial concentrate. Flagella obtained with this procedure showed in vivo and in vitro innate activating capacities similar to those of flagella produced at laboratory scale. This procedure is flexible, allowing an increase in production scale, an enhancement of flagellin yield and no requirement for expensive equipment.     

A very efficient method to cleave Lipid A and saccharide components in bacterial lipopolysaccharides.

A novel mild procedure for the selective cleavage of ketosidic linkages is developed using ceric ammonium nitrate (CAN) in anhydrous N,N-dimethylformamide. Its application to lipopolysaccharides (LPS) is very significant because in the so far investigated LPS, the connection between the Lipid A region and the oligo(poly)saccharide part is always a keto-sugar. This procedure has been tested on LPS of Escherichia coli which contains Kdo as a linker between Lipid A and OPS and on Acinetobacter haemoliticus which contains D-glycero-D-talo-2-octulopyranosonic acid (Ko) as a linker and it performed efficiently in both cases.     

Study of the binding between bacterial lipopolysaccharides and polymyxin by a bioluminescent method

For rating the interaction of lipopolysaccharides (LPS) with polymixin B (PmB) a bacterial bioluminescence is offered to be used. Bioluminescence level of bacteria decreases under free antibiotic amounts action. It is shown, that as a result of interaction with LPS antibiotic properties of PmB are reduced, and the intensity of bacterial bioluminescence is restored. The bioluminescence level in such system characterizes the LPS quantity. Kinetic properties of bacterial light emission at the presence of LPS and PmB are investigated as well as equilibrium state of the system. Kinetic and equilibrium constants describing this reaction are determined. The conditions of quantitative bioluminescent definition of LPS in an interval of concentration 0.166-10 micrograms/ml have been chosen and calibration curves are presented.     

A novel chromatographic procedure for purification of bacterial plasmids

A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up.     

Electrophoretic and mass spectrometric strategies for profiling bacterial lipopolysaccharides

Capillary electrophoresis (CE) is a high-resolution separation technique that has been widely used for trace analysis in biological samples. On-line capillary electrophoresis-electrospray mass spectrometry (CE-MS) was developed for the analysis of lipopolysaccharide (LPS) glycoforms from the gram-negative bacteria, Haemophilus influenzae. In this paper, we report on the application of CE-MS to characterize structural differences in O-deacylated LPS samples from H. influenzae strains Rd 11.7 and 375.1. The resolution capability of on-line CE-MS was first demonstrated by analysis of a complex LPS mixture from H. influenzae strain Rd 11.7. This strain contains a mixture of isomeric glycoforms differing in the number and positions of hexose moieties. Sialic acid containing glycoforms were also determined. Structural features of LPS from a lic1 mutant of H. influenzae strain 375 (375.1) were studied using on-line CE-MS/MS. With the separation provided by CE, two isomeric glycoforms differing in the location of phosphoethanolamine substituents were characterized by tandem mass spectrometry.     

A new method for purification of anti-glycosphingolipid antibody. Avian anti-hematoside (NeuGc) antibody

A new method for purification of anti-glycosphingolipid antibodies had been developed. N-Glycolylneuraminyl(alpha 2-3)lactosylceramide [hematoside (NeuGc)] could be hydrophobically bound on octyl-Sepharose 4B in the presence of 0.1 M KCl. The Sepharose gel coated with hematoside (NeuGc) was used as immunoadsorbent for affinity column chromatography to purify avian anti-hematoside (NeuGc) antibody. The procedure is very simple, reproducible and applicable to purification of almost all anti-glycosphingolipid antibodies. The glycosphingolipid used for the affinity chromatography could be recovered without any destruction by successive extraction of the gel with methanol and methanol/chloroform (1:2, v/v).     

The occurrence of glycine in bacterial lipopolysaccharides

Abstract The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [¹⁴C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100°C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.     

Structural and functional analyses of bacterial lipopolysaccharides

Bacterial lipopolysaccharides (LPSs) are powerful immunomodulators in infected hosts, and may cause endotoxic shock. Most of them share a common architecture but vary considerably in structural motifs from one genus, species, and strain to another. Cells of the innate immune response recognize evolutionarily conserved LPS molecular patterns of endotoxins and structural details thereby greatly influencing their response.     

Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

Background  

Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production.     

Rhamnose-binding lectins from steelhead trout (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic acid

The interaction between bacteria and three L-rham-nose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.     

A host lipase detoxifies bacterial lipopolysaccharides in the liver and spleen

Much of the inflammatory response of the body to bloodborne Gram-negative bacteria occurs in the liver and spleen, the major organs that remove these bacteria and their lipopolysaccharide (LPS, endotoxin) from the bloodstream. We show here that LPS undergoes deacylation in the liver and spleen by acyloxyacyl hydrolase (AOAH), an endogenous lipase that selectively removes the secondary fatty acyl chains that are required for LPS recognition by its mammalian signaling receptor, MD-2-TLR4. We further show that Kupffer cells produce AOAH and are required for hepatic LPS deacylation in vivo. AOAH-deficient mice did not deacylate LPS and, whereas their inflammatory responses to low doses of LPS were similar to those of wild type mice for approximately 3 days after LPS challenge, they subsequently developed pronounced hepatosplenomegaly. Providing recombinant AOAH restored LPS deacylating ability to Aoah(-/-) mice and prevented LPS-induced hepatomegaly. AOAH-mediated deacylation is a previously unappreciated mechanism that prevents prolonged inflammatory reactions to Gram-negative bacteria and LPS in the liver and spleen.