Composition of the fractions separated by polyacrylamide gel electrophoresis of the lipopolysaccharide of a marine bacterium.

The sugar composition of lipopolysaccharide (LPS) isolated from whole cells of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonas B-16, ATCC 19855), variant 3, of the lipid A, core, and side-chain fractions derived from it, and of the LPS fractions (LPS I, II, and III) obtained by subjecting it to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been determined. Conditions optimum for the release of constituent monosaccharides by hydrolysis were established. Sugars were quantitated by gas-liquid chromatography of their alditol acetate derivatives. Lipid A was detected by gel electrophoresis and by the spectral shift obtained with a carbocyanin dye. A comparison of the molar ratios of the various fractions suggest that LPS III is an LPS molecule lacking an O-antigenic side chain, whereas LPS I and II are LPS molecules differing in side-chain composition. LPS I may be a mixture of two LPS species. In double immunodiffusion experiments using anti-whole-cell serum, LPS I and II showed a homologous cross-reaction with isolated whole-cell LPS. LPS III as well as lipid A, core, and side-chain fractions failed to give rise to precipitin lines.     

Heterogeneity and Distribution of Lipopolysaccharide in the Cell Wall of a Gram-Negative Marine Bacterium

Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and III, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS III, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I.     

Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii.

Lipopolysaccharides (LPSs) isolated from phase I and phase II Coxiella burnetii (LPS I and LPS II, respectively) were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The yields of crude phenol-water extracts from phase I cells were roughly three to six times higher than those from phase II cells. Purification of LPSs by ultracentrifugation gave similar yields for both LPS I and LPS II. Purified LPS I and LPS II contained roughly 0.8 and 0.6% protein, respectively. The fatty acid constituents of the LPSs were different in composition and content, with branched-chain fatty acids representing about 15% of the total. beta-Hydroxymyristic acid was not detected in either LPS I or LPS II. A thiobarbituric acid-periodate-positive compound was evident in the LPSs; however, this component was not identified as 3-deoxy-D-mannooctulosonic acid by gas and paper chromatographies. LPS II contained D-mannose, D-glucose, D-glyceromannoheptose, glucosamine, ethanolamine, 3-deoxy-D-mannooctulosonic acid-like material, phosphate, and fatty acids. LPS I contained the unique disaccharide galactosaminuronyl glucosamine and nine unidentified components in addition to the components of LPS II. The hydrophobic, putative lipid A fraction of LPS I and LPS II contained the above constituents, but the hydrophilic fraction was devoid of ethanolamine. The LPS I disaccharide galactosaminuronyl glucosamine was found in both fractions of the acetic acid hydrolysates. Analysis of LPSs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that LPS II was composed of only one band, whereas LPS I consisted of six or more bands with irregular spacing. Ouchterlony immunodiffusion tests demonstrated that LPS I reacted with phase I but not with phase II whole-cell hyperimmune antibody, and LPS II reacted neither with phase I nor phase II hyperimmune antibody. From these results, it was concluded that the chemical structures of LPSs from C. burnetii were different from those of the LPSs of gram-negative bacteria; however, the LPS structural variation in C. burnetii may be similar to the smooth-to-rough mutational variation of saccharide chain length in gram-negative bacteria.     

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.     

Immunological Studies on Dermatophytes II. Serological Reactivities of Mannans Prepared from Galactomannans I and II of Microsporum quinckeanum, Trichophyton granulosum, Trichophyton interdigitale, Trichophyton rubrum, and Trichophyton schoenleinii

The contribution of terminal galactofuranose residues to the antigenic specificity and to cross-reactivity of galactomannans isolated from five species of dermatophytes, Microsporum quinckeanum, Trichophyton granulosum, T. interdigitale, T. rubrum, and T. schoenleinii, was investigated. Galactofuranose units were removed from galactomannans I and galactomannans II by mild acid hydrolysis. The resulting mannans were tested for serological reactivity with rabbit antiserum to M. quinckeanum by qualitative precipitation in gel and by quantitative complement-fixation analyses. Our results showed that, with this antiserum, the galactofuranose residues contributed greatly to the antigenic specificity and to cross-reactivity of the galactomannans II, but these residues were less significant as antigenic determinants in the galactomannans I. We have shown that mannans isolated from three Candida species reacted with rabbit antiserum to M. quinckeanum.     

The O18 antigens (lipopolysaccharides) of Escherichia coli. Structural characterization of the O18A, O18A1, O18B and O18B1-specific polysaccharides.

The O-specific polysaccharide moieties (PS) of the O18A, O18A1, O18B, and O18B1 antigens (lipopolysaccharides, LPS) consist of L-rhamnose (Rha), N-acetyl-D-glucosamine, D-galactose, and D-glucose in different molar ratios. By using chemical fragmentation, methylation, as well as one- and two-dimensional NMR spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In O18A-PS and O18A1-PS x = 2, whereas in O18B-PS and in O18B11-PS x = 3. In all four polysaccharides alpha-D-Galp (residue D) is substituted at O-3. This substituent L (residue E) is beta-D-GlcpNAc-(1 in O18A-PS and O18A1-PS and it is alpha-D-Glcp-(1 in O18B-PS and O18B1-PS. Whereas there is no further substituent on the main chain of the O18A and O18B polysaccharides, in O18A1-PS and O18B1-PS the alpha-D-GlcpNAc residue A is substituted with alpha-Glcp-(1 (residue F), which is linked to O-6 in O18A1-PS and to O-4 in O18B1-PS. These results show that the O18 antigen comprises a group of four related LPS (O18A and O18B, with their glucosylated forms O18A1 and O18B1). The results are discussed with respect to epitope definition and biochemical implications.     

Purification and characterization of glycosphingolipid-specific endoglycosidases (endoglycoceramidases) from a mutant strain of Rhodococcus sp. Evidence for three molecular species of endoglycoceramidase with different specificities

Two molecular species of endoglycoceramidase (designated as endoglycoceramidases I and II) were purified 32,700 and 43,000 times with overall recoveries of 4.8 and 2.9%, respectively, from a culture fluid of the mutant strain M-750 of Rhodococcus sp., cultivated in the absence of inducers (ganglioside). After being stained with Coomassie Brilliant Blue or a silver-staining solution, each purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The apparent molecular weights, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 55,900 for endoglycoceramidase I and 58,900 for endoglycoceramidase II, and their pIs were 5.3 and 4.5, respectively. both were capable of hydrolyzing the glucosylceramide linkage of ganglio-type, lacto-type, and globo-type glycosphingolipids to afford intact oligosaccharides and ceramides. Globo-type glycosphingolipids were strongly resistant to hydrolysis by endoglycoceramidase II in comparison with endoglycoceramidase I. Neither could hydrolyze gala-type glycosphingolipids, cerebrosides, sulfatides, glycoglycerolipids, or sphingomyelins. In addition to these two enzymes, the strain M-750 produced a third minor molecular species of endoglycoceramidase designated as endoglycoceramidase III. It was found capable of specifically hydrolyzing the galactosylceramide linkage of gala-type glycosphingolipids that were not hydrolyzable at all by endoglycoceramidases I or II. The molecular weights of the oligosaccharide and ceramide released from asialo GM1, incubated either in normal H2O or H2(18)O with the enzyme, were compared by fast atom bombardment-mass spectrometry. The result clearly indicated that both endoglycoceramidases I and II hydrolyze the glycosidic linkage between the oligosaccharide and ceramide. Thus, a systematic name of the endoglycoceramidase should be glycosyl-N-acyl-sphingosine 1,1-beta-D-glucanohydrolase.     

Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa.

Serotypes O2, O5, and O16 of Pseudomonas aeruginosa are chemically related, and the O antigens of their lipopolysaccharides share a similar trisaccharide repeat backbone structure. Serotype-specific monoclonal antibodies (MAbs) MF71-3, MF15-4, and MF47-4 against the O2, O5, and O16 serotypes, respectively, were isolated. MAb 18-19, which is cross-reactive with all strains of this chemically related serogroup, was also produced. When column chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated lipopolysaccharide (LPS) samples from each of the serotypes were probed with the MAbs in Western immunoblots, each of the serotype-specific MAbs interacted only with high-molecular-weight bands of the homologous LPS, with a minimum O-antigen chain length of at least 6 to 10 repeats. In contrast, cross-reactive MAb 18-19 was shown to interact in Western immunoblots with the entire LPS banding pattern except the fastest-running band, which lacks O antigen. Chemical modification of P. aeruginosa LPS by alkali treatment and carboxyl reduction abolished reactions between LPS and MAb 18-19, while reactions of modified LPS with serotype-specific MAbs were not affected. Therefore, cross-reactive MAb 18-19 likely recognizes the chemical backbone structure of the O repeat that is common to all three serotypes of the O2-O5-O16 group, while the O-specific MAbs appeared to recognize LPS epitopes that could be presented when 6 to 10 or more O-antigen repeat units are present on the LPS molecule. Thus, the O-specific LPS epitopes likely involve unique chemical structures, glycosidic linkages, and some order of folding of the O side chains.     

Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.

The lipopolysaccharide (LPS) molecule is an important virulence determinant in Klebsiella pneumoniae. Studies on the serotype O1 LPS were initiated to determine the basis for antigenic heterogeneity previously observed in the O1 side chain polysaccharides and to resolve apparent ambiguities in the reported polysaccharide structure. Detailed chemical analysis, involving methylation and 1H- and 13C-nuclear magnetic resonance studies, demonstrated that the O-side chain polysaccharides of serotype O1 LPS contained a mixture of two structurally distinct D-galactan polymers. The repeating unit structures of these two polymers were identified as [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] (D-galactan I) and [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] (D-Galactan II). D-Galactan I polysaccharides were heterogeneous in size and were detected throughout the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) profile of O1 LPS. In contrast, D-galactan II was confined to the higher-molecular-weight region. The structures of the two D-galactans were not influenced by simultaneous synthesis of a capsular K antigen. Apparently, neither of the D-galactans constitutes a common antigen widespread in Klebsiella spp. as determined by immunochemical analysis. Examination of the LPSs in mutants indicated that expression of D-galactan I can occur independently of D-galactan II. Transconjugants of Escherichia coli K-12 strains carrying the his region of K. pneumoniae were constructed by chromosome mobilization with RP4::mini-Mu. In these transconjugants, the O antigen encoded by the his-linked rfb locus was determined to be D-galactan I, suggesting that genes involved in the expression of D-galactan II are not closely linked to the rfb cluster.     

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.     

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus.

Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an O-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an O-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.     

Heterogeneity of Rhizobium lipopolysaccharides.

The lipopolysaccharides ( LPSs ) from strains of Rhizobium leguminosarum, Rhizobium trifolii, and Rhizobium phaseoli were isolated and partially characterized by mild acid hydrolysis and by polyacrylamide gel electrophoresis. Mild acid hydrolysis results in a precipitate which can be removed by centrifugation or extraction with chloroform. The supernatant contains polysaccharides which, in general, are separated into two fractions ( LPS1 and LPS2 ) by Sephadex G-50 gel filtration chromatography. The higher-molecular-weight LPS1 fractions among the various Rhizobium strains are highly variable in composition and reflect the variability reported in the intact LPSs (R. W. Carlson and R. Lee, Plant Physiol. 71:223-228, 1983; Carlson et al., Plant Physiol. 62:912-917, 1978; Zevenhuizen et al., Arch. Microbiol. 125:1-8, 1980). The LPS1 fraction of R. leguminosarum 128C53 has a higher molecular weight than all other LPS1 fractions examined. All LPS2 fractions examined are oligosaccharides with a molecular weight of ca. 600. The major sugar component of all LPS2 oligosaccharides is uronic acid. The LPS2 compositions are similar for strains of R. leguminosarum and R. trifolii, but the LPS2 from R. phaseoli was different in that it contained glucose, a sugar not found in the other LPS2 fractions or found only in trace amounts. Polyacrylamide gel electrophoretic analysis shows that each LPS contains two banding regions, a higher-molecular-weight heterogeneous region often containing many bands and a lower-molecular-weight band. The lower-molecular-weight bands of all LPSs have the same electrophoretic mobility, which is greater than that of lysozyme. The banding pattern of the heterogeneous regions varies among the different Rhizobium strains. In the case of R. leguminosarum 128C53 LPS, the heterogeneous region of a higher molecular weight than is this region from all other Rhizobium strains examined and consists of many bands separated from one another by a small and apparently constant molecular weight interval. When the heterogeneous region of R. Leguminosarum 128C53 LPS was cut from the gel and analyzed, its composition was found to be that of the intact LPS, whereas the lower-molecular-weight band contains only sugars found in the LPS2 oligosaccharide. In the case of R. leguminosarum 128C63 and R. trifolii 0403 LPSs, the heterogeneous regions are similar and consist of several band s separated by a large-molecular-weight interval with a the major band of these heterogeneous regions having the lowest molecular weight with an electrophoretic mobility near that of beta-lactoglobulin. The heterogeneous region from R. phaseoli 127K14 consists of several bands with electrophoretic mobilities near that of beta-lactoglobulin, whereas this region from R. trifolii 162S7 shows a continuous staining region, indicating a great deal of heterogeneity. The results described in this paper are discussed with regard to the reported properties of Escherichia coli and Salmonella LPSs.     

The Synthesis and Localization of Envelope Glycoproteins in Oocytes of Xenopus laevis using Immunocytochemical Methods

The site of origin and the mode of differentiation of the coelomic envelope (CE) in growing oocytes of Xenopus laevis were studied using the rabbit antiserum raised against the isolated envelope from oviposited eggs. The antiserum preabsorbed with egg extracts reacted with most components of CE glycoproteins detectable by SDS-PAGE, and stained specifically the CE of full-grown (st. VI) oocytes using indirect immunofluorescence methods. Transmission electron microscopy employing IgG-conjugated colloidal gold demonstrated that the CE antigens were distributed throughout the whole cytoplasm of st. I oocytes, and began to be deposited around the oocyte surface at late st. I. During st. II to VI the density of CE antigens in the oocyte cytoplasm decreased markedly, while the deposition of extracellular CE antigens increased gradually in association with the formation of a fibrillar network. The CE antigens were observed in and around the highly extended oocyte microvilli during st. II to IV, but were never found in follicle cells at any stages of oocyte growth. On western blot analyses, the extracellular CE components appeared first at st. II, and increased both in amount and number of bands during st. III - V to attain a typical electrophoretic profiles of well-developed CE. The cytosols of growing oocytes, however, possessed several antigenic components which were distinct from those of extracellular CE, suggesting the occurrence of intracellular precursor molecules for CE. The CEs of st. IV oocytes defolliculated and cultured in [³H] leucine contained certain CE components that expressed the radiolabel on fluorography. These results indicate that in Xenopus laevis the oocyte is directly involved in the synthesis and assembly, as well as secretion of CE with least contribution by the follicle cells.     

Comparative Serology of the Marine Fish Pathogen Vibrio anguillarum

The different serotyping systems, based on thermostable O antigens, reported for Vibrio anguillarum and V. ordalii were compared by quantitative agglutination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent silver staining or Western blotting (immunoblotting) of purified lipopolysaccharide (LPS), using polyclonal rabbit antisera. The results demonstrate that 16 different serotypes within V. anguillarum (designated O1 to O16) can be distinguished. Each of these serotypes is characterized by a distinct polysaccharide banding pattern, as revealed by silver-stained gels of purified LPS. The comparative analysis allowed a complete alignment of the different serotypes for the first three serovars: O1, O2, and O3. Moreover, immunoblotting showed that strains belonging to each of these serotypes had the same LPS banding pattern independent of the origin of the strain. Serotype O2 contains different subtypes, O2a and O2b. While no differences were apparent between these subgroups in silver-stained gels, they could be separated by quantitative agglutination (titer determination) or immunoblotting. V. ordalii, the former biotype II of V. anguillarum, strongly reacts with anti-V. anguillarum O2a antiserum. Strains of the two species can be separated on the basis of different LPS profiles in the high-molecular-weight region of silver-stained gels of purified LPS. The silver-stained LPS profiles of the different serotypes of V. anguillarum that have been established are provided for further comparison in the future.     

Comparative structural aspects of cation binding to phosphatidylserine bilayers

X-ray diffraction data recorded for monovalent and divalent cation complexes of a series of phosphatidylserines (PS) varying in chain length reveal a simple structural pattern. Only two bilayer structural types differing in hydrocarbon chain tilt but with similar polar group conformations are observed for (i) anhydrous acidic PS, (ii) anhydrous K+-PS, and (iii) Li+, Mg2+, Ca2+, Sr2+, Ba2+, and Pr3+ complexes of 'hydrated' PS. The X-ray diffraction data suggest that PS becomes dehydrated on complexing with Li+, Mg2+, Ca2+, and other divalent cations and adopts either the chain untilted (form I) or tilted (form II) bilayer structure.     

Chemical characterization of the lipopolysaccharides from enteropathogenic Escherichia coli O142 and O158.

Lipopolysaccharides (LPS) from two enteropathogenic strains of E. coli O142 and O158 were isolated by hot phenol-water extraction procedure. Polyacrylamide gel electrophoretic pattern of the LPS showed the typical ladder like pattern of smooth type of LPS. The LPS of E. coli O158 was found to contain L-rhamnose, D-glucose and N-acetyl-D-galactosamine as major constituents together with D-galactose, N-acetyl-D-glucosamine, L-glycero-D-manno-heptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) whereas LPS from E. coli O142 contained L-rhamnose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as major constituents together with D-glucose, D-galactose, N-acetyl-D-glucosamine, L-glycero-D-mannoheptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO). LPS was degraded by mild acid hydrolysis to yield a degraded polysaccharide fraction and an insoluble lipid-A fraction. The main fatty acids of the lipid-A fraction of the LPS were C12:O, C14:O, and 3-OH C14:O for O158 strain whereas E. coli O142 lipid-A consisted of C12:O, C14:O, 3-OH C14:O, and C16:O. The degraded polysaccharide fraction on gel permeation chromatography gave a high moleculer weight O-chain fraction and a core oligosaccharide and a fraction containing degraded sugars. The chemical composition of LPS and its fragmented products are reported in this communication.     

The type and yield of lipopolysaccharide from symbiotically deficient rhizobium lipopolysaccharide mutants vary depending on the extraction method

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.     

A novel Helicobacter pylori cell-surface polysaccharide

Helicobacter pylori bacteria colonize the gastric mucosa of more than half of the world's human population and its infection may instigate a wide spectrum of gastric diseases in the host. At the moment, there is no vaccine against H. pylori, a microorganism recognized as a category 1 human carcinogen, and treatment is limited to antibiotic management. Pioneering antigenic studies carried out by Penner and co-workers, which employed homologous H. pylori antisera specific for cell-surface lipopolysaccharide (LPS), revealed the presence of six distinct H. pylori serotypes (O1 to O6). Subsequent studies have shown that H. pylori serotype O1 expressed LPS with lengthy O-chain polysaccharide (PS) composed of Lewis blood-group structures ('Lewis O-chains'), serotype O3 LPS produced 'Lewis O-chains' attached to a heptoglycan domain, serotype O4 LPS possessed LPS with glucosylated 'Lewis O-chains' and serotype O6 LPS expressed the heptoglycan domain capped by a short 'Lewis O-chain'. These LPSs were terminated at the reducing-end by a core oligosaccharide and lipid A of conserved structures. With the intent of formulating a multivalent H. pylori LPS-based vaccine, we are studying the structural variability of H. pylori cell-surface glycans. Here, we describe the novel LPS structure produced by H. pylori serotype O2 that differed markedly from the typical H. pylori 'Lewis O-chain' structures, in that its main component was an elongated PS composed of alternating 2-, and 3-monosubstituted alpha-D-Glcp residues [-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]n. These findings revealed the bio-molecular basis for the observed serospecificity of H. pylori serotype O2, and that this unique bacterial PS must be included in the formulation of a multivalent LPS H. pylori vaccine.     

Structure and biological relationships of Coxiella burnetii lipopolysaccharides

Lipopolysaccharides (LPSs) extracted from nine strains of Coxiella burnetii were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and lethal toxicities in galactosamine-sensitized mice. The structure of a unique disaccharide of hydrolyzed phase I LPS was determined to be galactosaminuronyl-alpha (1-6)-glucosamine (GalNU-alpha (1-6)-GlcN, C12H22N2O10) with an Mr of 354. The Mr of LPSs of C. burnetii intra- and interspecific strains and the content of GalNU-alpha (1-6)-GlcN and two sugars, virenose and dihydrohydroxystreptose, were used as biochemical markers of truncated LPSs. Smooth-phase I LPS contained all three compounds, semi-rough-phase I LPS did not contain virenose, and rough-phase II LPS contained none of the three compounds. These analyses indicate that the intermediate to larger Mr LPSs require the addition of GalNU-alpha (1-6)-GlcN and dihydrohydroxystreptose to obtain the major (10.5 kDa), the intermediate (between 10.5 and 27 kDa), and the minor (23 kDa) LPS bands. The addition of virenose to the major and the minor bands produced the large Mr phase I LPSs. Extreme microheterogeneity in the banding profile ranging in Mr from the 2.5 to 10.5 kDa may be due to unidentified components, while the microheterogeneity in Mr of the 10.5-kDa and larger LPS bands is related to variations in the compounds described here. All of the LPSs were toxic in galactosamine-sensitized mice, albeit they were 100-1000-fold less toxic than Escherichia coli and Salmonella typhimurium endotoxin.     

Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4.

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS. Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography. The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc. Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M-H]- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: [equation: see text] Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.