Deoxycholate Polyacrylamide Gel Electrophoresis Research Articles

A glycomic approach reveals a new mycobacterial polysaccharide.

Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host-pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate-polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10-50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis.

Slr2019, lipid A transporter homolog, is essential for acidic tolerance in Synechocystis sp. PCC6803.

Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse environmental stress. In Synechocystis sp. PCC6803 (Synechocystis), lipopolysaccharide (LPS) may play a role in this protection. To examine the relationship between stress tolerance of Synechocystis and LPS, we focused on Slr2019 because Slr2019 is homologous to MsbA in Escherichia coli, which is related to LPS synthesis. First, to obtain a defective mutant of LPS, we constructed the slr2019 insertion mutant (slr2019) strain. Sodium deoxycholate-polyacrylamide gel electrophoresis indicated that slr2019 strain did not synthesize normal LPS. Second, to clarify the participation of LPS in acid tolerance, wild type (WT) and slr2019 strain were grown under acid stress; slr2019 strain growth was significantly weaker than WT growth. Third, to examine influences on stress tolerance, slr2019 strain was grown under various stresses. Under salinity and temperature stress, slr2019 strain grew significantly slower than WT. To confirm cell morphology, cell shape and envelope of slr2019 strain were observed by transmission electron microscopy; slr2019 cells contained more electron-transparent bodies than WT cells. Finally, to confirm whether electron-transparent bodies are poly-3-hydroxybutyrate (PHB), slr2019 strain was stained with Nile Blue A, a PHB detector, and observed by fluorescence microscopy. The PHB granule content ratio of WT and slr2019 strain grown at BG-11 pH 8.0 was each 7.18 and 8.41%. At pH 6.0, the PHB granule content ratio of WT and slr2019 strain was 2.99 and 2.60%. However, the PHB granule content ratio of WT and slr2019 strain grown at BG-11N-reduced was 10.82 and 0.56%. Because slr2019 strain significantly decreased PHB under BG-11N-reduced compared with WT, LPS synthesis may be related to PHB under particular conditions. These results indicated that Slr2019 is necessary for Synechocystis survival in various stresses.

S1640 Can Helicobacter pylori Lipopolysaccharide Lipid a Composition Affect Its Ability to Induce an Inflammatory Response Through Toll Like Receptor 4

Introduction H pylori recognition by the host is poorly understood, although there is evidence that lipid A recognition occurs via Toll-like receptor 4. H pylori produces two lipid A species, a minor E coli -like hexa-acylated species and a major tetra-acylated species. The latter is thought to be important for immune evasion, with many laboratory strains thought to lack the minor more immunogenic species which is present in clinical strains. Aim To assess whether changes in lipid A alter H pylori9s ability to induce an inflammatory response. Methods H pylori strains were repeatedly subcultured on blood agar and structural changes in the lipopolysaccharide were analysed using Deoxycholate Polyacrylamide Gel Electrophoresis (DOC-PAGE) and Gas Chromatography Mass Spectrometry (GC-MS). Additional ultracentrifugation removed contaminating lipoproteins and capsular polysaccharides. A monocyte/LPS challenge system was used to assess pro-inflammatory cytokine production by the different H pylori LPS preparations. Prediction of the orientations adopted by H pylori LPS structures within TLR4 receptor complex interactions were generated using the automated docking program AutoDock v4. Results H pylori LPS was not altered by subculturing, although purified laboratory and clinical strains were shown to have compositional differences. Fatty acids of carbon length 14 and 16 (indicative of hexa-acylated lipid A) were absent from laboratory strain H pylori lipid A following purification, but remained in the lipid A purified from clinical strains. Unpurified laboratory strain LPS induced greatest levels of cytokine production in monocyte/LPS challenge experiments followed by purified clinical strain LPS, with purified laboratory strain LPS inducing the lowest levels (Abstract 053). Modelling analysis confirmed that the tetra-acylated species was less effective than the hexa-acylated species at interacting with the TLR4 signalling complex. Conclusion Purification of H pylori LPS had a dramatic effect on inflammatory cytokine production indicating that contaminating lipoprotein and or capsular polysaccharide can impact the immune response. However, LPS derived from clinical isolates is significantly more immunogenic than laboratory strains. This further strengthens the evidence that TLR4-mediated immune responses are important in H pylori infections.

Isolation of lipids from photosystem I complex and its characterization with high performance liquid chromatography/electrospray ionization mass spectrometry.

A method for simultaneous analysis of lipids extracted from photosystem I complex was developed with high performance liquid chromatography/electrospray ionization mass spectrometry. The photosystem I complex was firstly solubilized and separated using deoxycholate polyacrylamide gel electrophoresis method after ultrasonic treatment of the sample (leaves of pea, Pisum sativum L.). The Photosystem I complexes were electrophoretically eluted from the deoxycholate polyacrylamide gel electrophoresis bands containing them, and the electron transport activity of the eluent measured as confirmation. Lipids, which were isolated from the complex having photosystem I activity, were separated and characterized with high performance liquid chromatography/electrospray ionization mass spectrometry. Five lipids, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylglycerol, sulphoquinovosyldiacylglycerol and phosphaditylcholine were found combining with photosystem I complex. Different species of these lipids were found in the ESI mass spectra and the compositions of the acyl groups in them were determined.

Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7.

The aromatic hydrocarbon-degrading bacterium, Pseudomonas putida G7, produces exopolymers of potential interest in biotechnological applications. These exopolymers have been shown to have significant metal-binding ability. To initiate the study of the metal-polymer interactions, we explored the physical and chemical nature of the P. putida G7 exopolysaccharide, a major component of the exopolymer. A capsular structure was observed by light microscopy surrounding both planktonic and attached cells in biofilms after immunofluorescence staining with polyclonal antiserum raised against planktonic cells. Further work with planktonic cells showed that the immunostained capsule remained associated with young (log phase) cells, whereas older (stationary phase) cells lost their capsular material to the external milieu. Visualization of frozen, hydrated stationary phase cells by cryo-field emission scanning electron microscopy (cryoFESEM) revealed highly preserved extracellular material. In contrast, conventional scanning electron microscopy (SEM) of stationary phase cells showed rope-like material that most probably results from dehydrated and collapsed exopolymer. Both capsular and released exopolymers were separated from cells, and the released extracellular polysaccharide (EPS) was purified. Deoxycholate-polyacrylamide gel electrophoresis (PAGE) and silver/alcian blue staining of the partially purified material showed that it contained both EPS and lipopolysaccharide (LPS). Further purification of the EPS using a differential solubilization technique to remove LPS yielded highly purified EPS. Gas chromatography-mass spectrometry revealed that the purified EPS contained the monosaccharides, glucose, rhamnose, ribose, N-acetylgalactosamine and glucuronic acid. The structural and chemical properties of the P. putida EPS described here increase our understanding of the mechanisms of toxic metal binding by this well-known Proteobacterium.

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 component different from endotoxin extracted from Prevotella intermedia ATCC 25611 activates lymphoid cells from C3H/HeJ mice and gingival fibroblasts from humans.

A novel immunobiologically active fraction was prepared from a phenol-water extract of Prevotella intermedia ATCC 25611 by Sephadex G-100 column chromatography. The fraction consisted mainly of carbohydrate and protein and was devoid of fatty acid. The fraction showed high-molecular-weight bands (10,000 to 12,000) on deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) and was scarcely active in a Limulus test. We designated the fraction Prevotella glycoprotein (PGP). The PGP fraction showed strong mitogenicity on splenocytes and cytokine-inducing activities on peritoneal macrophages from both C3H/HeJ and C3H/HeN mice, and it stimulated human gingival fibroblasts to produce cytokines. The activities of the PGP fraction were resistant to heat inactivation (100 degrees C for 1 h) and protease treatments and were scarcely inhibited by polymyxin B. In contrast, the purified lipopolysaccharide fraction (LPS-PCP) extracted from the same bacterium with a phenol-chloroform-petroleum ether mixture, which showed strong Limulus activity and a single low-molecular-weight band (approximately 3,000) on DOC-PAGE, lacked the activities on splenocytes and macrophages from C3H/HeJ mice and human gingival fibroblasts. The activities of the LPS-PCP fraction on cells from C3H/HeN mice were completely inhibited by polymyxin B. The LPS extracted from the same bacterium with hot phenol-water (LPS-PW) exhibited the properties of both the PGP fraction and the LPS-PCP fraction. These findings suggest that the unique bioactivities of the LPS-PW fraction of oral black-pigmented bacteria reported to date, which differed from those of the classical endotoxin, were derived from the PGP fraction and not from the LPS itself.

Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme)

The lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype 06 rough-type mutant A28 was isolated by a modified phenol-chloroform-petroleum ether extraction method. Deoxycholate-polyacrylamide gel electrophoresis indicated a single band with mobility similar to that of the complete core region of the wild-type parent serotype 06 (International Antigenic Typing Scheme) strain. Compositional analysis of the LPS indicated that the core oligosaccharide was composed of D-glucose (three units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (two units), 3-deoxy-D-manno-octulosonic acid (two units), L-alanine (one unit), and phosphate (two units). Under the mild conditions of hydrolysis with methanolic hydrogen chloride, a 7-O-carbamoyl substituent was observed on the second heptose residue. The glycan structure of the LPS was determined by employing one- and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry-based methods with a backbone oligosaccharide that was obtained from the LPS by deacylation, dephosphorylation, and reduction of the terminal glucosamine. On the basis of the results of the present study and our earlier work with the P. aeruginosa 06-derived core-defective mutant R5 (H. Masoud, E. Altman, J. C. Richards, and J. S. Lam, Biochemistry, 33:10568-10578, 1994), a structural model for the complete core oligosaccharide is proposed.

Human Mast Cell Chymase and Leukocyte Elastase Release Latent Transforming Growth Factor-β1 from the Extracellular Matrix of Cultured Human Epithelial and Endothelial Cells

Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-beta 1 (TGF-beta 1) to extracellular matrices and its release and activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-beta 1, its propeptide (beta 1-latency-associated protein), and latent TGF-beta-binding protein and incorporated latent TGF-beta 1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-beta 1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-beta 1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized M(r) 25,000 TGF-beta 1 but did not dissociate high M(r) latent TGF-beta 1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-beta 1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-beta from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4′ by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3′ were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.

The lipopolysaccharides of Rhodospirillum rubrum, Rhodospirillum molischianum, and Rhodopila globiformis

The cell wall lipopolysaccharides from three phototrophic species of the alpha1-group of Proteobacteria, Rhodospirillum rubrum, Rhodospirillum molischianum, and Rhodopila globiformis were isolated and chemically characterized. Sodium deoxycholate polyacrylamide gel electrophoresis patterns revealed that the lipopolysaccharides of all three species possess O-chains. They are composed of repeating units only in R. molischianum and R. globiformis. The presence of l-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate indicated core structures in all three lipopolysaccharides. Glucosamine was found as backbone amino sugar in lipid A of R. molischianum and R. rubrum, while R. globiformis has 2,3-diaminoglucose as backbone amino sugar. The latter species also differed from the two former ones in its content of hydroxy fatty acids (3-OH-14:0, 3-OH-16:0 in R. rubrum and R. molischianum and 3-OH-14:0, 3-OH-18:0 and 3-OH-19:0 (possibly iso- or anteisobranched) in R. globiformis).

Analysis of salmonella lipopolysaccharides by sodium deoxycholate—polyacrylamide gel electrophoresis

Lipopolysaccharides (LPSs) were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium deoxycholate (DOC) or sodium dodecyl sulphate (SDS) and the results obtained compared. Two-dimensional PAGE of the S form of LPS revealed that, with SDS, bands which appeared to be single after the one-dimensional experiment were resolved into several bands after two-dimensional electrophoresis. In the presence of DOC, however, a diagonal of single bands was obtained in one-dimensional electrophoresis indicating optimum resolution. The high quality of resolution by DOC-PAGE was constant for amounts of LPS up to 20 micrograms. Finally, DOC-PAGE does not require boiling of the samples in DOC, which may be an advantage over SDS-PAGE.

Sodium deoxycholate-polyacrylamide gel electrophoresis of lipopolysaccharides at low temperature.

Sodium deoxycholate (DOC)-polyacrylamide gel electrophoresis (PAGE) has previously been used to examine in detail the chemical heterogeneity of lipopolysaccharide (LPS) : It is now shown that DOC-PAGE carried out at 4°C can give reproducible banding patterns even when a large amount of LPS (20mg) is applied to the slab gel (4×150×300mm). By DOC-PAGE, we found that LPS obtained from E. coli UKT-B showed 14 silver-staining bands on gels but lacked the bands corresponding to a series of high-molecular-weight LPS usually observed in the S from LPS.These findings suggest that the DOC-PAGE at 4°C should be useful in fractionating LPS intactly to study LPS preparations with unknown structures.

Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3-diamino-2,3-dideoxyglucose

Lipopolysaccharides were isolated from two strains of Thiobacillus ferrooxidans and one strain each of Thiobacillus thiooxidans, Thiobacillus novellus and Thiobacillus sp. IFO 14570. Neutral sugars, 2-keto-3-deoxyoctonate, fatty acids and the rare 2,3-diamino-2,3-dideoxyglucose were detected in all lipopolysaccharides. Lipopolysaccharides of both T. ferrooxidans strains contained l-glycero-d-manno-heptose, whereas that of T. thiooxidans contained both l-glycero-d-manno-heptose and d-glycero-d-manno-heptose. On the other hand, heptoses were absent in lipopolysaccharides of T. novellus and Thiobacillus sp. IFO 14570. Lipid A of T. ferrooxidans and T. thiooxidans contained both glucosamine and 2,3-diamino-2,3-dideoxyglucose, in contrast, lipid A of T. novellus and the Thiobacillus sp. IFO 14570 most likely contain only 2,3-diamino-2,3-dideoxyglucose as backbone sugar. Deoxycholate polyacrylamide gel electrophoresis revealed S-type character for all lipopolysaccharides studied. The significance of the lipopolysaccharide composition for taxonomic and phylogenetic questions with regard to thiobacilli is discussed.

Lipopolysaccharides of chemolithotrophic bacteriaThiobacillus versutusand a relatedThiobacillusspecies

The lipopolysaccharides (LPSs) of two strains of Thiobacillus versutus and a Thiobacillus sp. strain were isolated and chemically analyzed. They contained neutral sugars, glucosamine, 2-keto-3-deoxyoctonate (KDO), and phosphorus, but were devoid of heptose. The fatty acids were characterized as amide-linked 3-OH-14:0 and 3-oxo-14:0, and ester-linked 3-OH-10:0. The lipid A backbones contain glucosamine as the only amino sugar. Deoxycholate-polyacrylamide gel electrophoresis (DOC-PAGE) showed that the LPSs have R-type character. The presence of amide-linked 3-OH-14:0 and 3-oxo-14:0 and of ester-linked 3-OH-10:0, the lack of heptose, and the R-type character of LPSs, indicate a structure of LPSs of T. versutus and Thiobacillus sp. strains similar to those found for the phylogenetically related Rhodobacter species and Paracoccus denitrificans.

Lipopolysaccharides of chemolithotrophic bacteria Thiobacillus versutus and a related Thiobacillus species

The lipopolysaccharides (LPSs) of two strains of Thiobacillus versutus and a Thiobacillus sp. strain were isolated and chemically analyzed. They contained neutral sugars, glucosamine, 2-keto-3-deoxyoctonate (KDO), and phosphorus, but were devoid of heptose. The fatty acids were characterized as amide-linked 3-OH-14:0 and 3-oxo-14:0, and ester-linked 3-OH-10:0. The lipid A backbones contain glucosamine as the only amino sugar. Deoxycholate-polyacrylamide gel electrophoresis (DOC-PAGE) showed that the LPSs have R-type character. The presence of amide-linked 3-OH-14:0 and 3-oxo-14:0 and of ester-linked 3-OH-10:0, the lack of heptose, and the R-type character of LPSs, indicate a structure of LPSs of T. versutus and Thiobacillus sp. strains similar to those found for the phylogenetically related Rhodobacter species and Paracoccus denitrificans.

Taenia taeniaeformis in mice: Passive transfer of protection with sera from infected or vaccinated mice and analysis of serum antibodies to oncospheral antigens

Passive transfer of immunity to Taenia taeniaeformis infection was investigated using sera either from mice protected against infection by vaccination with oncosphere antigen or from mice 28 days after infection (28 DPI) with eggs. Transfer of 0.2 ml serum from mice vaccinated with either freeze-thaw-sonicated oncospheres (TtO FTS) or solubilized oncospheres (TtO DOC) failed to provide protection against infection with larvae in the recipient mice despite the fact that the serum donors were completely protected against the development of any viable larvae from a challenge infection with eggs. Some transfer of protection was achieved using larger volumes of sera (0.25, 0.5 ml) from vaccinated mice and by using sera collected from vaccinated mice 7 days after a challenge infection with eggs. The levels of protection provided with these sera were however always inferior to that achieved by passive transfer with sera collected from mice 28 DPI. Anti-oncospheral antibody levels in the sera used for passive transfer experiments were analysed using an Enzyme Linked Immunosorbent Asay (ELISA) and by Western blot immunoassay of oncosphere antigens separated in deoxycholate polyacrylamide gel electrophoresis (DOC PAGE). Differences in the levels of passive protection transferred with sera from 28 DPI mice and vaccinated mice did not correlate with anti-oncospheral antibody responses detected in ELISA. A correlation was evident between passive protection and detection of antibodies against an oncospheral antigen having a relative mobility of 0.62 in 10% acrylamide DOC PAGE. Gel cut-outs containing this antigen did not vaccinate mice against infection and loss of important conformational determinants of this antigen is suggested as a possible explanation for this. A simple association was not evident between vaccination-induced protection against infection and the presence of serum antibodies capable of passively transferring protection. The relevance of these observations for the rational selection of antibody probes for in vitro analysis of vaccine antigens is discussed.