Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild-type strain PAO1 and derived rough-type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum-ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of D-glucose (four units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (Hep; two units), 3-deoxy-D-manno-octulosonic acid (Kdo; two units), L-alanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one-dimensional and two-dimensional (2D) NMR techniques in combination with MS-based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two-dimensional 31P (omega1)-half-filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild-type strain. The presence of a 7-O-carbamoyl substituent was observed on the second heptose. The structure of the core region of the O-chain-deficient LPS from P. aeruginosa serotype 05 is as follows: [structure: see text] where R1 is beta-D-Glcp-(1-->2)-alpha-L-Rhap-(1-->6)-alpha-D-Glcp-(1--> and R2 is alpha-D-Glcp-(1-->6)-beta-D-Glcp-(1->. A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.     

Structure of the cell wall polysaccharide in the food protein yeast Candida spec H. III. Characterization of different phosphate bonds in the mannan-protein-phosphate complex

The 31P-NMR spectra of the proteophosphomannan (PPM) and also that of mildly hydrolyzed PPM demonstrated phosphomonoester (in both preparations), acid labile and acid stable phosphodiester linkage, and polyphosphate. Decreasing in size by pronase digestion, separation, purification and characterization of the high and low molecular phosphates by 31P-NMR spectroscopy and chemical analysis revealed the mannan protein is phosphorylated in the N-glycosidically linked carbohydrate parts and in the O-glycosidically linked oligosaccharides. Another phosphate serves as a bridge between the serine of the protein and mannose, mannobioses and mannotrioses and between the threonine and a lipophilic acylglycerid unit. The origin of the polyphosphates has been discussed.     

Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal

A Vibrio cholerae O139 strain, MO10-T4, lacking capsular polysaccharide, produces a short-chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high-performance anion-exchange chromatography, one- and two-dimensional 1H-, 13C-, and 31P-NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS: [structure: see text] where PEtn stands for 2-aminoethyl phosphate, Fru for fructose, Hep for L-glycero-D-manno-heptose, and Kdo for 3-deoxy-D-manno-octulosonic acid; unless otherwise stated, the monosaccharide residues are D and present in the pyranose form. An O-acetyl group is present on a secondary position, tentatively O4 of the alpha-linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown. An O139-negative mutant strain, Bengal-2R, derived from V. cholerae O139, has also been investigated and shown to produce an O-antigen-lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10-T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2-aminoethyl phosphate, the O-acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10-T4 LPS, a short O-side chain is attached at position 3 of the 7-substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.-R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43-58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59-65.]     

Characterization of lipopolysaccharide-deficient mutants of Pseudomonas aeruginosa derived from serotypes O3, O5, and O6

Well-characterized rough mutants are important for the understanding of structures, functions, and biosynthesis of lipopolysaccharide (LPS) in gram-negative organisms. In this study, three series of Pseudomonas aeruginosa LPS-deficient mutants, namely PAC strains derived from serotype O3, AK strains derived from strain PAO1 (serotype O5), and serotype O6-derived mutants were subjected to biochemical analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining as well as immunochemical characterization using LPS-specific monoclonal antibodies. The O-side-chain deficiency among the O6-derived mutants was also examined, and three mutants, A28, R5, and H4, were subsequently chosen for the elucidation of component sugars of the core structure of serotype O6 LPS. LPS of strain A28 has L-rhamnose and proportionally higher amounts of D-glucose, a feature shared by the O5-derived mutant, strain AK1401 (previously demonstrated as a mutant with a core-plus-one O repeat). In contrast strains R5 and H4 were shown to be devoid of L-rhamnose and have low and undetectable amounts of D-glucose, respectively, which indicated their core deficiency. The LPS-deficient or -sufficient characteristics of the P. aeruginosa strains examined correlated will with serum sensitivity data. This report represents a comprehensive analysis of rough mutants derived from O3 and O5 strains that have been used by others in many studies and a first look at the core oligosaccharide region of serotype O6 LPS obtained with the O6-derived mutants generated in this study.     

PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance

Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity. Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial peptides through the two-component systems PhoP-PhoQ and PmrA-PmrB. Polymyxin resistance is encoded by the PmrA-PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4' phosphate of lipid A. Two PmrA-PmrB-regulated S. typhimurium loci (pmrE and pmrF) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A. One locus, pmrE, contains a single gene previously identified as pagA (or ugd) that is predicted to encode a UDP-glucose dehydrogenase. The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes. Genes immediately flanking this putative operon are also regulated by PmrA-PmrB and/or have been associated with S. typhimurium polymyxin resistance. This work represents the first identification of non-regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.     

Immunosuppressive therapy with microemulsion cyclosporine A shortens the hospitalization of pediatric liver transplant recipients

Lipopolysaccharide (LPS) from S. typhimurium on exposure to gamma-radiation resulted in decrease in toxicity and was less mitogenic, Silver stained profiles of irradiated LPS on polyacrylamide gels revealed complete loss of its heteropolysaccharides which was confirmed further by analysing lipid A and LPS from Salmonella minnesota Re mutants on SDS-PAGE. Glucosamine and 2-keto 3-deoxy-octonate(Kdo) contents were significantly decreased on treatment. Lipid A obtained by removal of heteropolysaccharides from LPS was less toxic on exposure to gamma radiations.     

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.     

Effect of acetylsalicylic acid on metabolism and contractility in the ischemic reperfused heart

The effect of acetylsalicylic acid (ASA) on high-energy phosphates (adenosine triphosphate: ATP, creatine phosphate: CrP, inorganic phosphate: Pi) and intracellular pH during myocardial ischemia and reperfusion was studied using phosphorus 31-nuclear magnetic resonance (31P-NMR) in the isolated rabbit hearts. Coronary flow, left ventricular systolic developed pressure (LV Dev.P) and left ventricular end-diastolic pressure (LVEDP) were also measured. Langendorff hearts perfused at 37 degrees C with the perfluorochemical emulsion Fluosol-43 were subjected to 15 min and 30 min of zero-flow ischemia and to 15 min of low-flow ischemia (coronary perfusion pressure = 20 mmHg) followed by 65 min of reperfusion (control, Group I). ASA (0.28 mmol/L) was infused either for the entire experimental period from beginning 45 min prior to ischemia (Group II) and infused immediately after reperfusion (Group III). During ischemia, Group II showed a significant suppression of the decrease in the ATP level and pH with both zero-flow and low-flow ischemia compared to those in the other groups, and moreover the increase in Pi and the decrease in CrP in low-flow ischemia were also suppressed. In Group III, the ATP level during reperfusion was significantly higher than that in Group I, but was not significantly different from that in 30 min zero-flow ischemia. In 30 min zero-flow ischemia, Pi, CrP and coronary flow after reperfusion in Group II tended to recover to preischemic values. There were no differences in LV Dev.P among the 3 groups. In conclusion, ASA has a protective effect on myocardial high-energy phosphates during ischemia and reperfusion in rabbit hearts.     

Isolation and characterization of two genes, waaC (rfaC) and waaF (rfaF), involved in Pseudomonas aeruginosa serotype O5 inner-core biosynthesis

Recent studies have provided evidence to implicate involvement of the core oligosaccharide region of Pseudomonas aeruginosa lipopolysaccharide (LPS) in adherence to host tissues. To better understand the role played by LPS in the virulence of this organism, the aim of the present study was to clone and characterize genes involved in core biosynthesis. The inner-core regions of P. aeruginosa and Salmonella enterica serovar Typhimurium are structurally very similar; both contain two main chain residues of heptose linked to lipid A-Kdo2 (Kdo is 3-deoxy-D-manno-octulosonic acid). By electrotransforming a P. aeruginosa PAO1 library into Salmonella waaC and waaF (formerly known as rfaC and rfaF, respectively) mutants, we were able to isolate the homologous heptosyltransferase I and II genes of P. aeruginosa. Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. Similarly, plasmid pCOREf1 was able to complement the Salmonella waaF mutant. Sequence analysis of the DNA insert of pCOREc2 revealed one open reading frame (ORF) which could code for a protein of 39.8 kDa. The amino acid sequence of the deduced protein exhibited 53% identity with the sequence of the WaaC protein of S. enterica serovar Typhimurium. pCOREf1 contained one ORF capable of encoding a 38.4-kDa protein. The sequence of the predicted protein was 49% identical to the sequence of the Salmonella WaaF protein. Protein expression by the Maxicell system confirmed that a 40-kDa protein was encoded by pCOREc2 and a 38-kDa protein was encoded by pCOREf1. Pulsed-field gel electrophoresis was used to determine the map locations of the cloned waaC and waaF genes, which were found to lie between 0.9 and 6.6 min on the PAO1 chromosome. Using a gene-replacement strategy, we attempted to generate P. aeruginosa waaC and waaF null mutants. Despite multiple attempts to isolate true knockout mutants, all transconjugants were identified as merodiploids.     

Structural study of a highly O-acetylated core of Legionella pneumophila serogroup 1 lipopolysaccharide

A core oligosaccharide was obtained after mild acid degradation of Legionella pneumophila serogroup 1 lipopolysaccharide (LPS). On the basis of chemical, GLC-MS, 1H, and 13C NMR spectroscopic data, it was found that the oligosaccharide obtained is a highly O-acetylated heptasaccharide having the following structure: [formula: see text] where Kdo is 3-deoxy-D-manno-octulosonic acid and QuiNAc is 2-acetamido-2,6-dideoxyglucose. In the LPS, the O-specific polysaccharide chain is linked to position 3 of the terminal rhamnosyl group and is cleaved during degradation of the LPS. The degradation also induced partial migration and partial removal of the O-acetyl group from the terminal rhamnosyl group which, together with the occurrence of the reducing Kdo residue in multiple forms, contributes to the heterogeneity of the isolated core oligosaccharide. No such highly O-acetylated core oligosaccharide has been reported so far for LPS of Gram-negative bacteria.     

The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.     

High-performance liquid chromatography of proteins

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.     

Definition of conditions for the detection of genotoxic chemicals in the adult rat-liver epithelial cell/hypoxanthine-guanine phosphoribosyl transferase (ARL/HGRPT) mutagenesis assay

1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configuration. 2. A bilayer configuration of the (lyso)phospholipids was also observed after treatment of erythrocyte ghosts with various phospholipases even in the case that 98% of the phospholipid was converted into lysophospholipid (72%) and ceramides (26%). 3. A slightly decreased order of the phosphate group of phospholipid molecules, seen as reduced effective chemical shift anisotropy in the 31P-NMR spectra, was found following the formation of diacyglycerols and ceramides in the membrane of intact erythrocytes. Treatment of ghosts always resulted in an extensive decrease in the order of the phosphate groups. 4. The results allow the following conclusions to made: a. Hydrolysis of phospholipids in intact red cells and ghosts does not result in the formation of non-bilayer configuration of residual phospholipids and lysophospholipids. b. Haemolysis, which is obtained by subsequent treatment of intact cells with sphingomyelinase and phospholipase A2, or with phospholipase C, cannot be ascribed to the formation of non-bilayer configuration of phosphate-containing lipids. c. Preservation of bilayer structure, even after hydrolysis of all phospholipid, shows that other membrane constitutents, e.g. cholesterol and/or membrane proteins play an important role in stabilizing the structure of the erythrocyte membrane. d. A major prerequisite for the application of phospholipases in lipid localization studies, the preservation of a bilayer configuration during phospholipid hydrolysis, is met for the erythrocyte membrane.     

Characterization of a novel branched tetrasaccharide of 3-deoxy-D-manno-oct-2-ulopyranosonic acid. The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter baumannii strain nctc 10303 (atcc 17904)

For the first time, the tetrasaccharide Kdoalpha2-->5Kdoalpha2-->5(Kdoalpha2-->4)Kdo (Kdo is 3-deoxy-D-manno-oct-2-ulopyranosonic acid) has been identified in a bacterial lipopolysaccharide (LPS), i.e. in the core region of LPS from Acinetobacter baumannii NCTC 10303. The LPS was analyzed using compositional analysis, mass spectrometry, and NMR spectroscopy. The disaccharide D-GlcpNbeta1-->6D-GlcpN, phosphorylated at O-1 and O-4', was identified as the carbohydrate backbone of the lipid A. The Kdo tetrasaccharide is attached to O-6' of this disaccharide and is further substituted by short L-rhamnoglycans of varying length and by the disaccharide D-GlcpNAcalpha1-->4D-GlcpNA (GlcpNA, 2-amino-2-deoxy-glucopyranosuronic acid). The core region is not substituted by phosphate residues and represents a novel core type of bacterial LPS. The complete carbohydrate backbone of the LPS is shown in Structure I as follows: where Rha is rhamnose. Except were indicated, monosaccharides possess the D-configuration. Sugars marked with an asterisk are present in non-stoichiometric amounts.     

Secretory immunoglobulins in colonic neoplasms

1. The phosphonium analogues of choline, phosphorylcholine, CDPcholine and phosphatidylcholine were synthesized chemically and characterized by 1H-NMR and 31P-NMR; in 1,2-distearoyl-DL-glycero-3-phosphorylphosphocholine, the 31P-NMR chemical shift of phosphonium relative to phosphate was--28.2 ppm. 2. A comparison was made of the rates of reaction of choline kinase, cholinephosphate cytidyltransferase, cholinephosphotransferase and phospholipase C on natural and phosphonium substrates. Enzyme reaction rates were similar for all but the cytidyltransferase, which exhibited a 3-fold preference for the normal substrate. 3. Weanling rats were maintained for 6 weeks on a diet in which choline was fully replaced by phospho[1,2-14C2]choline mixed with a trace of [Me-3H] choline. Incorporation of phosphocholine into liver lipids was detectable by 31P-NMR even in crude tissue homogenates. Choline-based phospholipids of liver, kidney, lung and brain were extracted, and phosphocholine incorporation calculated from 31P-NMR peak area ratios. The phosphatidylcholine analogues were separated by preparative thin-layer chromatography. Incorporation of phosphocholine ranged from 33% in lung phosphatidylcholine to 6% in kidney sphingomyelin. Variations in 14C/3H ratio between feed and phospholipid extracts indicated preferences for exogenous choline over phosphocholine varying from 1.3: 1 in brain to 3.2: 1 in liver. The results indicated that phosphocholine is a potentially useful 31P-NMR probe for the study of membrane lipids.     

NMR studies of the structure and stability of the 1 : 2 actinomycin D-d-pG-C complex in aqueous solution

Bacteriophage-resistant mutants isolated and classified in a previous study were examined for alterations in their lipopolysaccharide (LPS) composition, and properties likely to be affected by alterations in LPS composition were studied. It was found that many of the mutants of the Ktw (K2-resistance), Ttk (T2, T4, or K19 resistance), Bar (bacteriophage), Wrm (wide-range mutants), and miscellaneous resistance groups were altered in their response to a series of antibiotics and to two LPS-specific bacteriophages, C21 and U3. Furthermore, many of the bacteriophages to which these mutants were resistant adsorbed to LPS preparations. By direct sugar analysis of the mutant LPS preparations, it was shown that the mutants fitted into six distinct classes, which are readily derived from LPS core with a structure resembling that of Salmonella or Escherichia coli O100. A number of the mutants were shown to map between pyrE and mtl, which has been previously shown to be the site of a cluster of rfa genes in both Salmonella and E. coli. Outer membrane protein composition was studied in the above mutants using polyacrylamide gel electrophoresis. Some strains were shown to have alterations in the amount of major proteins. The nature of the bacteriophage receptors involved and the alterations leading to resistance are discussed.     

Non-lamellar structure and negative charges of lipopolysaccharides required for efficient folding of outer membrane protein PhoE of Escherichia coli

Lipopolysaccharides (LPS) are amphiphilic molecules in the outer leaflet of the bacterial outer membrane. Recently, an early role for LPS in the folding of outer membrane porin PhoE was demonstrated in vitro. In order to elucidate the molecular mechanism of LPS-protein interactions, folding of PhoE protein was studied with a large set of well characterized LPS chemotypes. We demonstrate that negative charges in the inner core region contribute to the high efficiency of folding of PhoE protein. In addition, the supramolecular structure of the LPS aggregate seems to be important. LPS with a lipid A part that prefers a lamellar or a direct micellar structure and a high state of order of its acyl chains is much less efficient to support folding as compared with LPS with lipid A that prefers a non-lamellar structure and a low acyl chain order. These in vitro data indicate that extensive interactions between the core and lipid A region of LPS with the protein are required to support protein folding. The LPS-PhoE binding might be promoted by the presence of hydroxy fatty acids in the lipid A moiety of LPS.     

Dual inhibitory effect of gangliosides on phospholipase C-promoted fusion of lipidic vesicles

The effect of a variety of gangliosides has been tested on the phospholipase C-induced fusion of large unilamellar vesicles. Bilayer composition was phosphatidylcholine:phosphatidylethanolamine: cholesterol (2:1:1 mole ratio) plus the appropriate amounts of glycosphingolipids. Enzyme phosphohydrolase activity, vesicle aggregation, mixing of bilayer lipids and mixing of liposomal aqueous contents were separately assayed. Small amounts ( < 1 mol %) of gangliosides in the lipid bilayer produce a significant inhibition of the above processes. The inhibitory effect of gangliosides increases with the size of the oligosaccharide chain in the polar head group. Inhibition depends in a nonlinear manner on the ganglioside proportion, and is complete at approximately 5 mol %. Inhibition is not due to ganglioside-dependent changes in vesicle curvature or size. Ganglioside inhibition of vesicle fusion is due to two different effects: inhibition of phospholipase C activity and stabilization of the lipid lamellar phase. Enzyme inhibition leads to a parallel decrease of vesicle aggregation and lipid mixing rates. Mixing of aqueous contents, though, is depressed beyond the enzyme inhibition levels. This is explained in terms of the fusion pore requiring a local destabilization of the lipid bilayer, the lamellar structure being stabilized by gangliosides. 31P-NMR and DSC experiments confirm the inhibitory effect of gangliosides in various lamellar-to-nonlamellar transitions.     

Evidence for a new type of outer membrane lipid in oral spirochete Treponema denticola. Functioning permeation barrier without lipopolysaccharides

A new class of outer membrane lipid (OML) was isolated from the oral spirochete Treponema denticola strain ATCC 33521 using a phenol/chloroform/light petroleum procedure normally applied for lipopolysaccharide extraction. In addition to chemical analysis, Fourier transform infrared (FTIR) spectroscopy was applied to compare the biophysical properties of OML with lipopolysaccharides (LPS) and lipoteichoic acids (LTA). Isolated OML fractions represent 1.4% of the total dry cell weight, are about 4 kDa in size, and contain 6% amino sugars, 8% neutral sugars, 14% phosphate, 35% carbazol-positive compounds, and 11% fatty acids (containing iso- and anteiso-fatty acyl chains). Rare for outer membrane lipids, OML contains no significant amount of 3-deoxy-D-manno-octulosonic acids, heptoses, and beta-hydroxy fatty acids. The fatty acyl chain composition, being similar to that of the cytoplasmic membrane, is quite heterogeneous with anteiso-pentadecanoic acid (12%), palmitic acid (51%), and iso-palmitic acid (19%) as the predominant fatty acids present. Findings of a glycerol-hexose unit and two glycerol-hexadecanoic acid fragments indicate a glycolipid membrane anchor typically found in LTA. There was also no evidence for the presence of a sphingosine-based lipid structure. The results of FTIR measurements strongly suggest that the reconstituted lipid forms normal bilayer structures (vesicles) expressing a high membrane state of order with a distinct phase transition as typical for isolated LPS. However, in contrast to LPS, OML of T. denticola has a lower Tm near 22 degreesC and a lower cooperativity of the phase transition. The results suggest a different kind of permeation barrier that is built up by this particular OML of T. denticola, which is quite different from LPS normally essential for Gram-negative bacteria.