Structural characterization of monophosphoryl lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide

Sixteen monophosphoryl Lipid A (MLA) homologs obtained from the lipopolysaccharides of Salmonella minnesota Re595 were separated by preparative thin layer chromatography into eight fractions. The components of these fractions were analyzed directly (or as structural analogs) and characterized by mass spectrometry. Molecular weights were determined by negative and positive ion fast atom bombardment mass spectrometry and component structures were assigned following a study of fragmentation and metastable ion kinetic energy spectrometry. One fraction (TLC-8) contained a single heptaacyl MLA of Mr = 1,954, a structure previously elucidated (Qureshi, N., Mascagni, P., Ribi, E., and Takayama, K. (1985) J. Biol. Chem. 260, 5271-5278). The remaining seven fractions contained 15 additional MLAs with decreasing acylation. Two of these components have been previously reported in S. minnesota and Salmonella typhimurium. Three of the eight TLC fractions (TLC-8, -7, -6) were found to be biologically active toward human platelets inducing their aggregation and secretion of serotonin. All tested fractions induced varying degrees of phosphorylation of a platelet protein of Mr = 47,000 (P47) reflecting protein kinase C activation (Grabarek, J., Her, G. R., Reinhold, V. N., and Hawiger, J. J. (1990) J. Biol. Chem. 265, 8117-8121).     

Radioprotective properties of detoxified lipid A from Salmonella minnesota R595

In the past, the toxicity of bacterial lipopolysaccharide (LPS) or its principal bioactive component, lipid A, has detracted from their potential use as radioprotectants. Recently, a relatively nontoxic monophosphoryl Lipid A (LAM) that retains many of the immunobiologic properties of LPS has been isolated from a polysaccharide deficient Re mutant strain of Salmonella minnesota (R595). The ability of the native endotoxic glycolipid (GL) from S. minnesota (R595) as well as diphosphoryl lipid A (LAD) and nontoxic monophosphoryl lipid A (LAM) derived from GL to protect LPS responsive (CD2F1 or C3H/HeN) and nonresponsive (C3H/HeJ) mice from 60Co gamma irradiation has been studied. Administration of GL, LAD, or LAM to CD2F1 or C3H/HeN mice (400 micrograms/kg) 24 h prior to exposure provided significant radioprotection. No protection was afforded to C3H/HeJ mice. Experiments were also conducted to determine the relative abilities of GL, LAD, and LAM to stimulate hematopoiesis as reflected by the endogenous spleen colony (E-CFU) assay. Protection was not correlated with the ability of these substances to increase E-CFUs or to induce colony-stimulating activity (CSA).     

Phase diagram of deep rough mutant lipopolysaccharide from Salmonella minnesota R595.

The structural polymorphism of deep rough mutant lipopolysaccharide--in many biological systems the most active endotoxin--from Salmonella minnesota strain R595 was investigated as function of temperature, water content, and Mg2+ concentration. Differential scanning calorimetry was used to determine the amount of bound water and the enthalpy change at the beta<==>alpha gel to liquid crystalline acyl chain melting. The onset, midtemperature Tc, and completion of the beta<==>alpha phase transition were studied with Fourier-transform infrared spectroscopy. Synchrotron radiation X-ray diffraction was used to characterize the supramolecular three-dimensional structures in each phase state. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The beta<==>alpha acyl chain melting temperature Tc lying at 30 degrees C at high water content (95%) increases with decreasing water content reaching a value of 50 degrees C at 30% water content. Concomitantly, a broadening of the transition range takes place. At still lower water content, no distinct phase transition can be observed. This behavior is even more clearly expressed in the presence of Mg2+. In the lower water concentration range (< 50%) at temperatures below 70 degrees C, only lamellar structures can be observed independent of the Mg2+ concentration. This correlates with the absence of free water. Above 50% water concentration, the supramolecular structure below 70 degrees C strongly depends on the [LPS]:[Mg2+] ratio. For large [LPS]:[Mg2+] ratios, the predominant structure is nonlamellar, for smaller [LPS]:[Mg2+] ratios there is a superposition of lamellar and nonlamellar structures. At an equimolar ratio of LPS and Mg2+ a multibilayered organization is observed. The nonlamellar structures can be assigned in various cases to structures with cubic symmetry with periodicities between 12 and 16 nm. Under all investigated conditions, a transition into the hexagonal II structure takes place between 70 and 80 degrees C. These observations are discussed in relation to the biological importance of LPS as constituent of the outer membrane of gram-negative bacteria and as potent inducer of biological effects in mammals.     

Neutralization of Shwartzman-inducing activity by antibodies recognizing the Re core or lipid A structures of lipopolysaccharide from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477

Antibodies recognizing the Re core or lipid A structures of lipopolysaccharide (LPS) derived from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477 were tested for the ability to neutralize the preparatory activity of endotoxin using the local Shwartzman reaction. Shwartzman-inducing activity of R595 LPS (Re-form) was strongly suppressed when the LPS was incubated with the rabbit anti-R595 antiserum or the purified IgG antibody which recognizes core region of the LPS. The antiserum also suppressed the preparatory activity of LPS from S. typhimurium SL1102 (Re) and Escherichia coli F515 (Re), but not that of either S. typhimurium LT-2 (S) LPS or R595 lipid A. Moreover, it was found that the murine monoclonal antibody (MAb), SmRe100G (IgG2a) which recognizes the core region of R595 LPS, significantly suppressed the preparatory activity of R595 LPS. Both conventional antibodies specific to R595 lipid A, which contains a 1,4'-bisphosphorylated beta-D-glucosaminyl-alpha-D-glucosamine disaccharide structure, and JCM1477 lipid A, which contains a monophosphorylated 3-amino-D-glucosamine disaccharide structure, neutralized the preparatory activity of homologous and a closely related lipid A, but not that of LPS. In addition, it was observed that MAb Sm5G (IgG2b) specific to enterobacterial lipid A preparations (especially R595 lipid A) neutralized the preparatory activity of R595 lipid A, although the effect was somewhat weak as compared with that of rabbit antiserum. These results suggest that anti-Re LPS antibody binding to the core of Re LPS is involved in suppressing the endotoxic activity of Re LPS, and that the direct binding of anti-lipid A antibody to some specific epitopes of lipid A is important in neutralizing the endotoxic activity.     

Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, Limulus amoebocyte lysate gelation) it expressed similar activity as Salmonella lipid A.     

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. III. Reappraisal of established methods

Established methods for analysis of components of lipopolysaccharides were assessed. Optimal release of glucosamine from lipopolysaccharide occurs after hydrolysis in 6 M hydrochloric acid at 100°C for 4 h and fatty acids are best released by treatment with boron trifluoride/methanol at 100°C for 6 h. The semicarbazide assay for 3-deoxy-d-manno-octulosonic acid was modified to give results comparable to those from the periodate/thiobarbituric acid method. It was concluded that each molecule of lipopolysaccharide from Salmonella minnesota R595 contains two octulosonic acid residues and only four fatty acids, on average. There are two amide-linked hydroxyacids, together with, on average, 0.5 residues of ester hydroxyacid and a total of 1.5 residues of ester-linked normal fatty acids. This conclusion differs from the accepted view of Salmonella lipid A, but is supported by NMR results.     

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide

We have reported here on the structural polymorphism of lipid A, the "endotoxic principle" of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg2+] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70 degrees C. These data were correlated with measurements of the beta----alpha phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges--in the absence of Mg2(+)-from 45 degrees C at high to 56 degrees C at low water content, and-at an equimolar content of Mg2(+)-from 52 degrees C at high to 59 degrees C at low water concentrations. In the gel phase-in which the lipid A acyl chains are more disordered than those from saturated phospholipids-cubic phases are adopted at high water content (greater than 60%) and at high [lipid A]:[Mg2+] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal HII state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg2+ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.     

Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium

Endotoxin extracted from the heptose-less mutant of Salmonella typhimurium was hydrolyzed in 0.1 N HCl in methanol/water (1:1, v/v) at 100 degrees C to yield lipid A, which was then fractionated on a Sephadex LH-20 column to yield a major monophosphoryl lipid A fraction. The monophosphoryl lipid A was further fractionated by preparative thin layer chromatography. This process yielded three major bands (TLC-1, -3, and -5) and two minor bands (TLC-7 and -9). The purity of these fractions was established by ion exchange and reverse phase high performance liquid chromatography. The thin layer fractions were analyzed by fast atom bombardment mass spectrometry. TLC-1 and -3 gave molecular ions (M-H)- at m/e 1730 and 1716, respectively. Both of these fractions contained beta-hydroxymyristic, lauric, and 3-myristoxymyristic acids in O-acyl linkages. The molecular formula and Mr of TLC-1 are C95H179O22N2P and 1731.16; those of TLC-3 are C94H177O22N2P and 1717.15. TLC-1 was a methyl homolog of TLC-3. The major component of TLC-5 (C80H151O22N2P and Mr = 1506.99) gave a molecular ion at m/e 1506 and contained two beta-hydroxymyristic acids and a lauric acid in the O-acyl linkages. The major component of TLC-7 (C66H125O19N2P and Mr = 1280.83) and the single component of TLC-9 gave molecular ions at m/e 1280 and 1098, respectively. TLC-7 contained lauric and beta-hydroxymyristic acids in the O-acyl linkages. TLC-9 (C54H103O18N2P and Mr = 1098.69) contained a single O-acylated beta-hydroxymyristate group. TLC-1 and -3 were nontoxic in the chick embryo lethality test and regressed established tumors in the syngeneic guinea pigs.     

Structure-function relationships in the interaction of alpha-thrombin with blood platelets.

Highly purified alpha-thrombin has been chemically modified in an attempt to determine which features of the molecule are important for normal platelet-thrombin interactions. Modifying agents included diisopropylphosphorofluoridate and 1-chloro-3-tosylamido-7-amino-L-2-heptanone, which modify serine and histidine, respectively, at the catalytic site, as well as N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, which modify a single tryptophan at or near the fibrinogen-binding site. Active site-directed modification did not appreciably affect the binding characteristics, but prevented platelet activation. In contrast, modification of tryptophan at the macromolecular substrate-binding site resulted in the loss of high affinity binding of thrombin to platelets, while low affinity binding was apparently unaffected. This modification altered but did not abolish the ability of thrombin to effect platelet aggregation and release of [14C]serotonin. These results suggest that residues at the catalytic site are not involved in binding and that the macromolecular substrate-binding site of alpha-thrombin participates in high affinity binding to platelets. These data are also consistent with the existence of at least two types of binding sites for thrombin on the platelet surface as well as more than one platelet-binding region on the thrombin molecule.     

Protective action of a vaccine made from Salmonella minnesota R 595 in the intranasal infection of mice with P. aeruginosa

The comparative study of heated corpuscular vaccines prepared from S. minnesota mutant R 595, chemotype Re, from S. minnesota strain SF 1111 with defective lipopolysaccharide and from P. aeruginosa strain PA 103 has been carried out. The vaccine prepared from the chemotype Re mutant, in contrast to the vaccine prepared from S. minnesota strain SF 1111, has been found to induce the development of active immunity (and the corresponding antiserum, passive immunity) to P. aeruginosa introduced intranasally into mice, as well as to stimulate the elimination of the cells of P. aeruginosa infective strain from the lungs of the mice. The potency of the vaccine prepared from the chemotype Re mutant has been found to be significantly no different from that of the vaccine prepared from P. aeruginosa strain PA 103.     

Fourier transform infrared spectroscopy characterization of the lamellar and nonlamellar structures of free lipid A and Re lipopolysaccharides from Salmonella minnesota and Escherichia coli.

The structural polymorphism of free lipid A and deep rough mutant lipopolysaccharide (LPS Re) from Salmonella minnesota strain R595 and Escherichia coli strain F515 was characterized by Fourier transform infrared (IR) spectroscopy. For this, the beta <--> alpha phase states and the three-dimensional supramolecular structures, the latter deduced from small-angle synchrotron radiation x-ray diffraction, were investigated at different water contents, Mg2+ concentrations, and temperatures. The analysis of the IR data for vibrations originating from the hydrophobic moiety shows that the beta <--> alpha acyl chain melting is strongly expressed only for the stretching and scissoring modes of the methylene groups. Vibrational groups originating from the interface region sense the acyl chain melting well (ester carbonyl bands) or only weakly (amide bands), and those resulting from the pure polar moiety not at all. From the x-ray data, the existence of lamellar (L), different cubic, and, for lipid A and LPS R595, also inverted hexagonal (HII) structures could be proven in the temperature range 20-80 degrees C with cubic <--> cubic and cubic <--> HII transitions for the Mg(2+)-free and L <--> HII transitions for the Mg(2+)-containing samples. These structural transitions can be characterized most readily by specific changes of the vibrational bands resulting from the interface region: the ester carbonyl and the amide bands. The magnitude of the changes corresponds to that of the structural rearrangement, i.e., is highest for the L <--> HII, lower for the cubic <--> HII, and lowest for the cubic <--> cubic transitions. The structural transitions are only marginally expressed for vibrational bands of the hydrophobic moiety. Similarly, the band contours of vibrations from the hydrophilic region are no indicators of the structural reorientations except for the carboxylate bands of LPS Re. Particularly the stretching vibrations of the phosphate groups are nearly completely invariant; the absolute values of their half bandwidths, however, differ significantly for lipid A and LPS Re, which seems to be of biological relevance. The ability of IR spectroscopy to detect supramolecular changes also beyond the measurability by x-ray diffraction, i.e., at water contents > 95 to 99.5%, is demonstrated.     

Isolation of adenosine 5'-diphosphate-L-glycero-D-mannoheptose, the assumed substrate of heptose transferase(s), from Salmonella minnesota R595 and Shigella sonnei Re mutants

From heptose transferase-less Re mutants of Salmonella minnesota and Shigella sonnei, a mixture of nucleotide-linked heptoses was isolated. After paper chromatography in different solvent systems, ADP derivatives of D-glycero-D-mannoheptose and L-glycero-D-mannoheptose could be isolated in pure form. The structure of ADP-L-glycero-D-mannoheptose was verified by analytical methods and by transformation of ADP-D-glycero-D-mannoheptose with ADP-D-glycero-D-mannoheptose-6-epimerase.     

Immunological activity and toxicity of a peroral vaccine made from mutant Re Salmonella minnesota

The possibility of the oral use of heated corpuscular vaccine prepared from S. minnesota mutant R 595 (chemotype Re) for protection against Pseudomonas aeruginosa has been studied. Oral immunization in 3 doses, each containing 10(9) cells of the vaccine strain, has been shown to protect mice from death after the intravenous injection of P. aeruginosa culture in a dose of 5 LD50 and induce a rise in the titers of antibodies to Re-glycolipid (Re-hemagglutinins). After multiple oral administration Re-vaccine shows low acute and chronic toxicity and induces local and systemic immunological transformation.     

Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595.

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.     

Chemical and ultrastructural differences in endotoxic glycolipids from Salmonella minnesota Re mutant extracted with various solvent systems

Endotoxic glycolipids ( ReGl ) extracted from the whole cells (WC) and cell walls of heptose-less Re mutant of Salmonella minnesota with hot phenol-water (PW), phenol-chloroform-petroleum ether (PCP), and chloroform-methanol (CM) were analyzed chemically and examined with an electron microscope. ReGl -PW-(WC) contained mannose and proteins as contaminants, ReGl -PCP(WC) consisted of an excess amount of amino compound (cadaverine), and ReGl -PCP(WC) consisted of proteins and cadaverine, in addition to the ReGl constituents. The ultrastructure of ReGl -PW(WC) resembled onions when stained with uranyl formate, and was spherical when stained with uranyl acetate, sodium phosphotungstate and ammonium molybdate, whereas those of ReGl -PCP(WC) and ReGl -CM(WC) were shaped like ribbons. However, the shadowed ultrastructures of ReGl -PCP(WC) and ReGl -CM(WC) showed small pieces of flat and wide sheets, respectively. ReGl -PCP(WC) re-extracted by the PW method was found to be converted into the onion-like structure which was similar to that of ReGl -PW(WC), while an intermediate form (fingerprint-like) was observed after re-extraction of ReGl -PW(WC) with PCP. It was strongly suggested that the ultrastructural arrangement of ReGl was dependent on the solvent systems used for extraction.     

Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus

Caulobacter crescentus CB15 is a dimorphic bacterium that is best known as a prokaryotic model for cell development. However, it is also being exploited in biotechnology, where the crystalline surface (S-layer) protein secretion system has been adapted for heterologous protein display or secretion. Because the S-layer attaches to the cell surface via lipopolysaccharide (LPS) and since the LPS represents a potential endotoxin contaminant of recombinant proteins, the lipid A component was examined in detail. LPS was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. The structure of the major lipid A of C. crescentus comprised the tetrasaccharide backbone alpha-D-GalpA-(1-->4)-beta-D-DAG-(1-->6)-alpha-D-DAG-(1-->1)-alpha-D-GalpA substituted with six fatty acids, and a molecular mass of 1875 (GalpA, galactopyranuronic acid; DAG, 2,3-diamino-2,3-dideoxyglucopyranose). No phosphate residues were detected. The major lipid A component had 12:0[3-O[Delta(5)-12:1(3-OH)]] and 12:0[3-O(Delta(5)-12:1)] fatty acyl chains at either the 3'- or the 2' positions of the distal subunit DAG B, and 12:0(3-OH) and 12:0[3,6-(OH)( 2)] fatty acyl chains at 3- and 2- positions of the reducing end subunit DAG A, respectively. In addition, several other variations in the structure were observed. The LPS was evaluated for TNF-alpha inducing activity and consistent with its unusual lipid A structure (relative to that of enteric bacteria), the activity was reduced by greater than 100-fold as compared to Escherichia coli ReLPS. This and other evidence suggests the potential application of this lipid A as a vaccine adjuvant or the suitability of Caulobacter displaying antigens for formulation of whole cell vaccines.     

Spectral studies on the interaction of iodine and anthracene sulfonate with bacterial phospholipids from different mutants of Salmonella minnesota.

Using spectrophotometric and spectrofluorometric techniques, the interaction of iodine and 2-anthracene sulfonate (ANS) with the phospholipids (PL) isolated from four genetically correlated Salmonella minnesota isolates viz., a smooth form (S), a deeply rough mutant (Rc) and two intermediate forms (Ra and Rb) were studied. Appearance of an isosbestic point and a new band in absorption spectra indicated charge-transfer (C-T) interaction of iodine with the PL through the formation of 1:1 complex. Stern-Volmer type fluorescence quenching of PL was observed with the addition of iodine to PL, while PL enhanced the fluorescence of anionic dye ANS. The values of the binding constants between iodine/PL and ANS/PL, measured by using suitable equations, showed a systematic gradation in the molecular properties of the PL in the membrane structure in smooth (S) and rough (Ra, Rb and Rc) mutants of Salmonella minnesota.