Salmonella minnesota

A number of amide- and ester-linked fatty acids and (/ )-3-hydroxy acids are components of the lipid A part in the LPS from Gram-negative bacteria. The acids have been tabulatedand the chemistry of lipid A summarized. The most common acids in lipid A from Enterobacteriaceae are the saturated 12 0,14 0, and 16 0, and the (/ )-3-hydroxy-14 0, The last is linked to N-2 and 0-3 of the 2-amino-2-deoxy-D-glucopyranosyl residues, and the others are ester-linked to the hydroxy acid, as in the lipid A (44) of Salmonella minnesota. Other linear and branched fatty acids, unsaturated acids, S)-2- and (/ )-3-hydroxy acids, and 3-oxotetradecanoic acid are components of lipid A from certain different species. In the lipid A from Rhizobium trifolii, 2,7-dihydroxyoctanoic acid is linked as amide to a 2-amino-2-deoxy-D-gl ucopy ranosy 1 residue. ... 

In a very elegant study, Droge and coworkers26 investigated the structures of the LPS from four Salmonella minnesota R-mutants designated mR595, mR3, mR7, and mR5. These mutants synthesize LPS that contains only lipid A and the inner core oligosaccharides,... 

Sugar Composition of LPS from Four Strains of Salmonella minnesota (Data as % of Sugar and, in Parentheses, as Molar Ratios)26... 

Determinations0 of KDO Content in Salmonella minnesota LPS, Using the Semicarbazide Assay88 and the TBA Assay11... 

Finally, Droge and coworkers26 showed that the 2-aminoethyl phosphate-substituted KDO group is the lateral KDO unit of the branched trisaccharide (see Fig. 7), as follows. LPS from Salmonella minnesota mR3 was subjected to periodate oxidation. This sample, together with a control that had not been oxidized, was then mildly hydrolyzed with acid (pH = 3.4) during 1 h at 100°. Following removal of lipid A, both samples were analyzed by gel-filtration on Sephadex G-10, and paper electrophoresis. As expected, the ninhydrin-positive material obtained from the control sample was identical with KDO 7-(2-aminoethyl phosphate) (17) as previously identified. This spot was absent from the periodate-treated sample. Instead, an almost neutral, ninhydrin-positive spot was observed. This material (compound 26) was eluted, subjected to reduction with sodium [3H]borohydride, and hydrolyzed under strongly acidic conditions (see Scheme 11). Fol-... 

Fig. 4.—Chemical structure of lipid A of the Salmonella minnesota Re mutant strain R595. For details see the text. See also the legend to Fig. 2. For substituents of the phosphate groups see Table I.

The occurrence of 3-deoxy-D-manno-octulosonic acid in lipopoly-saccharides has prompted its synthesis,537 together with the D-gal-octo158 and the D-gluco analogs.157 Although gas-liquid chromatography was successfully used to analyze the products of these syntheses, it has been reported that methanolysis of a bacterial endotoxin lipopolysaccharide failed to yield 3-deoxy-D-manno-octulosonic acid, presumably because of the lability of the latter to acid.381 However, Kasai and Nowotny have reported four peaks for the O-trimethylsilyl derivatives of 3-deoxy-D-manno-octulosonic acid obtained by methanolysis of the glycolipid from a Salmonella minnesota mutant.538 Reduced 3-deoxy-D-manno-octulosonic acid and its methyl ester have also been analyzed successfully as their acetates.339,539... 

Deoxy-D-manno-octulo- 5% Ucon LB-550 192 Salmonella minnesota glycolipid 538... 

Therapeutic vaccines were tested in BALB/c mice bearing TA3-Ha mammary carcinoma. The treatment consisted of 4 subcutaneous injections, at 3-6 days intervals, of Detox [a commercial preparation of cell wall skeletons from Mycobacterium phlei and non-toxic monophosphoryl lipid A from Salmonella minnesota (S. minnesota) in squalane oil and Tween 80 from Ribi Immunochemical research, Montana, USA] mixed with Thomsen-Friedenreich (TF) antigen coupled with KLH (Keyhole Limpet Hemocyanin) performed 5 days after the tumor cell injection. This vaccination achieved the survival of 25 % of the mice. Pretreatment of mice with cyclophosphamide in order to inhibit any suppressive response, increased survival to 50 % when the treatment began 5 days after tumor cell injection, and to 90 % when the treatment began 2 days after tumor cell injection. Both antibody as well as delayed-... 

Immunostimulatory adjuvants exert their effects predominantly at the cytokine level or through the activation of costimulatory signals. The type of response required for optimal protection depends on the pathogen. One class of immunostimulatory adjuvants is derived from the lipopolysaccharide of gram-negative bacteria. The most extensively evaluated member of this family, monophosphoryl lipid A (MPL), is obtained from Salmonella minnesota. MPL has been shown to induce the synthesis and release of cytokines, which promote the generation of specific immune responses. 

Figure 1. Schematic representation of the preparation of the lipid A backbone of Salmonella minnesota Re lipopolysaccharide. (Reproduced with permission from Ref 16. Copyright 1976, Eur. J. BiochemJ.

In a similar way it was demonstrated that in Salmonella minnesota Re LPS, KDO is bound to the nonreducing glucosamine residue in the lipid A backbone, presumably to position 3 (20). Recent experiments of our laboratory involving methylation analysis indicate that, in Proteus mirabilis, the polysaccharide component is linked to position 6 of the lipid A backbone (21). 

Substituents of the ester-linked phosphate group Lipopolysaccharide of a Re-mutant of Proteus mirabilis (R45) is similar to the lipopolysaccharide of the Salmonella minnesota Re-mutant (R595) in that it contains a phosphate to glucosamine ratio of approximately 2.4 2.0 ((21), Fig. 2). When the degradation procedure described for Salmonella, however, was applied to P. mirabilis lipopolysaccharide, an unexpected result was obtained (Fig. 2). 

Total fatty acids were liberated by subjecting Salmonella minnesota Re lipopolysaccharide (or free lipid A) to acidic (4 N HC1, 5 h, 100°C) followed by alkaline (1 N NaOH, 1 h, 100°C) hydrolysis. After extraction (chloroform), the free fatty acids were converted into their methyl esters (diazomethane) and analysed by combined gas-liquid chromatography/mass spectrometry. Alternatively, the fatty acids of lipid A are transesterified by treatment of lipopolysaccharide with methanolic HC1 (2 N HC1 in water-free CHaOH, 18 h, 85°C). By these procedures the following fatty acids were identified (in approximate amounts relative to 2 moles glucosamine) dodecanoic (12 0, 1.1 mole), tetradecanoic (14 0, 0.8 mole), hexadecanoic (16 0, 0.9 mole), 2-hydroxytetradecanoic (2-OH-l4 0, 0.1 mole), and 3-hydroxytetradecanoic acid (3-OH-14 0, 4 moles). In total, therefore, approximately 7 moles of fatty acids are present per mole of lipid A backbone. The stereochemistry of the hydroxylated fatty acids was determined by gas-liquid chromatography of their diastereomeric methoxyacyl-L-phenylethylamide derivatives (24). It was found that 2-hydroxyte-tradecanoic acid possesses the-Ts), and the predominating 3-hydroxytetradecanoic acid the (R) configuration. 

Kasai, N., Nowotny, A. Endotoxic glycolipid from a heptoseless mutant of Salmonella minnesota. J Bacteriol 94 (1967) 1824-1836. 

Peterson, A.A., McGroarty, E.J. High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli. J Bacteriol 162 (1985) 738-745. 

Qureshi, N., Mascagni, P., Ribi, E., Takayama, K. Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination. J Biol Chem 260 (1985) 5271-5278. 

Brandenburg, K. Fourier transform infrared spectroscopy characterization of the lamellar and non-lamellar structures of free lipid A and Re lipopolysaccharides from Salmonella Minnesota and Escherichia coli. Biophys J 64 (1993) 1215-1231. 

Brandenburg, K., Koch, M.H.J., Seydel, U. Phase diagram of lipid A from Salmonella minnesota and Escherichia coll rough mutant lipopolysaccharide. J Struct Biol 105 (1990) 11-21. 

Garidel, P., Rappolt, M., Schromm, A.B., Howe, J., Lohner, K., Andra, J., Koch, M.H., Brandenburg, K. Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity, Biochim Biophys Acta 1715 (2005) 122-131. 

Unger, F.M., Christian, R., Schulz, G., Waldstten F., Brade, H., Zhringer, U., Rietschel, E.T. Structure of the 3-Deoxyoctulosonate (Kdo-) Region of Lipopolysaccharide from Salmonella minnesota Re 595, Xllth International Carbohydrate Symposium, July 1-7, 1984, Utrecht, The Netherlands Abstract Book (1984) 348. 

Bruins, S.C., Stumacher, R., Johns, M.A., McCabe, W.R. Immunization with R mutants of Salmonella Minnesota. III. Comparison of the protective effect of immunization with lipid A and the Re mutant. Infect Immun 17 (1977) 16-20. 

Johns, M., Skehill, A., McCabe, W.R. Immunization with rough mutants of Salmonella minnesota. IV Protection by antisera to O and rough antigens against endotoxin. J Infect Dis 147 (1983)... 

Galanos, C., Luderitz, O., Freudenberg, M., Brade, L., Schade, U., Rietschel, E.T., Kusumoto, S., Shiba, T. Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A. Eur J Biochem 160 (1986) 55-59. 

Lipid A was investigated by FTIR, X-ray diffraction, and fluorescence techniques (Naumann et al., 1987). Analysis of the CH2 scissoring vibrations showed that lamellar structures exist at temperatures below 30 °C, but that no stable closed vesicles are formed. Orientation measurements of lipopolysaccharides prepared from E. coli and Salmonella minnesota by ATR-FTIR also showed a highly ordered gel phase. Besides, the transition from the gel pha.se a to the liquid crystalline phase was also studied (Brandenburg and Seydel, 1988). 

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