Doris Karibian

Structural and functional analyses of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPSs) are powerful immunomodulators in infected hosts, and may cause endotoxic shock. Most of them share a common architecture but vary considerably in structural motifs from one genus, species, and strain to another. Cells of the innate immune response recognize evolutionarily conserved LPS molecular patterns of endotoxins and structural details thereby greatly influencing their response.

Structure of the Bordetella pertussis 1414 endotoxin.

The endotoxin (lipopolysaccharide) of Bordetella pertussis, the agent of whooping cough, consists of a lipid A linked to a highly branched dodecasaccharide containing several acid and amino sugars. The elucidation of the polysaccharide structure was accomplished by first analyzing the structures of fragments obtained by hydrolysis and nitrous deamination and then piecing the fragments together. The fine structure of the antigenic distal pentasaccharide, presented here, was determined by chemical analyses as well as by high‐resolution nuclear magnetic resonance and mass spectrometry. The complete structure was reconstituted and confirmed by matrix‐assisted laser desorption/ionization mass spectrometry. The following structure was derived from the combined experimental data:

Novel variation of lipid A structures in strains of different Yersinia species1

The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C‐2′: this was dodecanoic acid (C12) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C14) in two other Y. enterocolitica and hexadecenoic acid (C16:1) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.

252CF-PLASMA DESORPTION MASS SPECTROMETRY OF UNMODIFIED LIPID A : FRAGMENTATION PATTERNS AND LOCALIZATION OF FATTY ACIDS

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.

252Cf-plasma desorption mass spectrometry applied to the analysis of endotoxin Lipid A preparations

Abstract Negative-ion plasma desorption mass spectra of underivatized Lipid A preparations, isolated from the endotoxins of 16 Enterobacterial and non-Enterobacterial strains, are presented. These heterogeneous preparations gave signals—in the range m/z 1000–2200—characteristic of the bacterial family from which the Lipid A was obtained. Molecular compositions based on chemical analyses were attributed to the most abundant ions. The information provided makes plasma desorption mass spectrometry a powerful tool in relating Lipid A structures to their numerous biological activities. The method is rapid and requires only microgram quantities of Lipid A.

Structural characterization of Bordetella parapertussis lipid A

Bordetella parapertussis like B. pertussis, is a causal agent of whooping cough but is not a strictly human pathogen. Because its endotoxin, a major structural component of the Gram-negative outer membrane, is an important virulence factor, we have analyzed the structure of its toxic lipid domain, in one rough and two smooth bacterial strains. Chemical analyses and mass spectra obtained before and after recently developed mild-alkali treatments revealed that the lipids A have the common bisphosphorylated β-(1→6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. All three strains have two major molecular species: a tetraacyl and a pentaacyl species. The rough strain is richer in a minor hexaacyl species. Acylation at the C-2, C-3, and C-3′ positions was different from that of the B. pertussis lipid A. The C-2 position carries a secondary hexadecanoic acid, the C-3 position is free, and the C-3′ position is substituted with hydroxydecanoic acid (not at C-3 as in B. pertussis), and the rough strain hexaacyl species carries a second secondary hexadecanoic acid. Like the lipid A of B. pertussis, the hydroxytetradecanoic acid at the C-2′ position was substituted by tetradecanoic acid.

Contribution of 252Cf-plasma desorption mass spectrometry to structural analysis of lipids A: examples of non-conservatism in lipid A structure

The great majority of lipids A studied so far have the enterobacterial lipid A-type skeleton: a bisphosphorylated glucosamine disaccharide, with fatty acids amidating the 2 amino groups and esterifying the C3 and C3′ positions. Differences between these lipids A occur in the nature and localization of the fatty acids. Such differences have been put forward as a possible taxonomic tool. It is now relatively easy to determine these differences using plasma desorption mass spectrometry (PDMS) if one knows the overall composition of the pure, native lipids A. We have used this technique to compare the lipids A of 2 or more species of several bacterial genera and found considerable conservatism within genera and sometimes between closely related genera (Salmonella and Escherichia ). An exception was Bordetella of which different species varied in the nature and/or the localization of their fatty acids. B. parapertussis , like B. pertussis, had a single C10OH but at a different location, and quite unusually had a non-hydroxylated fatty acid (C16 ) directly esterifying glucosamine I. On the other hand, 2 strains of B. bronchiseptica had a C12OH in place of the C 10OH of B. pertussis, whereas a third strain replaced the C 12OH by a C12, again a primary non-hydroxylated fatty acid. PDMS has allowed us to conclude that the combined pattern of fatty acids in lipids A is not a reliable taxonomic tool.

Use of mass spectrometry to compare three O-chain-linked and free lipopolysaccharide cores: differences found in Bordetella parapertussis

Plasma desorption mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry methods were used to investigate the molecular differences between lipopolysaccharide free core molecules and core molecules substituted by O-chains in Bordetella parapertussis, Salmonella ohio, and Escherichia coli 0119. The B. parapertussis analysis indicated a difference in mass of 569 amu corresponding to 3 distal sugars comprising terminal residues of heptose, galactosaminuronic acid, and, N-acetyl-N-methylfucosamine, a result supported by evidence from NMR and serology. No differences were evident in the analyses of cores in either S. ohio or E. coli O119, although the first O-chain unit carried by S. ohio core lacked a terminal glucose present in the residual O-chain repeating units.

Analusis by 252Cf plasma desorption mass spectrometry of Bordetella pertussis endotoxin after nitrous deamination

Abstract Endotoxic lipopolysaccharides (LPSs) are the major components of Gram-negative bacterial outer membrane. Like many amphipathic molecules, they pose problems of heterogeneity, purity, solubility, and aggregation. Nevertheless, PDMS has recently have been applied to unmodified endotoxins composed of LPS having uip to five sugar units in their saccharide chain. The B. Pertussis LPSs, most of which have a dodecasaccharide domain, ahve been analysed by classical methods and the masses of the separate lipid and saccharide domains determined after rupture of the bond linking them. However, the acid treatment employed for these and most chemical analyses can also modify structures in the vicinity of the bond. In order to investigate this biologically-important region, the endotoxin was treated to nitrous deamination, which shortens the saccharide chain to five sugars, but preserves the acid-labile region of the LPS. The PDM spectrum of this derivative, which required new conditions for its desorption, confirmed the structure analysis and demonstrated the presence of at least four molecular species.