RESUMO
Lipids A are the hydrophobic domains of bacterial endotoxic lipopolysaccharides. Since they are responsible for most of the biological activities (both pathogenic and beneficial) of endotoxins, the characterization of their structure is crucial to the understanding of their mode of action. However, the inadequacy of existing methods for preparing certain lipids A has prompted us to devise a new, mild procedure which gives intact products. Use was made of the special features of 252Cf-plasma desorption mass spectrometry for forming molecular ions from these species and giving qualitative and quantitative information from the primary mass spectrum.
Assuntos
Califórnio/química , Lipídeo A/análise , Bordetella pertussis/química , Cromatografia em Camada Fina , Escherichia coli/química , Espectrometria de Massas , Moraxella catarrhalis/química , Shigella flexneri/química , Açúcares Ácidos/químicaRESUMO
Smooth type endotoxins of Salmonella, Yersinia, and Escherichia were fractionated into long and short chain lipopolysaccharides by silica gel chromatography. Lipid A was prepared from the fractions and analyzed by plasma desorption mass spectrometry. Both Yersinia and Salmonella endotoxins had a large proportion of aminoarabinose-containing lipopolysaccharide molecular species that were found to be concentrated in the long chain fraction. In the Escherichia endotoxin, hypoacylated lipopolysaccharides (lacking the tetradecanoate and one of the four hydroxytetradecanoates) were found mostly in the short chain fraction. Possible implications of these results for the lipopolysaccharide biosynthetic pathway and for studies on the influence of sugar chain length on the biological effects of endotoxins are discussed.
Assuntos
Escherichia coli/química , Lipídeo A/química , Lipopolissacarídeos/química , Salmonella/química , Yersinia enterocolitica/química , Califórnio , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas/métodosRESUMO
A branched-chain hexasaccharide containing 3-deoxy-D-manno-oct-2-ulosonic acid was released by detergent-promoted hydrolysis from Bordetella pertussis endotoxin preparations that were first dephosphorylated with aqueous HF and then treated with nitrous acid. Its structure (2) [Formula: See text] was determined by chemical and physical methods. This hexasaccharide is present in all four lipopolysaccharides that make up the B. pertussis strain 1414 (phase 1) endotoxin preparations analysed, and is situated near to the hydrophobic domains. An analogous structure reported previously (ref 7) is erroneous and should be disregarded.
Assuntos
Bordetella pertussis/química , Endotoxinas/química , Lipopolissacarídeos/química , Oligossacarídeos/química , Fatores de Virulência de Bordetella , Sequência de Carboidratos , Desaminação , Cromatografia Gasosa-Espectrometria de Massas , Heptoses/análise , Hexosaminas/análise , Hexoses/análise , Ácidos Hexurônicos/análise , Hidrólise , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oxirredução , Ácido Periódico/química , Fosfoproteínas/química , Análise de SequênciaRESUMO
After treatment with aqueous, 50% hydrofluoric acid, a well-known dephosphorylating agent, the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO), an essential and characteristic constituent of endotoxins, can be readily demonstrated in reportedly KDO-deficient bacterial lipopolysaccharides.
Assuntos
Endotoxinas/análise , Açúcares Ácidos/análise , Lipopolissacarídeos/análiseRESUMO
Experiments were undertaken to localize in the lipopolysaccharide (LPS) the minimal structural determinants sufficient to initiate the signal leading to interleukin 1 (IL 1) secretion by human monocytes. Our results clearly demonstrated that this signal is triggered by structures present in the so-called inner-core region which chemically consists of 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) and heptose in many LPS of gram-negative bacteria. Thus, the isolated polysaccharide region of Bordetella pertussis endotoxin as well as fragments derived therefrom containing the reducing KDO unit were able to induce similar levels of IL1 induction as the native LPS. Similarly, the trisaccharide alpha-D-manno-heptopyranosyl-(1-3)-alpha-D-manno-heptopyranosyl -(1-5)-3 -deoxy-D-manno-octulosonic acid (hep-hep-KDO), representative for the inner-core region of a large number of enterobacterial LPS, was a very potent IL 1 inducer. Neither KDO monosaccharide, nor the alpha-(2-4)-linked 3-deoxy-D-manno-octulosonic acid disaccharide isolated from Salmonella rough-form LPS promoted the signal indicating that the minimal structure of endotoxin able to induce IL 1 secretion resides in the hep (1-5)-KDO disaccharide.
Assuntos
Dissacarídeos/imunologia , Endotoxinas/imunologia , Interleucina-1/biossíntese , Lipopolissacarídeos/imunologia , Monócitos/imunologia , Açúcares Ácidos/imunologia , Sequência de Carboidratos , Epitopos , Bactérias Gram-Negativas/imunologia , Humanos , Salmonella/imunologia , Relação Estrutura-AtividadeRESUMO
Specific binding of Bordetella pertussis and Neisseria meningitidis endotoxins to human monocytes and murine macrophages was demonstrated. Binding of B. pertussis endotoxin could be inhibited by endotoxins of Salmonella minnesota, Escherichia coli, and Klebsiella pneumoniae, the extent of inhibition being dependent on the origin of the lipopolysaccharides and on the origin of the mononuclear phagocytic cells. The binding of B. pertussis and N. meningitidis endotoxins which was mediated by the polysaccharide region of the endotoxins was serum dependent. The results indicated that the binding of endotoxin was promoted neither by natural antibodies directed against the endotoxin nor by proteins known to combine with endotoxins: immunoglobulins, albumin, or fibronectin; we have provided some evidence that complement components may play a role in the specific binding of endotoxins to the monocyte/macrophage membrane.