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Bordetella endotoxins show remarkable structural variability both among each other and in comparison to other gram-negative bacteria. Here we demonstrate that, in contrast to the common Bordetella pertussis laboratory strain and Tohama I derivative BP338, lipooligosaccharide from mouse challenge strain 18-323 is a poor inducer of inflammatory cytokines in human and murine macrophages, is greatly impaired in Toll-like receptor 4-mediated activation of nuclear factor-κB in transfected HEK-293 cells, and functions as a Toll-like receptor 4 antagonist. Comparison of lipid A and lipooligosaccharide structures of B. pertussis strains BP338 and 18-323 revealed that 18-323 (1) lacks the ability to modify its lipid A phosphate groups with glucosamine, (2) is distinct in its acylation at the C3' position of the lipid A diglucosamine backbone, and (3) expresses molecular lipooligosaccharide species that lack a terminal heptose. Our findings have important implications for interpreting previous studies of host defenses to B. pertussis infection in mice and in vitro.

A method for obtaining highly purified endotoxin (lipopolysaccharide [LPS]) in a few hours by repurification of commercial or laboratory preparations was devised. It avoids the use of phenol, which is not suitable for phenol-soluble lipopolysaccharides nor for some industrial purposes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry analysis confirmed the integrity of the purified LPSs. The purified products did not activate Toll-like receptor 2 (TLR2), nuclear oligomerization domain 1 (NOD1), or NOD2 but did activate TLR4. Applied to different lipopolysaccharides, the method also improved their mass spectra, thus facilitating their structural analysis.

Structural studies of Bordetella endotoxins (LPSs) have revealed remarkable differences: (i) between their LPSs and those of other bacterial pathogens; (ii) among the LPSs of the seven identified Bordetella species; and (iii) among the LPSs of some Bordetella strains. The lipid As have the "classical" bisphosphorylated diglucosamine backbone but tend to have fewer and species-specific fatty acid components compared to those of other genera. Nevertheless, three strains of B. bronchiseptica have at least three different fatty acid distributions; however, the recently identified B. hinzii and B. trematum LPSs had identical lipid A structures. The B. pertussis core is a dodecasaccharide multi-branched structure bearing amino and carboxylic groups. Another unusual feature is the presence of free amino sugars in the central core region and a complex distal trisaccharide unit containing five amino groups of which four are acetylated and one is methylated. The B. pertussis LPS does not have O-chains and that of B. trematum had only a single O-unit, unlike the LPSs of all the other species of the smooth-type. The O-chain-free cores of non-B. pertussis LPSs were always built on the B. pertussis core model but most were species-specifically incomplete. The LPS structures of three B. bronchiseptica strains were found to be different from each other. The O-chains of B. bronchiseptica and B. parapertussis were almost identical and had some features in common with B. hinzii O-chain. Serological analyses are consistent with the determined LPS structures.

The structure of Bordetella pertussis 1414 lipid A was investigated by classical methods of chemical analysis as well as plasma desorption mass spectrometry and fast atom bombardment mass spectrometry. Previous analysis showed that it contained a bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkage. The presence of two main molecular species as seen by thin-layer chromatography was confirmed by plasma desorption mass spectrometry, in which the larger signal was attributable to a molecular ion containing two glucosamine, two phosphate, one tetradecanoic acid, one hydroxydecanoic acid, and three hydroxytetradecanoic acid residues. The ion of the smaller signal was lighter by the mass of one hydroxytetradecanoic acid residue (226 Da). The fatty acids in ester linkage were localized by chemical and fast atom bombardment mass spectrometry analysis. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site for Kdo (3-deoxy-D-manno-octulosonic acid).

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.

Plasma desorption mass spectrometry has recently been used with success to characterize native, underivatized Re- to Rc-type endotoxins in terms of their constituent lipopolysaccharides. The spectra give masses for the major molecular species of lipopolysaccharide present from which their probable compositions could be inferred using the overall composition determined by chemical analyses. Moreover, the relative intensities of the signals are roughly proportional to the abundance of their corresponding molecular species. Native Rc-, Rb-, and Ra-type enterobacterial endotoxins with 5-10 core sugar units have been rendered amenable to plasma-desorption mass spectrometry analysis by improvement in their solubility and the use of cellobiose as an additive. The spectra of four Salmonella and Escherichia endotoxin preparations demonstrated heterogeneity in acylation and phosphorylation. Since these sources of heterogeneity are critical for many biological activities, the spectra underline the need to define the composition of each preparation of endotoxin used in structure-function studies.

Nine unmodified endotoxin preparations constituted of Re-, Rd-, and Rc-type lipopolysaccharides (2 to 5 glycoses), representing four species of enterobacteria were analyzed by 252Cf plasma desorption mass spectrometry. The constituent lipopolysaccharides were characterized by the ion pair: (M-H)- and its corresponding lipid fragment ion. The lipid fragment ion is produced by cleavage of the glycosidic bond of the 3-deoxy-D-manno-oct-2-ulosonic acid unit that substitutes O-6' of the glucosamin beta 1'-6glucosamine ("lipid A backbone") disaccharide of the lipid A moiety. These lipid fragment ions were identical to the (M-H)- ions seen in the spectra of homologous isolated lipid A preparations that were obtained by hydrolysis (pH 4.5, 100 degrees C) promoted by sodium dodecyl sulfate. Since the molecular components present in the endotoxin preparations analyzed are known, the ion pair (M-H)(-)-lipid fragment ion defines the molecular compositions of each individual lipopolysaccharide. Heterogeneity of the R-type endotoxin preparations analyzed was due almost exclusively to differing lipid A moieties. In three Salmonella minnesota 595 Re endotoxin preparations 10 different lipopolysaccharides were identified, only two of which were common to all three preparations. Of the nine lipopolysaccharides identified in two S. minnesota R7 endotoxin preparations, only two were present in both.

The nature of the binding sites for LPS on human monocytes was investigated using [3H] labeled intact LPS from Neisseria meningitidis and from Salmonella minnesota R7, and the [3H] labeled purified inner core region (PS-OMe) of S.m. R7 LPS. In the presence of serum, intact LPS from enterobacterial and nonenterobacterial strains bound to monocytes in a dose-dependent, saturable, and displaceable fashion. N.m. LPS and LPS from the enterobacterial strain of Escherichia coli 0111-B4 bound to the same sites on monocytes as assessed in competitive binding experiments. Specific binding of intact LPS to monocytes occurred through the CD14 molecule as shown by the ability of mAb and of F(ab')2 fragments of mAb directed against specific epitopes of CD14 to inhibit the binding of [3H]-LPS to cells and by the lack of binding of intact LPS to CD14-deficient cells from patients with paroxysmal nocturnal hemoglobinuria. Specific binding of LPS to monocytes was not mediated by the CD11/CD18 complex because mAb to the alpha and beta chains of the Leu-CAM molecules did not alter the binding of LPS to cells and because LPS did not inhibit the binding of labeled mAb to monocytes. [3H]-PS-OMe also bound in a dose-dependent and displaceable fashion to monocytes involving an unidentified, non-CD14, binding site on the cells. Binding of LPS to monocytes also involved nonsaturable binding sites for hydrophobic structures of LPS as evidenced in binding experiments performed in the absence of serum. These observations indicate that intact LPS may interact with the monocyte membrane in at least three ways including serum-dependent binding to CD14 and to a lectin-like receptor, and serum-independent hydrophobic interactions.

The endotoxin of Bordetella pertussis was cleaved by mild acidic hydrolysis to yield a polysaccharide (polysaccharide I, 15%), a glycolipid (63%) and lipid X (2%). Further treatment of the glycolipid with stronger acid released a second polysaccharide (polysaccharide II, 9%) and material similar to lipid A present in enterobacterial endotoxins. Both polysaccharides possess a single molecule of 3-deoxy-2-octulosonic acid as the reducing, terminal sugar. In polysaccharide II the octulosonic acid is phosphorylated in position 5 and presumably substituted in position 4; in polysaccharide I the octulosonic acid is not phosphorylated, but is substituted in position 5. Following treatment of the endotoxin with strong base, a fragment was isolated that contained bound, non-phosphorylated 3-deoxy-2-octulosonic acid, glucosamine phosphate and fatty acids. This indicated that polysaccharide I, like polysaccharide II, was bound to the lipid region of the endotoxin. The endotoxin structure thus defined is different from that proposed for the lipopolysaccharides of enterobacteria.

Due to the formation of micelles, severance of the hydrophilic (poly- or oligosaccharide) and hydrophobic ("Lipid A") domains of bacterial lipopolysaccharides at pH 3.4 or 4.5 and 100 degrees is slow and sometimes does not proceed at all; partially degraded fragments are usually formed. At pH 3.4 (100 degrees) in aqueous 1% sodium dodecylsulphate (SDS), both lipopolysaccharides of the Bordetella pertussis endotoxin are cleaved within 20-30 min, but 80% of the glycosidically bound phosphate present in the hydrophobic domain is lost. Other endotoxins behave similarly. At pH 4.5 (100 degrees) and in the absence of detergent, hydrolysis of the glycosidic bonds of 3-deoxy-D-manno-2-octulosonic acid residues of the B. pertussis endotoxin is negligible but, in aqueous 1% SDS, severance of the two regions of LPS 1 is complete within 1 h (that of LPS-2 requires 3-4 h), and the glycosidically bound phosphate of the isolated hydrophobic region is preserved. Comparison of the rate of acid-catalysed hydrolysis of the glycosidically bound phosphate present in this "isolated Lipid A" preparation with that of 2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-alpha- and -beta-D-glucopyranose 1-phosphates established that the former 1-phosphate was the alpha anomer.

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.

Endotoxin from fresly sedimented Bordetella pertussis cells, isolated by the phenol/water procedure when submitted to kinetically controlled, mild acidic hydrolysis released a polysaccharide (polysaccharide 1), a complex lipid (lipid X), and a glycolipid. When treated with somewhat stronger acid, the glycolipid yielded a second polysaccharide (polysaccharide 2) and another complex lipid (lipid A). The intact pertussis endotoxin had all the usual properties of endotoxins extracted from enteric bacteria. Lipid X and the intermediary glycolipid retained all the endotoxic properties of the unfractionated endotoxin. In lipid A, pyrogenicity was reduced to a very low level and toxicity and Shwartzman reactivity were absent; however, this fraction retained most of the endotoxin's antiviral activity, and its adjuvant power was considerably higher than that of the intact endotoxin. Lipid A elicited nonspecific resistance against challenge with certain bacteria, but not against others.

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.

The Yersinia genus includes human and animal pathogens (plague, enterocolitis) as well as non-pathogens. The lipopolysaccharide of the facultative pathogen Yersinia pseudotuberculosis has been implicated in the invasiveness of these bacteria. In this work, we have investigated the fine structure of the lipid A isolated from Y. pseudotuberculosis lipopolysaccharide using chemical analyses, gas chromatography/mass spectrometry, plasma desorption mass spectrometry, and matrix-assisted laser desorption mass spectrometry. Arabinose (Ara) and aminoarabinose (Ara-4N) esterified the phosphates as in Yersinia pestis lipid A. The acylation of Y. pseudotuberculosis lipid A differed from those found in Yersinia enterocolitica, Yersinia ruckeri, and Y. pestis lipopolysaccharides (LPSs): in the distribution of fatty acids between the two glucosamines in the fully acylated hexaacyl molecular species and by the acyloxyacyl substitution at position C-2′, where the Y. pseudotuberculosis lipid A has a C14OC16 making it closest to that of Y. pestis. [Copyright &y& Elsevier]