Helmut Brade

Bacterial Endotoxin: Chemical Constitution, Biological Recognition, Host Response, and Immunological Detoxification

The discovery of endotoxin dates from the late nineteenth century when Richard Pfeiffer, then working in Berlin, characterized endotoxins as heat-stable and cell-associated molecules (Westphal et al. 1977), thus distinguishing them from the heat-labile and proteinous exotoxins which are actively secreted by bacteria (Bhakdi et al. 1994). They were first found to be produced by Vibrio cholerae bacteria and later by Salmonella and Serratia. Endotoxins, due to their various potent biological activities soon attracted worldwide scientific interest. Initial chemical analyses of purified endotoxin indicated that it consists essentially of polysaccharide and lipid, and it was therefore termed lipopolysaccharide (LPS). Today the terms endotoxin (Wolff 1904) and lipopolysaccharide (Shear and Turner 1943) are used synonymously for the same molecule.

PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide

Platelet-activating factor (PAF) induces pulmonary edema and has a key role in acute lung injury (ALI). Here we show that PAF induces pulmonary edema through two mechanisms: acid sphingomyelinase (ASM)-dependent production of ceramide, and activation of the cyclooxygenase pathway. Agents that interfere with PAF-induced ceramide synthesis, such as steroids or the xanthogenate D609, attenuate pulmonary edema formation induced by PAF, endotoxin or acid instillation. Our results identify acid sphingomyelinase and ceramide as possible therapeutic targets in acute lung injury.

The chemical structure of bacterial endotoxin in relation to bioactivity

Lipopolysaccharides (LPS) constitute the O-antigens and endotoxins of Gram-negative bacteria. Whereas both the polysaccharide and lipid portion of LPS contribute to the pathogenic potential of this class of bacteria, it is the lipid component (lipid A) which determines the endotoxic properties of LPS. The primary structure of lipid A of various bacterial origin has been elucidated and Escherichia coli lipid A has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique structural arrangement and conformation. Such analyses have furthermore provided insight into the determinants required for lipid A binding to and activation of human target cells. Present research efforts aim at the molecular characterization of the specificity, modulation and biomedical consequences of the interaction of lipid A with host cells.

A Lethal Role For Lipid A In Salmonella Infections

Salmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a high threshold level, commonly called the lethal load. Despite much speculation, no direct evidence has been available to substantiate a role for any candidate bacterial components in causing death. One of the most likely candidates for the lethal toxin in salmonellosis is endotoxin, specifically the lipid A domain of the lipopolysaccharide (LPS) molecule. Consequently, we have constructed a Salmonella mutant with a deletion–insertion in its waaN gene, which encodes the enzyme that catalyses one of the two secondary acylation reactions that complete lipid A biosynthesis. The mutant biosynthesizes a lipid A molecule lacking a single fatty acyl chain and is consequently less able to induce cytokine and inducible nitric oxide synthase (iNOS) responses both in vivo and in vitro. The mutant bacteria appear healthy, are not sensitive to increased growth temperature and synthesize a full‐length O‐antigen‐containing LPS molecule lacking only the expected secondary acyl chain. On intravenous inoculation into susceptible BALB/c mice, wild‐type salmonellae grew at the expected rate of approximately 10‐fold per day in livers and spleens and caused the death of the infected mice when lethal loads of approximately 108 were attained in these organs. Somewhat unexpectedly, waaN mutant bacteria grew at exactly the same rate as wild‐type bacteria in BALB/c mice but, when counts reached 108 per organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 109 per organ were attained, when approximately 10% of the mice died. Most of the animals carrying these high bacterial loads survived, and the bacteria were slowly cleared from the organs. These experiments provide the first direct evidence that death in a mouse typhoid infection is directly dependent on the toxicity of lipid A and suggest that this may be mediated via pro‐inflammatory cytokine and/or iNOS responses.

STRUCTURAL ANALYSIS OF THE LIPOPOLYSACCHARIDE FROM CHLAMYDIA TRACHOMATIS SEROTYPE L2

The lipopolysaccharide (LPS) of Chlamydia trachomatis L2 was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. From a total of 5 × 104 cm2 of infected monolayers, 22.3 mg of LPS were obtained. Compositional analysis indicated the presence of 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo), GlcN, phosphorus, and fatty acids in a molar ratio of 2.8:2:2.1:4.5. Matrix-assisted laser-desorption ionization mass spectrometry performed on the de-O-acylated LPS gave a major molecular ion peak at m/z 1781.1 corresponding to a molecule of 3 Kdo, 2 GlcN, 2 phosphates, and two 3-hydroxyeicosanoic acid residues. The structure of deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide was determined by 600 MHz NMR spectroscopy as Kdoα2→8Kdoα2→4Kdoα2→6d-GlcpNβ1→6d-GlcpNα 1,4′-bisphosphate. These data, together with those published recently on the acylation pattern of chlamydial lipid A (Qureshi, N., Kaltashov, I., Walker, K., Doroshenko, V., Cotter, R. J., Takayama, K, Sievert, T. R., Rice, P. A., Lin, J.-S. L., and Golenbock, D. T. (1997) J. Biol. Chem. 272, 10594–10600) allow us to present for the first time the complete structure of a major molecular species of a chlamydial LPS.

Germline antibody recognition of distinct carbohydrate epitopes.

High-resolution structures reveal how a germline antibody can recognize a range of clinically relevant carbohydrate epitopes. The germline response to a carbohydrate immunogen can be critical to survivability, with selection for antibody gene segments that both confer protection against common pathogens and retain the flexibility to adapt to new disease organisms. We show here that antibody S25-2 binds several distinct inner-core epitopes of bacterial lipopolysaccharides (LPSs) by linking an inherited monosaccharide residue binding site with a subset of complementarity-determining regions (CDRs) of limited flexibility positioned to recognize the remainder of an array of different epitopes. This strategy allows germline antibodies to adapt to different epitopes while minimizing entropic penalties associated with the immobilization of labile CDRs upon binding of antigen, and provides insight into the link between the genetic origin of individual CDRs and their respective roles in antigen recognition.

Identification of a Novel Heptoglycan of α1→2-Linkedd-glycero-d-manno-Heptopyranose CHEMICAL AND ANTIGENIC STRUCTURE OF LIPOPOLYSACCHARIDES FROMKLEBSIELLA PNEUMONIAE SSP. PNEUMONIAE ROUGH STRAIN R20 (O1−:K20−)

 In a preliminary investigation (Süsskind, M., Müller-Loennies, S., Nimmich, W., Brade, H., and Holst, O. (1995) Carbohydr. Res. 269, C1–C7), we identified after deacylation of lipopolysaccharides (LPS) from Klebsiella pneumoniae ssp. pneumoniaerough strain R20 (O1−:K20−) as a major fraction the oligosaccharide, Gal p A β 1 → 6 threo hex 4 enurono p Glcp β 1 → 4 He 3 ↑ 1   →   3 Hep p α 1 7         ↑         Hep p α 1       p p α 1 → 5 Kd 4 ↑ Kdo α 2 o α 2 → 6 Glc 4 ↑ P p N β 1 → 6 Glc p N α 1 → P STRUCTURE   1 where Kdo was 3-deoxy-d-manno-oct-2-ulopyranosonic acid and Hepp was manno-heptopyranose. The presence of the threo-hex-4-enuronopyranosyl residue indicated a substituent at O-4 of the second GalA residue linked to O-3 of the second l,d-Hep residue, which had been eliminated by treatment with hot alkali. We now report the complete structure of lipopolysaccharide, which was elucidated by additional characterization of isolated core oligosaccharides and analysis of the lipid A. The substituent at O-4 of the second GalpA isd-GlcpN, which in a fraction of the LPS is substituted at O-6 by three or four residues ofd-glycero-d-manno-heptopyranose (d,d-Hepp). The complete carbohydrate backbone of the LPS is as follows, D , D , D He 2 ↑ D , D Hep p * α 1   D , D He 2 ↑ p p α 1 D He 2 ↑ p p α 1 Gal p A * β 1 → 6 Glc p β 1 → 4 L , D Hep 3 ↑ p p * α 1 → 6 Glc p N α 1 → 4 Gal p A α 1 → 3 L , D Hep p α 1 7         ↑         L , D Hepp α 1       K p α 1 → 5 Kdo 4 ↑ Kdo α 2 4         ↑         do α 2 *         α 2 → 6 Glc 4 ↑ P p N β 1 → 6 Glc p N α 1 → P STRUCTURE   2 (l-glycero-d-manno-heptopyranose;l,d-Hepp), where all hexoses possess the d-configuration. Sugars marked with an asterisk are present in nonstoichiometric amounts. The structure is unique with regard to the presence of an α1→2-linkedd-glycero-d-manno-heptoglycan (oligosaccharide), which has not been described to date, and does not contain phosphate substituents in the core region. Fatty acid analysis of lipid A identified (R)-3-hydroxytetradecanoic acid as sole amide-linked fatty acid and (R)-3-hydroxytetradecanoic acid, tetradecanoic acid, small amounts of 2-hydroxytetradecanoic acid, hexadecanoic acid, and traces of dodecanoic acid as ester-linked fatty acids, substituting the carbohydrate backboned-GlcpN4Pβ1→6d-GlcpNα1P. The nonreducing GlcN carries four fatty acids, present as two 3-O-tetradecanoyltetradecanoic acid residues, one of which is amide-linked and the other ester-linked to O-3′. The reducing GlcN is substituted in a nature fraction of lipid A by two residues of (R)-3-hydroxytetradecanoic acid, one in amide and the other in ester linkage at O-3. Two minor fractions of lipid A were identified; in one, the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing GlcN is esterified with hexadecanoic acid, resulting in 3-O-hexadecanoyltetradecanoic acid, and in the second, one of the 3-O-tetradecanoyltetradecanoic acid residues at the nonreducing GlcN is replaced by 3-O-dodecanoyltetradecanoic acid. Thus, the complete structure of LPS is as shown in Fig.1. After immunization of BALB/c mice, two monoclonal antibodies were obtained that were shown to be specific for the core of LPS fromK. pneumoniae ssp. pneumoniae, since they did not react with LPS or whole-cell lysates of a variety of other Gram-negative species. Both monoclonal antibodies could be inhibited by LPS but not by isolated oligosaccharides and are thus considered to recognize a conformational epitope in the core region.

Critical Role for Interleukin-1β (IL-1β) during Chlamydia muridarum Genital Infection and Bacterial Replication-Independent Secretion of IL-1β in Mouse Macrophages

ABSTRACT Recent findings have implicated interleukin-1β (IL-1β) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1β is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1β expression during a chlamydial genital infection. In the present study, IL-1β−/− mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1β both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1β secretion in WT mice, the major producers of IL-1β in vivo are F4/80+ macrophages and GR-1+ neutrophils, but not CD45− epithelial cells. Although elicited mouse macrophages infected with Chlamydiamuridarum in vitro secrete minimal IL-1β, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichiacoli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1β from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1β secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1β secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1β secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1β mRNA observed in vivo in this cell type.

Methylation analysis of the heptose/3-deoxy-D-manno-2-octulosonic acid region (inner core) of the lipopolysaccharide from Salmonella minnesota rough mutants.

A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy-D-manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd2P-, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di-O-acetyl and 2,4,6-tri-O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd2P- LPS, the hydrolysis step involved 0.1M trifluoroacetic acid at 100 degrees for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:02 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave 5-O-acetyl-3-deoxy-1,7,8-tri-O-methyl-2,4,6-tri-O-trideuteriomethyl++ +-D- glycero-D-talo/galacto-octitol and 1,5-di-O-acetyl-2,3,4,6,7-penta-O-methyl-L-glycero-D-manno-heptitol++ +. These results prove the presence of a (2----4)-linked KDO disaccharide in Re LPS and show that the core region of Rd2P- LPS contains a terminal alpha-L-glycero-D-manno-heptopyranosyl group and a non-substituted, a 4-O-, and a 4,5-di-O-substituted pyranosidic KDO residue in the molar ratios 1:1:0.2:1.

Isolation and chemical analysis of 7-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-l-glycero-d-manno-heptose as a constituent of the lipopolysaccharides of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli and Vibrio cholerae

Methyl 7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-hepto+ ++ pyranoside (1) was released from the lipopolysaccharide of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli by methanolysis and isolated by high-voltage paper electrophoresis. Its chemical structure was determined by chemical analysis, deamination with nitrous acid, g.1.c.-m.s., and 1H- and 13C-n.m.r. spectroscopy performed on its acetylated derivative. The disaccharide moiety of 1 was also detected in lipopolysaccharides of Vibrio cholerae.