Lore 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.

IDENTIFICATION OF A NOVEL DENDRITIC CELL-LIKE SUBSET OF CD64(+)/CD16(+) BLOOD MONOCYTES

Human monocytes (Mo) consist of a major subset of Fcγ‐receptor I (CD64)‐positive typical low accessory phagocytes, and a minor CD64– DC‐like subset with high T cell‐accessory and IFN‐α‐releasing activity. Both populations also differentially express CD16 (Fcγ‐receptor III). Double labeling with anti‐CD64 and anti‐CD16 mAb, as performed here, identified four different subsets. The CD64– subset consists of CD64– / 16+ cells with high antigen‐presenting cell (APC) function and macrophage‐like phenotype, and a CD64– / 16– subset of less active APC but which exhibits a higher mixed lymphocyte reaction (MLR) stimulating and IFN‐α‐producing capacity, possibly resembling plasmacytoid dendritic cell type II (DC2) blood precursors. As well as the majority of CD64+ cells that appeared CD64+ / 16– and represent typical low‐accessory, CD14high Mo, we could identify and describe a novel minor subset of CD64+ / 16+ cells which is unique in combining typical DC and Mo characteristics in the same cell. These are high IL‐12 production, high accessory capacity for antigen‐ or allogen‐activated lymphocytes, and high expression of HLA‐DR, CD86, and CD11c.

A Single Nucleotide Exchange in the wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia coli Strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strains LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.

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.

Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy-d-manno-octulosonate-containing synthetic antigens☆

Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivative of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective allyl glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, igM) recognizing a terminal Kdop group was shown to require for its binding the alpha-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7 - C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG3), which recognizes a (2--4)-linked Kdo disaccharide, was shown to require for its binding the alpha-anomeric configuration of both residues. The isomer having a reducing beta-Kdo residue was significantly less active, and that with a terminal beta-Kdo group was completely inactive. The OH-5 group of the reducing residue was shown to be not important for the specificity of this antibody, since it could be replaced by a hydrogen atom without loss of serological reactivity. The alpha-(2--8)-linked Kdo disaccharide was strongly cross-reactive with its (2--4)-linked isomer. The antibody recognized also parts of the 2-amino-2-deoxy-D-glucose backbone of lipid A.

Bacterial lipopolysaccharides: relationship of structure and conformation to endotoxic activity, serological specificity and biological function.

Gram-negative bacteria express in their cell envelope various amphiphilic macromolecules among which the lipopolysaccharides (LPS) are of special significance for bacterial viability and the interaction of bacteria with host organisms. Together with phospholipids and proteins, lipopolysaccharides form the outer membrane of gram-negative bacteria. This outer membrane has an asymmetric architecture, i.e., lipopolysaccharides are located exclusively in the outer leaflet through which the bacterial cell interacts with its environment.

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity

Lipopolysaccharide (LPS) from the outer membrane of Gram‐negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the ‘endotoxic principle’ of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition.

Antibody WN1 222-5 mimics Toll-like receptor 4 binding in the recognition of LPS

Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.73-Å resolution structure of the unique endotoxin-neutralizing protective antibody WN1 222-5 in complex with the core region shows that it recognizes LPS of all E. coli serovars in a manner similar to Toll-like receptor 4, revealing that protection can be achieved by targeting the inner core of LPS and that recognition of lipid A is not required. Such interference with Toll-like receptor complex formation opens new paths for antibody sepsis therapy independent of lipid A antagonists.