Ernst Th. Rietschel

Invited review: Bacterial lipopolysaccharides and innate immunity

Bacterial lipopolysaccharides (LPS) are the major outer surface membrane components present in almost all Gram-negative bacteria and act as extremely strong stimulators of innate or natural immunity in diverse eukaryotic species ranging from insects to humans. LPS consist of a poly- or oligosaccharide region that is anchored in the outer bacterial membrane by a specific carbohydrate lipid moiety termed lipid A. The lipid A component is the primary immunostimulatory centre of LPS. With respect to immunoactivation in mammalian systems, the classical group of strongly agonistic (highly endotoxic) forms of LPS has been shown to be comprised of a rather similar set of lipid A types. In addition, several natural or derivatised lipid A structures have been identified that display comparatively low or even no immunostimulation for a given mammalian species. Some members of the latter more heterogeneous group are capable of antagonizing the effects of strongly stimulatory LPS/lipid A forms. Agonistic forms of LPS or lipid A trigger numerous physiological immunostimulatory effects in mammalian organisms, but — in higher doses — can also lead to pathological reactions such as the induction of septic shock. Cells of the myeloid lineage have been shown to be the primary cellular sensors for LPS in the mammalian immune system. During the past decade, enormous progress has been obtained in the elucidation of the central LPS/lipid A recognition and signaling system in mammalian phagocytes. According to the current model, the specific cellular recognition of agonistic LPS/lipid A is initialized by the combined extracellular actions of LPS binding protein (LBP), the membrane-bound or soluble forms of CD14 and the newly identified Toll-like receptor 4 (TLR4)*MD-2 complex, leading to the rapid activation of an intracellular signaling network that is highly homologous to the signaling systems of IL-1 and IL-18. The elucidation of structure-activity correlations in LPS and lipid A has not only contributed to a molecular understanding of both immunostimulatory and toxic septic processes, but has also re-animated the development of new pharmacological and immunostimulatory strategies for the prevention and therapy of infectious and malignant diseases.

Lipopolysaccharides of Gram-Negative Bacteria

Publisher Summary Lipopolysaccharides (LPS) form a large, unique class of macromolecules representing a characteristic attribute of gram-negative bacteria. Associated with proteins, they are located in the outer leaflet of the outer membrane of the bacterial cell. In this exposed position on the cell surface, lipopolysaccharides are involved in the interaction of the cell with the environment. Thus, contact of the bacterium with the immune system leads to the stimulation of specific antibodies directed predominantly against determinant structures of the lipopolysaccharide. Hence, lipopolysaccharides represent the main surface antigens of gram-negative bacteria. The chapter describes general aspects of the O-specific chains and the core, and principles of their biosynthesis. The O-specific chains of lipopolysaccharides are made up of repeating units of identical oligosaccharides. These units usually contain different constituents, thus the O chain represents a heteropolysaccharide. The chapter discusses the structure of Salmonella lipopolysaccharides (lipid A), and its biosynthesis. Lipid As of other gram-negative bacteria, recently investigated is also described. The smallest lipid A substructure exhibits antigenicity, mitogenicity, lethal toxicity, (weak) pyrogenicity, and (weak) complement reactivity, but strong Limulus lysate activity. Some aspects of the biological properties of lipopolysaccharides are also discussed. In Gram-positive and Gram-negative bacteria, cell wall components other than lipopolysaccharide may also be endowed with endotoxin-like activities. Like lipopolysaccharide, these constituents are amphipathic in nature (probably with the exception of murein, though it may contain lipoprotein). Thus, the studies indicate that at least some lipid A activities are not restricted to one specific structure, but are rather connected with general physicochemical properties.

Molecular mechanisms of endotoxin activity

Endotoxin (lipopolysaccharide, LPS), a constitutent of the outer membrane of the cell wall of gramnegative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intra-cellular signal transduction pathways.

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.

Chemical structure of glycosphingolipids isolated from Sphingomonas paucimobilis

Two novel glycosphingolipids were isolated fromSphingomonas paucimobilis and their structures were completely elucidated. The glycosyl portion of the glycosphingolipid consists of an α‐D‐Manp‐[1→2)‐α‐D‐Galp‐(1→6)‐α‐D‐GlcpN‐(1→4)‐α‐D‐GlcpA‐R tetrasaccharide. The hydrophobic residue R was found to be heterogeneous with respect to the dihydrosphingosine residue.Erythro‐1,3‐dihydroxy‐2‐amino‐octadecane anderythro‐1,3‐dihydroxy‐2‐amino‐cis‐13,14‐methyleneoctadecane were identified in comparable amounts. Both dihydrosphingosine derivatives were quantitatively substituted by an (S)‐2‐hydroxymyristic acid in amide linkage.

Measurement of chemiluminescence in freshly drawn human blood

SummaryThe present investigations were undertaken to find out whether chemiluminescence measurements of stimulated granulocytes can be carried out in freshly drawn blood and — because of the ease of the method — be introduced into routine diagnostics.Blood was drawn from the cubital vein of healthy volunteers at various times and under various conditions. Subsequently the zymosan induced and luminol amplified chemiluminescence was recorded and analyzed. It could be demonstrated that variations existed between individuals which can, however, be minimized when photon counts obtained under standard conditions were related to the number of granulocytes present in the blood samples. It could be further demonstrated that also platelets are activated by zymosan as well and that they, contribute to the total chemiluminescence by a share of about 5%. Platelet chemiluminescence can effectively be suppressed by aspirin. Opsonising factors in plasma (presumably antibodies and/or complement) play a decisive role in the intensity and kinetics of blood chemiluminescence. Measurements of zymosan induced chemiluminescence in freshly drawn unfractionated and fractionated blood seem to be especially suited to monitor and analyze deviations and defects of the cellular and humoral defence mechanisms.ZusammenfassungDie vorliegenden Untersuchungen bilden die Grundlage, um die im Prinzip sehr einfache Messung der Chemilumineszenz stimulierter Granulocyten in frisch gewonnenem Vollblut durchzuführen und damit für die einfache Routine-Diagnostik anwendbar zu machen.Gesunden Freiwilligen wurde zu verschiedenen Zeiten und unter verschiedenen Bedingungen kubital Venenblut entnommen und anschließend die durch Zymosan induzierte und durch Luminol verstärkte Chemilumineszenz analysiert. Die Chemilumineszenz-Kurven verschiedener Individuen zeigten Variationen, die jedoch verringert werden, wenn die unter Standardbedingungen gemessenen Photonen auf die Zahl der im Blut vorhandenen Granulocyten bezogen werden. Es zeigte sich ferner, daß Blutplättchen ebenfalls durch Zymosan aktiviert werden und zu etwa 5% an der gesamten Chemilumineszenz beteiligt sind. Die Plättchen-Chemilumineszenz läßt sich indes durch Zusatz von Aspirin nahezu vollständig ausschalten. Von entscheidender Bedeutung für die Intensität und Kinetik der im Vollblut gemessenen Chemilumineszenz ist die Anwesenheit opsonierender Plasma-Faktoren (Antikörper und/oder Komplement).Die Messung der Chemilumineszenz im Vollblut erscheint uns geeignet, sowohl Abweichungen der zellulären wie der humoralen Abwehrfunktionen rasch und einfach zu erkennen.

Lipopolysaccharide‐binding protein mediates CD14‐independent intercalation of lipopolysaccharide into phospholipid membranes

Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CD14‐independent whereas at high LPS concentrations it is CD14‐independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CD14‐independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide‐binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14‐independent cell activation at high endotoxin concentrations.

Chemical structure and biological activities of lipid A's from various bacterial families

The endotoxic principle of lipopolysaccharides (LPS) is localized in their lipid A component. Biological effects of LPS on, for instance, body temperature, blood pressure, and blood picture, are also induced by free lipid A. In contrast to the great variability of the 0-specific chains, the chemical structure of lipid A is much more constant. It is common for Salmonella and similar for other genera of the Enterobacteriaceae. Recently, a number of lipid As have been recognized that exhibited distinct structural features compared with Enterobacteriaceae. These lipid As were found to be also distinct with regard to some of their biological properties.

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.

Prostaglandin and thromboxane synthesis in a pure macrophage population and the inhibition, by E-type prostaglandins, of chemiluminescence

Cell populations consisting predominantly of macrophages emit chemiluminescence when they are phagocytosing zymosan particles [ 1,2] or bacteria [ 1,3,4] . This chemiluminescence is considered indic- ative of the generation of reactive species of oxygen (Os-, HzOz, OH’), which are formed as intermediates during the stepwise reduction of molec,ular oxygen by one-electron donors, and of singlet O2 (A02) generated during the disproportionation of Os-. These species emit light in the presence of polyunsaturated fatty acids, polysaccharides or easily oxidizable substances like luminol [5]. As these species of oxygen are con- sidered important in the killing mechanism of phago- cytic cells [6] chemiluminescence may be viewed as a manifestation of their microbicidal activity [7] . Inde- pendently it has been reported that macrophages form and release prostaglandins (PCs) [8] under con- ditions comparable to those that evoke chemilumi- nescence. PGs have been implicated as mediators in inflammation and cellular immunity [9-l l] . We have studied chemiluminescence, PG formation and their possible interrelationship in macrophages cultivated from bone marrow precursor cells of mice. By morphological criteria these cells are not con-