Bacterial LPS represents one of the primary targets of the host innate immune system to recognize a Gram-negative bacterial infection. Upon LPS encounter, the subsequent innate immune response is characterized by the release of proinflammatory mediators (including TNF and IL-6), which is beneficial in initiating and orchestrating the elimination of the infection by means of the innate as well as the adaptive immunity. However, in the case of an excessive (systemic) exposure to LPS, the body will react with a systemic inflammatory reaction leading to multi-organ failure, with a high risk of death, a condition often referred to as septic shock.
Positioned in the outer membrane of Gram-negative bacteria, LPS serves as a most critical component guaranteeing both the structural and functional membrane integrity.
Introduction 14
Biochemically, the molecule was identified to consist of lipid and carbohydrate moieties, thereby defining the common name 'lipopolysaccharide'. The term 'endotoxin' is also still in use. Structurally, all known LPS variants share a common architecture, comprising three major building blocks, i.e. the Lipid A portion, a core polysaccharide as well as the O-polysaccharide (Fig. 1.2A). Modifications of this basic structure give rise to a huge range of variants in the different bacterial strains. LPS molecules containing all three sub-structures are referred to as 'wild type' or 'smooth' LPS. In contrast, molecules that lack the O-polysaccharide are known as 'rough' mutants, which can be further classified according to the level of completeness of their core polysaccharide structures. This way, rough LPS mutants are termed from 'Ra', with a complete core, to 'Re', having only the basic sugar residues attached to their Lipid A portion (see also below). All together, these structural variants are generally termed LPS 'chemotypes'. The classification as to smooth (S) and rough (R) derives from the appearance of the bacterial colonies made by strains expressing either of the two LPS versions as their major cell wall components. The discrimination as to S and R chemotypes is, however, better based on molecular differences in the LPS – and these structural versions come with distinct functional properties.
The Lipid A portion of the molecule was shown to be the carrier of the endotoxic activity, as synthetic Lipid A preparations exhibited biological activities identical to those of E.coli Lipid A (Tanamoto et al., 1984; Galanos et al., 1985). This portion of the LPS molecule is typically composed of a bisphosphorylated diglucosamine backbone which is substituted with up to four acyl chains. These acyl chains can be further substituted with fatty acids leading to a Lipid A that carries up to seven acyl substituents. Depending on the bacterial strain, these fatty acid substitutions vary by number, length, order and saturation.
In terms of biological activity, it appears that E.coli Lipid A, with its hexa-acylated and diphosphorylated diglucosamine backbone (Fig. 1.2B), represents the structure optimally recognized by the respective mammalian receptors and that any modification of this 'ideal' structure will result in reduced endotoxicity (Rietschel et al., 1994).
The core polysaccharide represents a relatively defined carbohydrate structure, with only a limited number of different sugars being incorporated. This consequently results in a high degree of conservation among bacterial strains, regarding this partial element.
Structurally, the core polysaccharide can be formally divided into an inner and an outer core, the latter being generally more variable by composition. The inner core is especially characterized by the presence of rather unusual sugars, such as
3-deoxy-D-manno-Introduction 15
octulosonic acid (Kdo) and heptose. Kdo is found in almost every known LPS. It links the core polysaccharide to the carbohydrate backbone of Lipid A. It might be this particular function – assuring bacterial viability – which makes the Kdo residue an indispensible constituent of virtually any LPS structure. Indeed, an essential role is supported by findings where the smallest saccharide component found in naturally occurring bacteria consisted of only one to three Kdo residues (Brade et al., 1987; Helander et al., 1988).
GlcN
Fig. 1.2: Structure of LPS. (A) Principle structure of 'wild type' LPS as divided into O-polysaccharide, core polysaccharide and Lipid A portion. Gal, galactose; GlcN, glucosamine; GlcNac, N-acetyl-glucosamine; Glu, glucose; Hep, heptose; Kdo, 2-keto-3-desoxyoctonate; P, phosphate. (B) Chemical structure of E.coli Lipid A as having the format widely believed to be optimally recognized by mammalian TLR4. Adapted from Erridge et al. (2002).
The O-polysaccharide consists of 1 to 50 repeating units with each of them being composed of 1 to 8 sugar residues. Thereby, a given bacterial strain shows individual O-polysaccharide characteristics by virtue of the set of sugar units (monosaccharides), their sequence and chemical linkage, substitutions and ring formats. By combination, these variables lead to an almost limitless diversity of O-polysaccharide structures, which is
Introduction 16
reflected by the appearance of hundreds of serotypes for particular Gram-negative species.
Due to its positioning at the outermost part of the LPS molecule, the O-polysaccharide is also the major target for host antibody responses, which is the reason for its alternative name 'O-antigen'.
At this point, it should be stressed that, despite the fact of Lipid A being the carrier of the molecules' endotoxic activity, the nature and number (length) of the attached sugar chains both have considerable impact on modulating this activity (Erridge et al., 2002).
Indeed, several studies already demonstrated some relationship between LPS structures (chemotypes) and function (Gangloff et al., 2005; Jiang et al., 2005b; Huber et al., 2006).
These observations were, however, made on mast cells and extra-neural macrophages.
With the growing understanding of differences in the TLR4 organization by individual cell types and a concomitant variation in agonist action, the situation may differ for microglia, which have not been studied yet in this regard. Thus, some of the 'rules' of chemotype signaling though TLRs, namely TLR4, may reveal variation when focusing on other cell types.
Aim of the Study 17