Introduction to LPS and endotoxin

A common feature of the outer envelope of Gram Negative bacteria, including chlamydiae, is that they contain lipolysaccharide (LPS) [or, more correctly, lipooligosaccharide]. These LPSs are commonly known as ENDOtoxins because they are an ENDOgenous component of the envelope and because of the wide variety of adverse [and also beneficial] reactions they trigger. Crucial to endotoxic activity is the lipid component, Lipid A , whose activity has long been known to be determined by the number, type and distribution of fatty acids [Hauschildt et al., 2000]. Endotoxins are potent stimulators of innate immunity. They are mitogenic for B cells and activated macrophages, induce the release of pro-inflammatory cytokines [Heine et al., 1993], and also fever producing agents such as interleukin-1 ; They may also trigger physiological cascades leading to disseminated intravascular coagulation and / or septic shock . However Netea et al., 2003b have argued that septic shock may be due to infection-related underproduction of critical proinflammatory cytokines, rather than the more accepted view of endotoxin induced overproduction.

Endotoxic activity may be measured in various animal models, including fever production or the Shwartzman reaction in rabbits, by their lethality in galactosamine-sensitised mice and by the sensitive limulus lysate test in which endotoxin induces gel formation in lysates derived from the Limulus polyphemus horseshoe crab.

The basic structure of LPS was established in the classic studies of Otto Luderitz and Karl Westphal exemplifying a strong German tradition of carbohydrate and LPS biochemistry which continues for chlamydiae in the of Helmut and Lore Brade and their colleagues at Borstel. The core consists of Lipid A , in which fatty acid chains are linked to two glucosamine residues, buried in the hydrophobic interior of the envelope. To this is linked a core branched-chain oligosaccharide region containing 3-deoxy-d-manno-2-octulosonic acid (KDO). These together constitute a so-called "rough" LPS [from enterobacterial colony morphology] which is pharmacologically active and which may be structurally and immunologically diverse due to the wide variety of sugar linkages which are possible. Bacteria with so-called "smooth" LPS have a variable length polysaccharide side chain of up to 40 sugars, some of which may be extremely uncommon sugars (eg tyvelose in typhoid bacilli). The resultant smooth chains, with its unique components, may enhance the survival of bacteria such as typhoid in the blood stream by preventing the binding of preexisting antibody to the immunologically common core oligosaccharide region underneath, making the organism less susceptible to complement-mediated lysis or to phagocytosis. Rough LPS is an important trigger of the alternative pathway of complement activation. Despite this common structure, LPS from different bacteria varies enormously in its ability to trigger endotoxic activity in the host.

Structure of Chlamydial LPS.

Early studies established that chlamydial LPS was immunologically related to rough enterobacterial LPS and particularly to _ Acinetobacter calcoaceticus_ [Brade & Brunner, 1979] and that it was the basis of the group specific chlamydial complement fixation test. However structural studies using the analytical techniques then available were impaired by the difficulties of producing sufficient chlamydial LPS for investigation. C. trachomatis serovar L2 LPS had the typical components of: D-glucosamine; long-chain 3-hydroxy fatty acids; 2-keto-3-deoxyoctonic acid and phosphate in a molar ratio of approximately 2:5:3:2.6. In this respect it resembled a rough enterobacterial LPS of the Re chemotype. However for the first time they also found 3-hydroxydocosanoic acid (3-OH C22:0) as an LPS constituent [Nurminen et al., 1985]. Brade et al., 1986 compared the antigenic properties of C. trachomatis and C. psittaci LPS and identified two antigenic determinants, one of which was chlamydia genus specific and the other which was cross reactive with S. minnesota Re LPS. The real break-through for structural studies came when Fran Nano by chance cloned a chlamydial glycosyl transferase enzyme that, in E. coli, generated a hybrid LPS expressing the chlamydial genus specific LPS determinant as well as E. coli determinants [Nano & Caldwell, 1985]. This hybrid LPS could be bulk produced, permitting structural resolution of the chlamydial group specific determinant. Thus by 1987 it had been determined that the chlamydial group specific epitope consisted of a trisaccharide of the sequence KDOp-(2----8)-KDOp-(2----4)-KDO. The structure was unique to chlamydiae in that two of the KDO residues were linked through a 2.8-linkage [Brade et al., 1987].

Confirmation of this came from elegant chemical synthesis of the proposed sugar structures by Paul Kosma and his team in Vienna, working in collaboration with the Borstel group. They synthesised the putative di-, tri-, tetra and pentasaccharides which were copolymerized with acrylamide or adsorbed on red blood cells to create test antigens to probe the epitopic specificities of monoclonal antibodies directed against natural chlamydial LPS. In conjunction with this, chemical and 600 MHz NMR studies and mass spectrometry were used to further define the structures, their linkages and conformation [Bock et al., 1992; Fu et al., 1992; Holst et al., 1994; Kosma et al., 1988; 1990; 1994]. This was a major undertaking; the first complete structure, for the LPS of C. trachomatis L2, which incorporated studies of the fatty acid acylation of Lipid A by Quireshi et al., 1997 and others was described only in 1999 [Rund et al., 1999]. The glucosamine disaccharide backbone of the Lipid A of C. trachomatis L2 is substituted with a complex mixture of fatty acids with ester or amide linkage with no ester-linked hydroxy fatty acids. Within C. trachomatis, the fatty acids vary slightly from batch to batch and within serovars L2, F or E [Heine et al., 2003; Nurminen et al., 1985; Brade et al., 1986; Quireshi et al., 1997].

The carbohydrate moiety of C. trachomatis serovars L2 and E consists of Kdoalpha2-->8Kdoalpha2-->4Kdoalpha2-->6D-GlcpNbeta1 -->6D-GlcpNalpha 1,4'-bisphosphate [Heine et al., 2003; Rund et al., 1999]. That of Chlamydophila psittaci 6BC consisted of two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The carbohydrate backbone of the lipid A of C. psittaci LPS is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter as in C. trachomatis exclusively present in an amide linkage [Rund et al., 2000]. Earlier suggestions that there might be a smooth form of chlamydial LPS [Lukacova et al., 1994], particularly in egg grown C. trachomatis L1 and in C. psittaci 6BC, have not so far been substantiated.

Endotoxic activity of chlamydial LPS.

Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha is associated with endotoxic shock, an often fatal complication of Gram negative bacterial infection, [though rarely of chlamydial infection]. LPS in the bloodstream rapidly binds to a serum LPS binding protein, (LBP). The complex interacts with CD14, a differentiation antigen at monocyte surfaces, leading to TNF-alpha production [Wright et al., 1990].

It has been known for considerable time that chlamydial LPS shows only weak endotoxic activity in terms of lethality, fever production or Shwartzman reactivity but they are active in B cell mitogenicity and in the induction of prostaglandin E2 from macrophages [Brade _ et al_., 1986]. Ingalls et al., 1995 compared the endotoxic activities of whole C. trachomatis EB and purified LPS with whole Salmonella minnesota R595 or with S. minnesota R595 LPS and lipooligosaccharide from Neisseria gonorrhoeae. C. trachomatis LPS and whole EB induced the release of tumor necrosis factor alpha from whole blood ex vivo. C. trachomatis LPS was capable of inducing the translocation of nuclear factor kappa B in a Chinese hamster ovary fibroblast cell line transfected with the LPS receptor CD14. However, C. trachomatis was approximately 100-fold less potent at inducing an inflammatory cytokine response than S. minnesota or N. gonorrhoeae. It was suggested this might explain why, C. trachomatis genital tract infection is more likely to be asymptomatic than the corresponding N. gonorrhoeae infection. LPS antagonists completely inhibited the tumour necrosis factor alpha-inducing activity of whole C. trachomatis EB, this suggests that the inflammatory cytokine response may be mediated primarily through chlamydial LPS [Ingalls et al., 1995].

Highly purified LPS from C. trachomatis serovars L2 or E and C. psittaci 6BC were found to be at least 100 to 1000 times less endotoxic than smooth enterobacterial LPS [Heine et al., 2003]. C. psittaci LPS was even less active than C. trachomatis LPS. Chlamydial LPS like other bacterial LPS used toll-like receptor TLR4 but not TLR2 for signalling in HEK 293 cells. Toll receptors are thought to be part of a relatively ancient immune system that play a central role in the recognition of microbial molecular patterns and they are expressed at the appropriate sites to interact with these molecules [ For a review see Imler & Hoffmann, 2001]. The extracellular domain of the toll receptors includes a leucine rich repeat region while the intracellular region has an interleukin-1 receptor domain. On activation, toll receptors induce cytokines and Main.ArchiveDocsBiologyImunolProtectCostim. Human TLR2 dimerises with TLR6 and recognizes bacterial peptidoglycans, lipoproteins, lipopolysaccharides etc. However chlamydial LPS was unusual in requiring CD14 for efficient transfer to the TLR4 signalling receptor [Heine et al., 2003]. Bone marrow derived dendritic cells, were also stimulated by C. trachomatis serovar L2 LPS to secrete tumour necrosis factor-alpha (TNF) via TLR4 but not TLR2. In contrast, whole micro organism C. trachomatis L2 induced TNF secretion independently of TLR4. Moreover secretion of TNF induced by whole C. trachomatis L2 EB occurred independently of LPS (as shown by the failure of polymyxin to inhibit) or of TLR4 [Prebeck et al., 2003]. This would be consistent with the observation of Netea et al., 2003 that non LPS components of C. pneumoniae were capable of stimulating cytokine production through TLR2-dependent pathways. At the first CBRS meeting in Memphis in 2003, Robin Ingalls reported that in macrophages TLR2 is recruited to the phagosome and seems to be involved in sifting phagosome contents for microbial components. TLR2 is present at high amounts at the cell membrane and localises to the Golgi area. In cells infected with C. trachomatis, TLR2 is enriched in the cytoplasm around the inclusion membrane and may form part of an intracellular signalling system. Robin suggested that _ C. trachomatis_ probably activates epithelial cells primarily via TLR2. This is an MyD88 dependent process and the activation requires replicating bacteria. Chlamydia also have TLR4 ligands, but do not activate TLR4 well. Key questions are what is the chlamydial TLR2 ligand and what is the TLR2-dependent signalling mechanism in response to chlamydiae? These new data suggest that the role of chlamydial LPS in inducing cytokine responses may be more complex than was hitherto realised.

[MEW] June 2003

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