Facing the continuous daily invasion of microorganisms, the human organism requires sophisticated and efficient defence mechanisms to protect from infections. In this context, the initial recognition of invading bacteria by the innate immune system is crucial for the subsequent induction of inflammatory responses, activation of the adaptive immune system and elimination of the pathogen.
1.2.1 Pattern associated molecular patterns
The innate immune system is designed to recognize highly conserved motifs unique to microorganisms that are not associated with human cells. These motifs are termed
“pathogen associated molecular patterns” (PAMPs). The main immune stimulatory
lipopolysaccharide (LPS) (189, 224) and lipoteichoic acid (LTA) (111, 185), respectively.
Other parts of the bacterial cell wall like peptidoglycan (PGN) and its breakdown products the muropeptides as well as lipoproteins (114, 254, 255, 259, 279), heat shock proteins, especially heat shock protein 60 kD (hsp60) (270) and bacterial DNA (143), also represent potent stimuli for activation of the innate immune system.
The basic structure of LPS of Gram-negative bacteria consists of a polysaccharide moiety attached to a lipid anchor, named lipid A (226). The polysaccharide moiety is made up of a core and a polymer of repetitive oligosaccharide molecules, called antigen. The O-antigens are variable among a bacterial genus and often also between bacterial strains and determine the serological specificity. The part of the LPS molecule, which has been shown to be responsible for cytokine induction, is the lipid A, which is highly conserved, but variations in the amount and design of fatty acids occur between species (37, 72, 225, 256). The counterpart to LPS in Gram-positive bacteria is the LTA, which is anchored in the cytosolic membrane and reaches through the murein sacculus (68). LTA is made up of a lipophilic glycolipid anchor with typically two fatty acids linked to a hydrophilic glycerophosphate backbone, which is substituted with D-alanine and N-acetyl-glucosamine (186). It has been shown that for biological activity like cytokine induction, the LTA anchor is indispensable, but the backbone with its substitutions is further increasing potency (54, 187).
1.2.2 Pattern recognition receptors
Innate immune cells, especially monocytes, are well equipped with pattern recognition receptors (PRR), which enable them to recognize different PAMPs. The most prominent PRR are the Toll-like receptors (TLR) (2, 4, 159) and the recently identified NOD (nucleotide-binding oligomerisation domain) proteins (39, 83).
NOD proteins, including NOD1 and NOD2, are cytosolic proteins that have been initially thought to recognize LPS (125), but it has been shown recently that they enable intracellular recognition of muropeptides, which are usually co-extracted with LPS during purification (40, 80). NOD1 and NOD2 consist of three distinct domains, a C-terminal one with a leucine-rich repeat (LRR), which is critical for ligand recognition, a central nucleotide binding site which mediates oligomerization and one or two N-terminal caspase recruiting domains (CARD), which are involved in regulation of apoptosis and activation of inflammatory responses (122, 212). NOD1 is expressed in virtually all tissues (123) and
recognizes muropeptides that contain meso-diaminopimelic acid (40), a component of PGN. Meso-diaminopimelic acid is common for Gram-negative bacteria but also occurs in a few Gram-positive bacteria. NOD2, which is expressed only in monocytes (94) and Paneth cells (155), is a PRR for muramyl dipeptide (MDP) (81), a major constituent and typical breakdown product of PGN, which is present in almost all bacterial cell walls. Only recently, a frame shift mutation in the NOD2 gene 3020insC, which leads to the truncation of the terminal LRR and thus to a protein which cannot detect MDP, has been associated with the occurrence of Crohn’s disease suggesting a major role of NOD2 in intestinal immunity (81, 126).
To date, ten members of the TLR family have been identified in humans. TLR are transmembrane receptors, expressed primarily on leukocytes but also on some other cells such as endothelial or epithelial cells. While most of the TLR are expressed on the cell surface, TLR3 and TLR9 are located intracellularly (156). The individual TLRs recognize distinct structural components of pathogens and their stimulation directly results in signaltransduction, induction of gene expression and inflammatory responses (3). TLR3 has been shown to recognize dsRNA of viruses (7), whereas TLR9 recognizes unmethylated CpG motifs of bacterial as well as viral DNA (108). The prototypical LPS from enterobacteria is recognized by TLR4 together with MD-2 and the glycosylphosphatidylinositol anchored CD14 (214, 251), while in general, TLR2 mediates responses to LTA (157), lipoproteins (188) and PGN (61). However, some LPS exist, which are, in comparison to the classical LPS from enterobacteria, characterised by differences in the lipid A moiety, like that from Bacteroides fragilis, Leptospira interrogans or Porphyromonas gingivalis that are known to be relatively weak immune stimuli and to act via TLR2 (64, 163). Nevertheless, it is currently discussed, whether these initial findings were due to contaminating lipoproteins, since more recent work also reports TLR4 dependence of these LPS (50, 53). Furthermore, for immune activation by hsp60, which has been reported to depend on TLR2 and TLR4 (252, 264), recent evidence suggests that some of the reported cytokine effects may be due to contaminating LPS (262).
1.2.3 Immune recognition of Chlamydiaceae
All chlamydial species are Gram-negative and are supposed to express a family-specific LPS epitope. So far, the LPS of C. trachomatis and C. psittaci have been isolated, but only in the case of C. trachomatis the LPS structure has been analyzed (106, 233). The
chlamydial Lipid A is highly hydrophobic due to the presence of unusual, long-chain fatty acids (142, 220) and its immune stimulatory potential is 100- to 1000-fold weaker compared to LPS from classical enterobacteria (106, 121). The TLR-dependence of C.
trachomatis LPS has not been clarified so far, since it was first described to be TLR4 dependent (216), but a recent study by Erridge et al. demonstrated that it is TLR2 dependent (64). C. trachomatis and C. pneumoniae are closely related bacteria and it is assumed that the structure of the O-antigen is comparable between the species, but no information on the lipid A structure is available so far. To date, the role of the chlamydial LPS in chlamydial infections is not clear, since both TLR2 and TLR4 are necessary for full immune activation in response to C. pneumoniae and C. trachomatis (47, 51, 195, 216, 217, 283). In addition, chsp60 has been suggested to be a potent inducer of inflammatory responses in endothelial cells and macrophages and a stimulator of vascular smooth muscle cell proliferation via TLR4 (25, 240). Moreover, there are also hints for TLR independent target cell activation (198), suggesting the existence of additional receptors.
Since C. pneumoniae are obligate intracellular bacteria, the cytoplasmatic NOD proteins, which are implicated in intracellular pattern recognition (35, 124), qualify as candidates for the recognition of C. pneumoniae. Only recently, it was shown that NOD1 plays a dominant role in C. pneumoniae-induced interleukin (IL)-8 release in endothelial cells (203). However, it is not clear, which structure of C. pneumoniae may interact with the NOD proteins. NOD proteins have so far been associated with recognition of different types of PGN building blocks (40, 80, 81). Although recent studies suggest a functional PGN pathway in Chlamydiaceae (112, 173), a clear cut biochemical evidence for the chlamydial PGN-synthesis or presence of PGN in Chlamydiaceae is missing (41, 70).
However, Chlamydiaceae are sensitive to antibiotics that inhibit PGN-synthesis (191). This phenomenon has been referred to as the “chlamydial PGN paradox”.