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- MATERIALS AND METHODS
The endotoxic activities of lipopolysaccharides (LPS) isolated from different strains of rhizobia and rhizobacteria (Bradyrhizobium, Mesorhizobium, and Azospirillum) were compared to those of Salmonella enterica sv. Typhimurium LPS. The biological activity of all the examined preparations, measured as Limulus lysate gelation, production of tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and nitrogen oxide (NO) induction in human myelomonocytic cells (line THP-1), was considerably lower than that of the reference enterobacterial endotoxin. Among the rhizobial lipopolysaccharides, the activities of Mesorhizobium huakuii and Azospirillum lipoferum LPSs were higher than those of the LPS preparations from five strains of Bradyrhizobium. The weak endotoxic activity of the examined preparations was correlated with differences in lipid A structure compared to Salmonella.
Soil bacteria belonging to the rhizobium lineage are able to fix atmospheric nitrogen during symbiosis with legume plants. Bacteria from the genus Bradyrhizobium induce nitrogen-fixing nodules on the roots of cultivated (Glycine max and Glycine soya) and wild-growing legumes (1, 2). M. huakuii induces the formation of nodules on the roots of Astragalus sinicus (3). A. lipoferum represents plant-growth-promoting rhizobacteria which colonize the root surface and are not able to penetrate root cells. They live in association with roots of grasses, cereals, and other monocotyledonous plants (4, 5).
Lipopolysaccharide, as an integral component of the cell walls of Gram-negative bacteria, plays an essential role in the proper development of symbiotic relationships (6). LPS, together with Omp proteins, is responsible for the asymmetric structure and semi-permeability of outer membranes. This is important for the appropriate morphogenesis and functionality of bacteroids, endosymbiotic forms of rhizobia which perform nitrogen fixation (7). LPS may play a role in the protection of rhizobia against plant defense response mechanisms. Suppression of systemic acquired resistance or hypersensitivity reaction has been shown during infection of plant tissues by microsymbionts (8–10).
Most pathogenic bacteria possess LPSs displaying endotoxic activity against host organisms. Lipid A, the part of LPSs that anchors the whole macromolecule in the outer membrane, is the centre of endotoxicity. The fine structure of enterobacterial lipid A has been identified as a glycolipid comprised of a β-(1,6)-linked glucosaminyl disaccharide substituted by two phosphate groups at positions C-1 and C-4 and six fatty acid residues with two acyloxyacyl moieties with a distinct location (Fig. 1) (11, 15, 16).
The activity of lipid A in LPSs is a result of its ability to recognize TLR4 on the surface of macrophages and endothelial cells. TLR4, acting in association with MD-2, recognizes LPS, which is extracted from the bacterial membrane and transferred to the TLR4-MD-2 complex by two accessory proteins: LPS binding protein and cluster of differentiation 14 (17,18). Activation of TLR4 receptors initiates a signaling cascade, resulting in the biosynthesis by macrophage cells of diverse mediators of inflammation (TNF, IL-1β or IL-6) (11). In the case of excessive release of cytokines, either clearing of local infection or a septic shock reaction may take place. It has been proved that the presence of phosphate groups and two acyloxyacyl moieties at distinct positions is needed for the activation of TLR4 receptors followed by the triggering of an endotoxin response in human immune cells (16, 19). Lipids A, which are significantly different from enterobacterial lipid A, are usually weakly toxic or nontoxic. This is the case with lipids A isolated from the LPSs of R. leguminosarum and R. etli (20), R. Sin-1 (21), and M. loti (22). The backbone of rhizobial lipid A is composed either of GlcpN or GlcpN3N disaccharide. Lipid A containing GlcpN can be modified by oxidation of the reducing GlcpN to 2-aminogluconate, as has been found in the LPSs of some Rhizobium species. The backbone may be substituted by phosphate, uronic acids, or other components, and is linked to an oligosaccharide core through a ketosidic bond formed by O-6 of the distal amino sugar and 3-deoxy-d-manno-oct-2-ulosonic acid residue (7). The amino groups of GlcpN3N and GlcpN, and the C-3 position of GlcpN are substituted by 3-hydroxy fatty acids. The hydroxyl groups may be further acylated either by nonpolar or (ω-1)-hydroxylated fatty acids, forming acyloxyacyl moieties (13, 14, 23–25). A comparison of the detailed structure of some rhizobial lipids A and the enterobacterial endotoxin shows that rhizobial lipids A are unusual. According to Urbanik-Sypniewska et al. (22), Vandenplas et al. (21) and Tsukushi et al. (26) some Sinorhizobium and Mesorhizobium strains possess varied endotoxic activity. Here, we report an investigation of the toxicity of lipopolysaccharides containing lipids A with unusual structures (see: 12–14).
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- MATERIALS AND METHODS
Minute amounts of LPS released from the surface of enteric bacteria are an early signal of infection for animal immune systems. A majority of host cells recognize traces of an endotoxin through the CD14-MD2-TLR4 protein complex. On the other hand, appearance of LPSs originating from non-enterobacterial species does not trigger a massive response from the host innate immune system (16, 37). All rhizobial LPSs have lipids A with unusual structures. Features which place these lipids A in the atypical group include the presence of very long chain fatty acids hydroxylated at penultimate positions (i.e. 27-octacosanoic acid); partial or complete absence of phosphate residues, which are replaced by uronic acid or neutral sugars; or proximal backbone amino sugar which has been oxidized to 2-aminogluconate (38).
All rhizobial lipopolysaccharides (lipids A) studied till now, with the single exception of S. meliloti (26), exhibit low endotoxic activity. Most experiments concerning the biological properties of these LPSs have been carried out on animal (mouse) models or using murine spleen leukocytes, monocytes, or a mouse leukemic monocyte macrophage cell line (RAW 264.7) (22, 26, 39). The biological properties of the LPS isolated from Sinorhizobium Sin-1 are the only ones to have been tested on a human monocytic cell line (Mono Mac 6) (21). However, in most cases, the responses of the murine immune system have been similar to, or identical with, those of the human one.
The biological activity of the LPSs examined in the present paper, measured as their ability to induce production of the cytokines TNF, IL-1β, and IL-6, and release of NO from human myelomonocytic cells (THP-1), demonstrates that the LPSs from the five Bradyrhizobium strains and from M. huakuii, and A. lipoferum exhibit significantly less endotoxic potency than Salmonella LPS. Gelation of LAL occurred at an LPS concentration of 0.1 μg/mL for B. japonicum and B. yuanmingense LPSs, and of 0.01 μg/mL in the case of B. elkanii, Bradyrhizobium sp. (Lupinus), and B. liaoningense. These results indicate that Bradyrhizobium LPSs are 1000–10,000 times weaker endotoxins than are enterobacterial LPS. For M. huakuii and A. lipoferum LPSs, gelation was observed at 0.1 ng/mL, which indicates that these endotoxins are 10 times weaker than the standard LPSs. Thus, our studies lead to the conclusion that all the examined LPSs are weak endotoxins and probably have low lethality for animals (22).
The differences between the examined strains and the standard endotoxin in biological activities of the LPS preparations were reflected in differences in the structure of lipid A, the centre of the endotoxic properties of the whole LPS molecule. The relationship between lipid A structure and its biological activity has been extensively studied, and the factors regulating the immunological activity of LPS identified. Among them, phosphate residues and the number, type, and distribution of fatty acids in lipid A are the most important (40). For proinflammatory activity, an enterobacterial lipid A that contains six fatty acids, of which two nonpolar ones are asymmetrically located creating two acyloxyacyl moieties, is required. Lipid A deprived of one fatty acid residue is about 100-fold less toxic, whereas lipid A analogues carrying only four primary fatty acids completely lack agonistic activity (16,41).
M. huakuii produces a naturally heterogenic lipid A, in particular due to the occurrence of hexa-acyl, penta-acyl, and tetra-acyl subspecies (13). The monophosphorylated subfraction of this lipid A occurs mainly as penta-acyl and hexa-acyl, containing, apart from 27-hydroxyoctacosanoic fatty acid, one eicosanoic moiety. The unphosphorylated subfraction of the lipid A is represented mainly as the hexa-acyl fraction. Thus, the presence of a large proportion of lipid A molecules with a lower degree of acylation might be a strong factor in the reduced biological activity of this LPS preparation. In addition, the presence of an unusual, very long chain hydroxylated fatty acyl (27-hydroxyoctacosanoic), which is typical of rhizobial lipids A, might affect toxicity, possibly by handicapping accommodation in the active site of the MD-2 receptor. The impaired toxicity of mesorhizobial lipid A may also result from reduced substitution by the ester-linked phosphate residue (50% of total). The C-1 position of the reducing end of the backbone in this lipid A is occupied by a galacturonic acid unit. The presence of two phosphate groups (at positions C-1 and C-4) in the lipid A greatly affects the endotoxic activity of enterobacterial LPS (40, 42). Removal of one of the phosphate groups reduces the biological activity of the enterobacterial endotoxin almost 100-fold, and monophosphoryl lipid A is a weak activator of the human innate immune response. Furthermore, the deletion of phosphate from the C-1 position in Salmonella Minnesota not only weakens the affinity of the ligand but also induces a structural rearrangement of the TLR4-MD-2-adaptor multimer receptor (16). Our results are also in agreement with the findings by Tsukushi et al. (26) and Urbanik-Sypniewska et al. (22), whose studies of the endotoxic properties of M. loti lipopolysaccharides have shown that LPSs from bacteria belonging to the genus Mesorhizobium are very weak endotoxins.
As has been described recently, A. lipoferum lipid A is completely lacking in phosphate, but contains galacturonic acid linked to the diglucosamine backbone at position C-1. This lipid A is heterogeneous in relation to the acylation pattern (12). Among the pool of lipid A molecules, at least three subfractions have been identified (penta-, tetra-, and tri-acylated lipids A). A. lipoferum lipid A does not contain any very long chain fatty acids. Thus, it seems that a lack of phosphate and a low degree of acylation play crucial roles in reduction of the toxicity of this lipid A.
The structure of lipid A isolated from B. elkanii LPS has been described in detail by Komaniecka et al. (14). This lipid A is completely lacking in any negatively charged residues. The GlcpN3N disaccharide backbone is further substituted by three mannopyranose residues, forming a pentasaccharide. Although B. elkanii lipid A is homogenous in the number of fatty acids and contains six acyl residues, two of them are unusual, being very long (ω-1)-hydroxylated secondary fatty acids, with a chain length ranging from 26 to 33 carbon atoms (14). Our current data suggest that the structure of B. japonicum lipid A is similar to that published for B. elkanii (unpublished data). Thus, it seems that bradyrhizobial lipids A are unusual high-molecular-mass molecules with weak endotoxic activity because of the presence of a large hydrophobic part, which probably blocks the active site of the TLR4 receptor and prevents it from forming the TLR4-MD-2-LPS complex.