Division of Microbiology, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan1
Author for correspondence: Ken-ichi Tanamoto. Tel: +81 3 3700 9484. Fax: +81 3 3700 9484. e-mail: tanamoto{at}nihs.go.jp
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ABSTRACT |
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Keywords: lipopolysaccharide, LPS, biological activity of lipid A, endotoxin
Abbreviations: FCS, fetal calf serum; LAL, Limulus amoebocyte lysate; PMA, phorbol myristate acetate; TNF-, tumour necrosis factor alpha
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INTRODUCTION |
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We have determined the chemical structure of lipid A isolated from Comamonas testosteroni (Iida et al., 1996 ), which is an aerobic, Gram-negative rod and is known to grow on testosterone. The proposed chemical structure of the lipid A is shown in Fig. 1
. It is of interest because it contains a relatively short-chain (R)-3-hydroxydecanoic acid as a main acyl residue instead of (R)-3-hydroxytetradecanoic acid, which is the main constituent of enterobacterial lipid As. A short-chain fatty acid possibly plays a role in the low endotoxicity of the lipid A from Rhodopseudomonas species. In addition, the C. testosteroni lipid A has the following two characteristics concerning the position of substitution of fatty acids. First is the lack of a second acylation of the 3-hydroxy fatty acid attached at the 3' position. Second is the substitution of the hydroxyl group of the 3-hydroxy fatty acid attached at position 2. Both chemical characteristics are also considered to control the biological activity of lipid A.
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In the present study, the endotoxic activity of the chemically well-defined lipid A isolated from C. testosteroni LPS was examined in the expectation that this would provide further information on the above points.
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METHODS |
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Preparation of C. testosteroni LPS and lipid A.
Preparation of lipid A from C. testosteroni was described by Iida et al. (1996) . The LPS was extracted by the hot phenol/water method (Westphal et al., 1952
), and purified by enzyme treatments (Westphal & Jann, 1965
) and repeated ultracentrifugation (105000 g, 3 h, 6 times). Lipid A was obtained from the LPS by hydrolysis with 1% acetic acid at 100 °C for 1·5 h and was purified by gel permeation chromatography using a column (3 cmx100 cm) of Sephadex LH-20 (Pharmacia) with chloroform/methanol (1:1, v/v) as the eluent (with a flow rate of 24 ml h-1).
The proposed chemical structure is shown in Fig. 1. The lipid A was solubilized by converting it into a triethylamine form and this was used for biological assay.
Limulus amoebocyte lysate (LAL) gelation assay.
Activation of the proclotting enzyme of the horseshoe crab Limulus was estimated colorimetrically by measuring the absorbance of p-nitroaniline released from a synthetic substrate in a quantitative assay. The assay was performed in 96-well Costar flat plates at 37 °C for 30 min, and the absorbance of the chromogen was measured at 405 nm using a microplate reader (Thermo Max; Molecular Devices). Pyrogen-free water was used to dilute the test samples.
Lethal toxicity test.
The lethality test was performed according to the method described by Galanos et al. (1979) using 1020-week-old C57BL/6 female mice (Nihon SLC). Test samples in 0·1 ml pyrogen-free water were injected intravenously immediately after intraperitoneal administration of 12 mg D-galactosamine in 0·5 ml pyrogen-free PBS (l-1: 8 g sodium chloride, 0·2 g potassium chloride, 1·15 g anhydrous disodium phosphate and 0·2 g anhydrous monopotassium phosphate; Nissui Pharmaceutical).
Mitogenic activity (Tanamoto et al., 1984 ).
BALB/c female mice (610 weeks old; Nihon SLC) were killed by cervical dislocation. Spleen cells were isolated, mashed gently, passed through a wire grid and suspended in serum-free Iscoves modified Dulbeccos medium containing L-glutamine and HEPES buffer (pH 7·4) supplemented with penicillin (100 units ml-1) and streptomycin (100 µg ml-1). The cells were washed with the medium three times and adjusted to 4x106 cells ml-1 in Iscoves medium. They were cultured in 96-well microplates (Corning) containing 200 µl cell suspension in each well after the addition of test samples for 48 h at 37 °C in a humidified environment in the presence of 5% (v/v) CO2. [3H]thymidine [0·2 µCi (7·4 kBq) per culture; Amersham] was added, and the mixture incubated further for 24 h. The cells were harvested on glass fibre filters, and the radioactivity incorporated into the cells was measured in toluene-based scintillation fluid (5 ml) with a scintillation counter (LSC-700 instrument; Aloka). Data are expressed as mean±SD c.p.m. of triplicate determinations.
Induction of TNF- release from mouse peritoneal macrophages.
The mice were killed by cervical dislocation. Mouse peritoneal macrophages were obtained by washing the peritoneal cavity with 5 ml Iscoves medium (Tanamoto, 1994 ). One millilitre aliquots of a cell suspension adjusted to 2x106 cells ml-1 in Iscoves medium were cultured in 24-well Costar plates at 37 °C with 5% CO2 for 3 h. Macrophages were allowed to adhere to the plate. After the cells had been washed three times with PBS, 1 ml of Iscoves medium was added to each well and the cells were incubated with the test sample for 6 h.
Induction of TNF- and NO release from macrophage-like J774-1 cells.
J774-1 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 50 µM 2-mercaptoethanol, 5 mM HEPES, penicillin (100 units ml-1) and streptomycin (100 µg ml-1) in a 5% CO2 atmosphere at 37 °C. The cells were harvested by scraping with a cell scraper (Costar) and suspended in fresh medium. The cells (1x106 cells ml-1 in each well of 24-well dishes) were allowed to adhere to the plastic for 3 h at 37 °C, washed twice with medium and incubated for an additional 4 and 72 h with the stimulant for TNF- and NO induction, respectively.
Induction of TNF- release from THP-1 cells.
THP-1 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µM 2-mercaptoethanol, 5 mM HEPES, penicillin (100 units ml-1) and streptomycin (100 µg ml-1) in a 5% CO2 atmosphere at 37 °C. The cells (2x105 cells ml-1 in each well of 24-well dishes) were prepared for the experiments by adding 100 ng PMA ml-1, which had been stored as a 1 mg ml-1 solution in DMSO and diluted with the medium immediately prior to use (Golenbock et al., 1991 ), and 0·1 µM 1,25-dihydroxy vitamin D3 to cell suspensions in RPMI 1640 medium with 10% FCS (Kitchens et al., 1992
). The cell suspensions were allowed to differentiate and adhere to the plastic for 72 h at 37 °C. After washing twice with medium, the cells were incubated for an additional 24 h with the stimulant.
TNF- assay.
The supernatant of each culture obtained was transferred to a plastic tube, the cells were centrifuged at 600 g, and the supernatant was stored at -80 °C until used to determine TNF-. The TNF-
produced was measured by cytotoxicity assay against L929 murine fibroblast cells. L929 cells were grown in tissue culture flasks in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-mercaptoethanol, 5 mM HEPES, penicillin (100 units ml-1) and streptomycin (100 µg ml-1). Cells were detached with trypsin, washed and resuspended in medium at 4x105 cells ml-1; 100 µl aliquots were then plated in 96-well flat-bottomed plates (Corning). After incubation for 35 h at 37 °C in 5% CO2, 50 µl actinomycin D (4 µg ml-1) in RPMI medium was added to each well, followed by 50 µl of test sample (final volume: 200 µl per well). The results are expressed as means±SD of triplicate determinations.
NO assay.
NO produced in the supernatant was measured as a stable form of nitrite by using Griess reagent (Green et al., 1982
). Briefly, 100 µl quantities of test sample were mixed with the same volume of Griess reagent [1:1 (v/v); 0·1% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride in H2O/1% (w/v) sulfanilamide in 5% (v/v) H2PO4] in a 96-well plate, and the absorbance was then read at 550 nm with a microplate reader (Molecular Devices).
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RESULTS |
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DISCUSSION |
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Usually, the natural lipid As obtained from LPS by acid hydrolysis are very heterogeneous in their number of fatty acids and phosphates. Although slight heterogeneity was also recognized in the lipid A of C. testosteroni used in the present study with regard to the degree of phosphorylation and acylation, it was highly homogeneous as a natural product as was shown by the data of liquid secondary ion-MS (Iida et al., 1996 ). Data on structure/activity relationships obtained from natural lipid A cause confusion owing to heterogeneity, and the biological study of this highly homogeneous lipid A is expected to give more reliable information.
The lipid A from C. testosteroni has been shown to have several interesting structural characteristics. Although its backbone structure and phosphorylation pattern is identical to that of enterobacterial lipid As (Mayer & Weckesser, 1984 ), the fatty acid composition and distribution pattern of acyloxyacyl residues differs as follows. First, C. testosteroni lipid A contains a short-chain fatty acid, (R)-3-OH C10:0, as the main acyl residue, instead of (R)-3-hydroxytetradecanoic acid, a common constituent of enterobacterial lipid As. Second, acyloxyacyl residues are present at positions 2 and 2' of the lipid A backbone. Third, the hydroxyl group of the fatty acid attached to position 3' was unsubstituted, whereas it is esterified by a second acyl residue in enterobacterial lipid As. (R)-3-OH C10:0 has been found in lipid As of some other bacterial species, for example Rhodocyclus tenuis (Tharanathan et al., 1983
), Rhodobacter capsulata (Omar et al., 1983
), Rhodobacter sphaeroides (Strittmatter et al., 1983
) and some species of Pseudomonas (Kulshin et al., 1991
). Of these, Rb. capsulata and Rb. sphaeroides lipid As have been tested for their biological activity and were found to express no toxicity (Galanos et al., 1971
; Qureshi et al., 1991
; Strittmatter et al., 1983
; Omar et al., 1983
). It is therefore assumed that the short-chain fatty acids contribute to the low toxicity. The present study on the biological properties of the lipid A of C. testosteroni, however, demonstrated that, in comparison with S. minnesota R595 lipid A used as a control, it exhibits similar or even higher activity in all biological assays tested, including LAL gelation activity, lethal toxicity in galactosamine-sensitized mice, TNF-
induction activity towards murine peritoneal macrophages, J774-1 cells and human THP-1 cells, and mitogenicity towards murine spleen cells. These results indicate that the short-chain 3-hydroxy fatty acid linked directly to the lipid A backbone is not responsible for the low toxicity of the lipid A. Rather, the substitution of a relatively longer-chain fatty acid (C16) as acyloxyacyl residue and/or the lesser substitution of hydroxyl groups of 3-hydroxy fatty acids are responsible for the low endotoxicity of the lipid A. The lower activity of the lipid A possessing relatively longer fatty acids was suggested in S. minnesota-type lipid A using chemically synthesized material (Galanos et al., 1986
; Kanegasaki et al., 1986
; Kotani et al., 1986
), as well as lipid As from Porphyromonas gingivalis (Ogawa, 1994
; Tanamoto et al., 1997b
).
The results also revealed that second acylation of the 3-hydroxy fatty acid attached at the 3' position is not essential for manifestation of endotoxic activity, because the hydroxyl group of the acyl residue was not substituted in C. testosteroni lipid A. Since this position is usually substituted in most enterobacterial LPS, but was found to be free in non-toxic Rhodopseudomonas lipid A, this chemical characteristic has also been suspected to be the cause of its non-toxicity.
Furthermore, the results revealed that substitution of the hydroxyl group of the 3-hydroxy fatty acid attached to position 2 does not affect the manifestation of endotoxic activity. Recently, we have found that Salmonella-type heptaacylated lipid A is inactive in human cells, whereas E. coli type lipid A is equally active towards both mouse and human cells (Tanamoto & Azumi, 2000 ). The only chemical difference between the two lipid As is the substitution of the hydroxyl residue of the 3-hydroxy fatty acid attached to the position 2 for reducing glucosamine. Therefore, the lipid A from C. testosteroni is the ideal structure for assessing the role of the substitution of this position as well. The results indicate that the substitution of this position is not the reason for the species specificity. Taken together, these facts suggest that the species specificity, in addition to the structure/activity relationship, is not regulated by a single factor, but is more complex. Toll-like receptor 4 is now widely accepted to mediate recognition of LPS with the help of CD14 and LPS-binding protein. However, we still do not know the structural basis for how these molecules control the potency of LPS, in relation to the chemical structure of lipid A. This is an important point for the further understanding of the structure/activity relationship of LPS and remains to be investigated.
Thus, the present study provides important information on the relationship between the chemical structure and biological activity of lipid A.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 July 2000;
revised 8 December 2000;
accepted 10 January 2001.
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