Endotoxic properties of lipid A from Comamonas testosteroni

Ken-ichi Tanamoto1, Takatoshi Iida1, Yuji Haishima1 and Satoko Azumi1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The lipid A from Comamonas testosteroni has been isolated and its complete chemical structure determined [Iida, T., Haishima, Y., Tanaka, A., Nishijima, K., Saito, S. & Tanamoto, K. (1996). Eur J Biochem 237, 468–475]. In this work, the relationship between its chemical structure and biological activity was studied. The lipid A was highly homogeneous chemically and was characterized by the relatively short chain length (C10) of the 3-hydroxy fatty acid components directly bound to the glucosamine disaccharide backbone by either amide or ester linkages. The lipid A exhibited endotoxic activity in all of the assay systems tested (mitogenicity in mouse spleen cells; induction of tumour necrosis factor alpha release from both mouse peritoneal macrophages and mouse macrophage-like cell line J774-1, as well as from the human monocytic cell line THP-1; induction of nitric oxide release from J774-1 cells; Limulus gelation activity and lethal toxicity in galactosamine-sensitized mice) to the same extent as did ‘Salmonella minnesota’ lipid A or Escherichia coli LPS used as controls. The strong endotoxic activity of the C. testosteroni lipid A indicates that the composition of 3-hydroxydecanoic acid is not responsible for the low endotoxicity of the lipid A observed in members of the genus Rhodopseudomonas, as has previously been suggested. Furthermore, both the lack of a second acylation of the 3-hydroxy fatty acid attached at the 3' position, and the substitution of the hydroxyl group of the 3-hydroxy fatty acid attached at position 2, do not affect the manifestation of endotoxic activity or species specificity.

Keywords: lipopolysaccharide, LPS, biological activity of lipid A, endotoxin

Abbreviations: FCS, fetal calf serum; LAL, Limulus amoebocyte lysate; PMA, phorbol myristate acetate; TNF-{alpha}, tumour necrosis factor alpha


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lipid A is now widely accepted as the biologically active centre of bacterial endotoxin (Lüderitz et al., 1982 ; Homma et al., 1985 ). Although numerous data have been accumulated regarding the relationship between the chemical structure and biological activity of lipid A, many problems still remain unsolved. Given the fact that most naturally occurring lipid As from various Gram-negative bacteria express similar endotoxic features despite their chemical diversity, the chemical structure required for activity does not seem to be very specific. On the other hand, several lipid A species exhibit dramatically low, or no endotoxicity despite the similarity of their chemical structure to the active form (Galanos et al., 1971 ; Qureshi et al., 1991 ; Strittmatter et al., 1983 ; Omar et al., 1983 ; Tanamoto et al., 1997b ). These findings indicate that the biological activity of lipid A is controlled by specific structural factors, which are still undefined. In addition, we cannot simply distinguish the active from the inactive structure of lipid A since responses can vary between assay systems. Some lipid As or lipid A analogues exhibit strong typical endotoxic activity without showing any activity in the other assay systems (Tanamoto, 1995 ; Troelstra et al., 1997 ). Cells from different species express diverse responses against some lipid A structures. This was proven by lipid A precursor and Salmonella lipid A, both of which are strong activators in almost all endotoxin activities but are completely inactive in human cells (Golenbock et al., 1991 ; Tanamoto & Azumi, 2000 ). Furthermore, endotoxin-non-responsive mice were found to respond to a specific LPS (Tanamoto et al., 1997a ; Tanamoto, 1999 ). Recently, using chemically synthesized pure material, we also proved that by a one-point chemical modification the biologically active lipid A changed to a nontoxic and antagonistic structure (Tanamoto, 1998 ). These facts indicate that individual cells (the receptor molecules) discriminate between slight differences in chemical structures of lipid A. Taking all of this into consideration, it is evident that the study of the chemical structure/activity relationship is of great importance for the exact understanding of the real action of endotoxin, as well as for the development of potential antagonists for the treatment of endotoxic diseases.

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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Fig. 1. Proposed chemical structure of C. testosteroni lipid A. The molecule consists of 1,6-ß-linked glucosamine disaccharide which carries (R)-3-O-(hexadecanoyl)-decanoic acid, (R)-3-hydroxydecanoic acid, (R)-3-O-(dodecanoyl)-decanoic acid and (R)-3-hydroxydecanoic acid at positions 2, 3, 2' and 3', respectively, and phosphate groups at positions 1 and 4'.

 
Besides these interesting structural features, the C. testosteroni lipid A is highly homogeneous compared to other naturally produced lipid As, as was shown by liquid secondary ion-MS (Iida et al., 1996 ).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Materials.
Recombinant tumour necrosis factor alpha (TNF-{alpha}) standards and rabbit polyclonal antisera against murine TNF-{alpha} were obtained from Asahi Kasei Kogyo. THP-1 and J774-1 cell lines were obtained from the Japanese Cancer Research Resources Bank (JCRB). Rabbit IgG was obtained from Zymed Laboratories. RPMI 1640 medium with glutamine and Iscove’s modified Dulbecco’s medium were the products of Gibco Laboratories. Phorbol myristate acetate (PMA), 1,25-dihydroxy vitamin D3 and D-galactosamine were purchased from Sigma. Quantitative Limulus assay reagent (Endospecy) was obtained from Seikagaku Kogyo. Pyrogen-free water was a product of Hikari Seiyaku. LPS from ‘Salmonella minnesota R595 and Escherichia coli 03K2a2b:H2 was extracted by the aqueous phenol method (Galanos et al., 1969 ). Lipid A was obtained as an insoluble substance after 1% acetic acid treatment of LPS at 100 °C for 90 min (Galanos et al., 1971 ).

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 10–20-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 (6–10 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 Iscove’s modified Dulbecco’s 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 Iscove’s 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-{alpha} 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 Iscove’s medium (Tanamoto, 1994 ). One millilitre aliquots of a cell suspension adjusted to 2x106 cells ml-1 in Iscove’s 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 Iscove’s medium was added to each well and the cells were incubated with the test sample for 6 h.

Induction of TNF-{alpha} 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-{alpha} and NO induction, respectively.

Induction of TNF-{alpha} 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-{alpha} 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-{alpha}. The TNF-{alpha} 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 3–5 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).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mitogenicity of C. testosteroni lipid A
The mitogenic activities of C. testosteroni lipid A were tested on murine spleen cells of LPS-responsive BALB/c mice by measuring the c.p.m. of [3H]thymidine incorporated into the cells. As shown in Fig. 2, C. testosteroni lipid A exhibited comparable mitogenicity on splenocytes of BALB/c mice to that of lipid A from ‘S. minnesota’ R595 used as a control. A significantly higher activity was induced by LPS of C. testosteroni. The background count with no lipid A was 666±19 c.p.m. (mean±SD in triplicate wells).



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Fig. 2. Mitogenic responses of murine spleen cells to C. testosteroni LPS and lipid A. Spleen cells from BALB/c mice were suspended in serum-free Iscove’s medium at 4x106 cells ml-1; 200 µl aliquots of the suspension were plated in 96-well tissue culture dishes and reciprocally diluted mitogen in 10 µl water was added. After culturing the cells for 48 h, [3H]thymidine (0·2 µCi per well) was added. After further culture for 24 h, cells were harvested and the radioactivity incorporated was measured. The results are expressed as mean±SD c.p.m. in triplicate experiments. The background count with no lipid A was 669±19 c.p.m. {bullet}, ‘S. minnesota’ R595 lipid A; {square}, C. testosteroni LPS; {blacksquare}, C. testosteroni lipid A.

 
Induction of TNF-{alpha} release by C. testosteroni lipid A from mouse peritoneal macrophages
TNF-{alpha} released from mouse peritoneal macrophages into the medium after stimulation with C. testosteroni LPS and lipid A was measured. As shown in Fig. 3, the cells from BALB/c mice started to secrete TNF-{alpha} at a concentration of 0·1 ng ml-1 after stimulation by the lipid A. The induction of TNF-{alpha} release from macrophages by the lipid A increased dose-dependently, and maximum TNF-{alpha} production (15 ng ml-1) was observed at the highest concentration tested, 10 µg ml-1. The activity was almost the same as that of ‘S. minnesota’ R595 lipid A in regard to both the minimum stimulation dose needed to produce TNF-{alpha} and the maximum TNF-{alpha} production. A relatively higher activity was induced by LPS of C. testosteroni. In order to determine whether the cytotoxic activity against L929 cells was due to TNF-{alpha}, supernatants from a macrophage culture were incubated for 12 h with polyclonal rabbit antiserum to TNF-{alpha}, with nonspecific IgG used as the control. The polyclonal antibody to TNF-{alpha} completely abolished the cytotoxicity of the supernatants in all the samples tested (data not shown).



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Fig. 3. Induction of TNF-{alpha} release from murine peritoneal macrophages by C. testosteroni LPS and lipid A. Peritoneal macrophages from C3H/HeN mice were used for TNF-{alpha}-inducing assays. Macrophages (2x106 ml-1) were incubated in serum-free Iscove’s medium with various concentrations of LPS or lipid A. After 6 h incubation at 37 °C, the supernatants were examined for TNF-{alpha}. The results are expressed as means±SD of triplicate wells. {bullet}, ‘S. minnesota’ R595 lipid A; {square}, C. testosteroni LPS; {blacksquare}, C. testosteroni lipid A.

 
Induction of TNF-{alpha} and NO release by C. testosteroni lipid A from J774-1 cells
The ability of C. testosteroni lipid A as well as LPS to induce TNF-{alpha} and NO from mouse macrophage-like J774-1 cells was compared with that of ‘S. minnesota’ R595 lipid A used as a control. The cells are very sensitive to stimulation by all of these preparations. Significant production of both TNF-{alpha} and NO started at a concentration of 1 ng ml-1 (Fig. 4a) and 10 ng ml-1 (Fig. 4b), respectively, and increased dose-dependently. No significant difference was observed between these preparations in the stimulation of the cells.



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Fig. 4. Induction of TNF-{alpha} and NO release from J774-1 cells by C. testosteroni LPS and lipid A. J774-1 cells (1x106 cells ml-1 in each well of 24-well dishes) were incubated in RPMI 1640 medium supplemented with 10% FCS with the stimulant. After 4 and 72 h of incubation at 37 °C, the supernatants were examined for TNF-{alpha} (a) and NO (b), respectively. The results are expressed as means±SD of triplicate wells. {bullet}, ‘S. minnesota’ R595 lipid A; {square}, C. testosteroni LPS; {blacksquare}, C. testosteroni lipid A.

 
Induction of TNF-{alpha} release by C. testosteroni lipid A from THP-1 cells
It is suggested that human cells respond to lipid A in a different way from murine cells, as was seen in the response to lipid A precursor and ‘S. minnesota’ type lipid A. To determine the ability of C. testosteroni lipid A to activate human cells, human monocyte-macrophage cell line THP-1 was examined for TNF-{alpha} production in response to C. testosteroni lipid A using E. coli LPS and ‘S. minnesota’ R595 lipid A as a positive and negative control, respectively. As shown in Fig. 5, the cells were stimulated by E. coli LPS at a concentration of 10 ng ml-1 and produced 0·5 ng TNF-{alpha}ml-1, but were not stimulated by ‘S. minnesota’ lipid A at all, even at a concentration of 10 µg ml-1. The cells produced a comparable amount of TNF-{alpha} in the medium after stimulation with the same concentration of C. testosteroni lipid A to that after stimulation by E. coli LPS.



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Fig. 5. Induction of TNF-{alpha} release from human THP-1 cells by C. testosteroni LPS and lipid A. Human THP-1 cells (2x105 cells ml-1 in each well) were incubated with 100 ng ml-1 PMA and 0·1 µM 1,25-dihydroxyvitamin D3 for 72 h in RPMI medium containing 10% FCS at 37 °C. After an additional 24 h incubation with 10 µl test sample, the supernatants were assayed for TNF-{alpha}. Values represent the mean±SD concentration of TNF-{alpha} from triplicate experiments. {triangleup}, E. coli LPS;{bullet}, ‘S. minnesota’ R595 lipid A; {blacksquare}, C. testosteroni lipid A.

 
Chromogenic LAL test of C. testosteroni lipid A
LAL gelation activity of C. testosteroni lipid A was assayed colorimetrically by measuring the absorbance of p-nitroaniline released from a synthetic substrate. As shown in Fig. 6, C. testosteroni lipid A activated the cascade of the clotting system of the horseshoe crab at a concentration of 6·25 pg ml-1, and linear dose dependency was obtained up to 12·5 pg ml-1. The activity was higher than the ‘S. minnesota R595 lipid A control. When the dose of each sample required to obtain an absorbance of 0·2 is compared, C. testosteroni lipid A (8·0 pg ml-1) exhibited 2·4-fold higher activity than that of ‘S. minnesota’ lipid A (19·0 pg ml-1).



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Fig. 6. Limulus gelation activity of C. testosteroni LPS and lipid A. Limulus amoebocyte lysate was incubated with test samples for 30 min and the absorbance of the chromogen released was then measured. {bullet}, ‘S. minnesota’ R595 lipid A; {blacksquare}, C. testosteroni lipid A.

 
Lethal toxicity in galactosamine-sensitized mice
Lethal toxicity was assayed using galactosamine-sensitized C57BL/6 mice. The results are shown in Table 1. In this test system, C. testosteroni lipid A showed 100% death at a dose of 0·01 µg per mouse, and 17% death at 0·001 µg per mouse. S. minnesota’ R595 lipid A used as a control was 83% lethal at a dose of 0·01 µg per mouse and 29% at 0·001 µg per mouse. Thus, C. testosteroni lipid A exhibited comparable toxicity to that of ‘S. minnesota lipid A.


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Table 1. Lethal toxicity of C. testosteroni LPS and lipid A for galactosamine-sensitized C57BL/6 mice

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present study, we examined the biological activity of the free form of lipid A from C. testosteroni, which was recently isolated and characterized chemically (Iida et al., 1996 ).

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-{alpha} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Ministry of Education, Science and Culture (08670329 to K.T.).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Galanos, C., Lüderitz, O. & Westphal, O. (1969). A new method for the extraction of R lipopolysaccharides. Eur J Biochem 9, 245-247.[Medline]

Galanos, C., Lüderitz, O. & Westphal, O. (1971). Preparations and properties of antisera against the lipid A component of bacterial lipopolysaccharides. Eur J Biochem 24, 116-122.[Medline]

Galanos, C., Freudenberg, M. A. & Reutter, W. (1979). Galactosamine-induced sensitization to the lethal effects of endotoxin. Proc Natl Acad Sci U S A 76, 5939-5943.[Abstract]

Galanos, C., Lüderiz, O., Freudenberg, M., Brade, L., Schade, U., Rietschel, E. Th., Kusumoto, S. & Shiba, T. (1986). Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A. Eur J Biochem 160, 55-59.[Abstract]

Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K. & Raetz, C. R. H. (1991). Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes. J Biol Chem 266, 19490-19498.[Abstract/Free Full Text]

Green, L. C., Wanger, D. A., Glogowsky, J., Sipper, P. L., Wishnok, J. S. & Tannenbaum, S. R. (1982). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126, 131-138.[Medline]

Homma, J. Y., Matsuura, M., Kanegasaki, S. & 11 other authors (1985). Structural requirements of lipid A responsible for the functions: a study with chemically synthesized lipid A and its analogues. J Biochem 98, 395–406.[Abstract]

Iida, T., Haishima, Y., Tanaka, A. & Tanamoto, K. (1996). Chemical structure of lipid A isolated from Comamonas testosteroni lipopolysaccharide. Eur J Biochem 237, 468-475.[Abstract]

Kanegasaki, S., Tanamoto, K., Yasuda, T. & 10 other authors (1986). Structure-activity relationship of lipid A: comparison of biological activities of natural and synthetic lipid A’s with different fatty acid compositions. J Biochem 99, 1203–1210.[Abstract]

Kitchens, R. L., Ulevitch, T. R. J. & Munford, R. S. (1992). Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway. J Exp Med 176, 485-494.[Abstract]

Kotani, S., Takada, H., Takahashi, I. & 7 other authors (1986). Low endotoxic activities of synthetic Salmonella-type lipid A with an additional acyloxyacyl group on the 2-amino group of beta (1–6) glucosamine disaccharide 1–4'-bisphosphate. Infect Immun 52, 872–884.[Medline]

Kulshin, V. A., Zaringer, U., Lindner, B., Lager, K., Dmitriev, B. A. & Rietschel, E. Th. (1991). Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides. Eur J Biochem 198, 697-704.[Abstract]

Lüderitz, O., Freudenberg, M., Galanos, C., Lehmann, V., Rietschel, E. Th. & Shaw, D. H. (1982). Lipopolysaccharides of gram-negative bacteria. Curr Top Membr Transp 17, 79-151.

Mayer, H. & Weckesser, J. (1984). ‘Unusual’ lipid A’s: structures, taxonomical relevance and potential value for endotoxin research. In Handbook of Endotoxins , pp. 221-241. Edited by E. Th. Rietschel. Amsterdam/New York/Oxford:Elsevier.

Ogawa, T. (1994). Immunobiological properties of chemically defined lipid A from lipopolysaccharide of Porphyromonas (Bacteroides) gingivalis. Eur J Biochem 219, 737-742.[Abstract]

Omar, A. S., Flammann, H. T., Borowiak, D. & Weckesser, J. (1983). Lipopolysaccharide of two strains of the phototrophic bacterium Rhodopseudomonas capsulata. Arch Microbiol 134, 212-216.[Medline]

Qureshi, N., Honovich, J. P., Hara, H., Cotter, R. J. & Takayama, K. (1991). Diphosphoryl lipid A obtained from the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides is an endotoxin antagonist in mice. Infect Immun 59, 441-444.[Medline]

Strittmatter, W., Weckesser, W., Salimath, P. V. & Galanos, C. (1983). Nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023. J Bacteriol 155, 153-158.[Medline]

Tanamoto, K. (1994). Induction of prostaglandin release from macrophages by bacterial endotoxin. Methods Enzymol 236, 31-41.[Medline]

Tanamoto, K. (1995). Dissociation of endotoxic activities in a chemically synthesized lipid A precursor after acetylation. Infect Immun 63, 690-692.[Abstract]

Tanamoto, K. (1998). Production of nontoxic lipid A by chemical modification and its antagonistic effect on LPS action. Prog Clin Biol Res 397, 269-280.[Medline]

Tanamoto, K. (1999). Induction of lethal shock and tolerance by Porphyromonas gingivalis lipopolysaccharide in D-galactosamine-sensitized C3H/HeJ mice. Infect Immun 67, 3399-3402.[Abstract/Free Full Text]

Tanamoto, K. & Azumi, S. (2000). Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of LPS action on human line cells. J Immunol 164, 3149-3156.[Abstract/Free Full Text]

Tanamoto, K., Galanos, C., Lüderitz, O., Kusumoto, S. & Shiba, T. (1984). Mitogenic activities of chemically synthesized lipid A analogues and suppression of mitogenicity of lipid A. Infect Immun 44, 427-433.[Medline]

Tanamoto, K., Azumi, S., Haishima, Y., Kumada, H. & Umemoto, T. (1997a). The lipid A moiety of Porphyromonas gingivalis LPS specifically mediates the activation of C3H/HeJ mice. J Immunol 158, 4430-4436.[Abstract]

Tanamoto, K., Azumi, S., Haishima, Y., Kumada, H. & Umemoto, T. (1997b). Endotoxic properties of free lipid A from Porphyromonas gingivalis. Microbiology 143, 63-71.[Abstract]

Tharanathan, R. N., Weckesser, J., Strittmatter, W. & Mayer, H. (1983). Structural studies on the D-arabinose containing lipid A from Rhodospirillum tenue 2761. Eur J Biochem 136, 175-180.[Abstract]

Troelstra, A., Antal-Szalmas, P., de Graff-Miltenburg, L. A. M., Weersink, A. J. L., Verhoef, J., Van Kessel, K. P. M. & Van Strijp, J. A. G. (1997). Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes. Infect Immun 65, 2272-2277.[Abstract]

Westphal, O. & Jann, K. (1965). Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr Chem 5, 83-91.

Westphal, O., Lüderitz, O. & Bister, F. (1952). Über die Extraktion von Bakterien mit Phenol/Wasser. Z Naturforsch 76, 148-155.

Received 7 July 2000; revised 8 December 2000; accepted 10 January 2001.



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