By
From the * William Harvey Research Institute, St. Bartholomew's and the Royal London School of
Medicine and Dentistry, London EC1M 6BQ, United Kingdom; the Department of Pharmacology
and Pathophysiology, Utrecht Institute for Pharmaceutical Science, Utrecht University, 3584 CA
Utrecht, The Netherlands; and the § Department of Molecular Biology and Biotechnology, University of
Sheffield, Sheffield S10 2TN, United Kingdom
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Abstract |
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The incidence of septic shock caused by gram-positive bacteria has risen markedly in the last
few years. It is largely unclear how gram-positive bacteria (which do not contain endotoxin)
cause shock and multiple organ failure. We have discovered recently that two cell wall fragments of the pathogenic gram-positive bacterium Staphylococcus aureus, lipoteichoic acid (LTA)
and peptidoglycan (PepG), synergize to cause the induction of nitric oxide (NO) formation,
shock, and organ injury in the rat. We report here that a specific fragment of PepG, N-acetylglucosamine--[1
4]-N-acetylmuramyl-L-alanine-D-isoglutamine, is the moiety within the
PepG polymer responsible for the synergism with LTA (or the cytokine interferon
) to induce
NO formation in the murine macrophage cell line J774.2. However, this moiety is also present
in the PepG of the nonpathogenic bacterium Bacillus subtilis. We have discovered subsequently
that S. aureus LTA synergizes with PepG from either bacterium to cause enhanced NO formation, shock, and organ injury in the rat, whereas the LTA from B. subtilis does not synergize
with PepG of either bacterium. Thus, we propose that the structure of LTA determines the
ability of a particular bacterium to cause shock and multiple organ failure (pathogenicity), while
PepG acts to amplify any response induced by LTA.
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Introduction |
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Aresurgence of nosocomial gram-positive infections was first noticed in the early 1980s. Today, between one third and half of all cases of sepsis are caused by gram-positive organisms, and it is likely that the incidence of gram-positive sepsis will continue to rise and predominate in the years to come (1, 2). Gram-positive organisms do not contain LPS (or endotoxin), which is the cell wall component of gram-negative bacteria responsible for the initiation of gram-negative septic shock. However, gram-positive bacteria can cause septic shock and multiple organ failure without causing endotoxemia (3, 4), and endotoxin is not always found in the serum of patients with septic shock (1, 2). The cell wall of gram-positive bacteria contains lipoteichoic acid (LTA)1 and peptidoglycan (PepG), which themselves can activate leukocytes, stimulate the generation of proinflammatory cytokines, and hence, cause a moderate systemic inflammatory response syndrome (5).
The endogenous vasodilator autacoid nitric oxide (NO) is generated by three different isoforms of NO synthase (NOS), two of which are expressed constitutively (eNOS in endothelium, nNOS in brain), the third (iNOS) induced by endotoxin (LPS) or cytokines (9). There is now substantial evidence that an enhanced formation of NO by iNOS contributes to the circulatory failure (hypotension, and vascular hyporeactivity to vasoconstrictors) and possibly the organ injury associated with endotoxemia (10).
LTA is a macroamphiphile, equivalent to LPS in gram-negative bacteria, containing a substituted poly-glycerophosphate backbone attached to a glycolipid (for a review,
see reference 13). The glycolipid content in LTA resembles
the bacterial membrane composition, which usually varies
in a genus-specific manner (13, 14). LTA from Staphylococcus aureus can cause a moderate induction of iNOS (15)
which (in murine macrophages) requires the activation of
tyrosine kinases and NF-B (17). Although LTA from S. aureus can cause moderate hypotension in the rat, LTA
(unlike S. aureus itself) does not cause multiple organ failure
or death in this species (16). However, we have discovered
recently that LTA and PepG act in synergy to release TNF-
and IFN-
, to induce iNOS, and to cause shock and multiple organ failure in anesthetized rats (18, 19). The mechanism by which LTA and PepG act in synergy is not clear.
In addition, it is not known which of the structural components of PepG (or LTA) is essential for the observed synergism.
PepG is a large polymer that provides stress resistance and shape-determining properties to bacterial cell walls. This polymer contains long sugar chains of two alternating sugar derivatives, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), which are highly cross-linked by peptide subunits and bridges (see Fig. 1). The peptide subunit (or stem peptide) consists of alternating L- and D-amino acids, up to four or five in length, and is connected to the COOH group of NAM. Among different bacterial species, the structure of the sugar chains is highly preserved, while the composition of the peptide subunits varies.
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This study was designed to investigate which of the
structural elements of PepG are essential to synergize with
LTA to cause the induction of iNOS in macrophages, and
shock and multiple organ failure in vivo. Two distinct
gram-positive organisms were used: S. aureus, a bacterium
most commonly implicated in gram-positive sepsis (1), and
Bacillus subtilis, a nonpathogenic bacterium. We have identified a structural moiety present in the PepG polymer of
both S. aureus and B. subtilis that is essential to synergize with LTA or IFN- to induce iNOS in macrophages. Furthermore, we provide evidence that the origin of LTA but
not PepG determines whether the cell wall components
from a gram-positive organism synergize to induce iNOS
and multiple organ failure.
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Materials and Methods |
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Bacterial Cell Wall Components.
Cell walls from vegetative cells of B. subtilis and S. aureus were prepared as described previously (20). Secondary polymers (teichoic and teichiuronic acids) were removed from the PepG by treatment of cell walls (10 mg dry wt/ml) with 48% vol/vol hydrofluoric acid at 4°C for 24 h. The pure PepG was then washed six times, by centrifugation (14,000 g, 5 min) and resuspension in distilled water, until the pH was neutral before storage atCell Culture Experiments.
Murine macrophages (J774.2; The European Collection of Animal Cell Cultures, Salisbury, UK) were cultured in 96-well plates with DME (200 µl/well) containing 10% FCS (GIBCO BRL, Paisley, UK) and L-glutamine (4 mM). Rat aortic smooth muscle cells (RASMC, A10 cell line; The European Collection of Animal Cell Cultures) were cultured in 96-well plates with RPMI 1640 medium (200 µl/well) containing 10% FCS and L-glutamine (4 mM). The 96-well plates containing the cells were then incubated at 37°C in a humidified incubator until the cells reached confluence. Cultured cells were activated by the isolated bacterial cell wall components or fragments, and after 24 h, the accumulation of nitrite in the supernatant was measured as an indication of iNOS activity. Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) was added to the culture medium, and the difference in OD between 550 and 650 nm was measured. The nitrite concentrations were calculated using standard solutions of sodium nitrite prepared in culture medium (23). Cell viability was assessed by the mitochondrial reduction of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan (23). After removal of supernatant for nitrite measurements, cells were incubated with MTT (0.4 mg/ml) for 30 min at 37°C. Medium was removed by aspiration, cells were solubilized in DMSO, and the reduction of MTT was measured by spectrometry at 550 nm. Unless otherwise stated, there were no significant changes in the cell viability after various treatments of either macrophages or RASMC.Immunoblot (Western Blot) Analysis for iNOS.
Western blot analysis was used to determine the expression of iNOS protein. After exposure of J774.2 macrophages to the relevant cell wall components, cells were suspended in homogenization buffer comprised of Tris-HCl (50 mM), EDTA (10 mM), Triton X-100 (1% vol/ vol), and the protease inhibitors pepstatin A (50 µM), leupeptin (0.2 mM), and PMSF (1 mM). The homogenate was boiled for 5 min with gel loading buffer (Tris 20 mM, EDTA 2 mM, SDS 2% wt/vol, glycerol 20% vol/vol, 2-ME 10% vol/vol, and bromphenol blue 2 mg/ml, pH 6.8) in a ratio of 1:1 (vol/vol). Total protein equivalents for each sample were resolved on SDS/7.5% vol/ vol polyacrylamide gels and transferred to nitrocellulose. The membranes were incubated for 20 h at 4°C with a rabbit polyclonal antibody raised to murine macrophage iNOS (24). Bands were detected using a peroxidase-conjugated anti-rabbit IgG and 3,3'-diaminobenzidine or enhanced chemiluminescence (Amersham International, Little Chalfont, Buckinghamshire, UK). The expression of immunoreactive iNOS was quantified by scanning densitometry (imaging densitometer model GS-700; Bio-Rad Laboratories, Hemel Hempstead, Hertfordshire, UK).In Vivo Experiments.
Male Wistar rats (240-300 g; Glaxo Wellcome PLC, Greenford, Middlesex, UK) were anesthetized with thiopentone sodium (120 mg/kg, i.p.). The trachea was cannulated to facilitate respiration, and the rectal temperature was maintained at 37°C using a homeothermic blanket system (Harvard Apparatus Ltd., Edenbridge, Kent, UK). The right carotid artery was cannulated and connected to a pressure transducer (SensoNor 840; SensoNor A.S., Horten, Norway) for the measurement of mean arterial blood pressure and heart rate (MacLab 8; AD Instruments, Hastings, UK). The jugular vein was cannulated for the administration of compounds. Cardiovascular parameters were allowed to stabilize, and at time 0, animals received (a) S. aureus LTA (3 mg/ kg), (b) B. subtilis LTA (3 mg/kg), (c) B. subtilis PepG (1 mg/kg), (d) S. aureus LTA (3 mg/kg) and B. subtilis PepG (1 mg/kg), (e) B. subtilis LTA (3 mg/kg) and B. subtilis PepG (1 mg/kg), or (f) saline (0.3 ml 0.9% NaCl). Hemodynamic parameters were measured for 6 h. The pressor response to norepinephrine was assessed 15 min before and every hour after the administration of the bacterial cell wall components. At the end of the experiment at 6 h, a blood sample was obtained for the measurement of markers of organ failure. Serum concentrations of creatinine, urea (indicators of the development of renal dysfunction), and alanine aminotransferase (ALT, an indicator of the development of liver injury) (25, 26) were analyzed by a contract laboratory for veterinary clinical chemistry (Vetlab Services, West Sussex, UK). Lungs were collected and stored (Lung iNOS Activity Assay.
The activity of iNOS in lung homogenates was determined by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in the absence of free calcium. Frozen lungs were homogenized on ice in a buffer composed of Tris-HCl (50 mM), EDTA (0.1 mM), EGTA (0.1 mM), 2-ME (12 mM), and PMSF (1 mM), pH 7.4. Lung homogenates (~100 µg protein) were incubated in the presence of [3H]L-arginine (7.5 kBq per tube of 100 µl), L-arginine (10 µM), NADPH (1 mM), calmodulin (300 IU/ml), tetrahydrobiopterin (5 µM), L-valine (50 mM), and EDTA (1 mM) for 30 min at 25°C in Tris-buffer. Reactions were stopped by addition of 1 ml ice-cold Hepes buffer (pH 5.5) containing EGTA (2 mM) and EDTA (2 mM). After separation using Dowex 50W (sodium form) columns, the eluted [3H]L-citrulline activity was measured by scintillation counting. Experiments performed in the absence of NADPH determined the extent of [3H]L-citrulline formation independent of iNOS activity.Materials and Drug Solutions.
Unless otherwise stated, all compounds were obtained from Sigma Chemical Co. L-[2,3,4,5-3H]arginine hydrochloride was obtained from Amersham International, and tetrahydrobiopterin (6R-L-erythro-5,6,7,8-tetra-hydrobiopterin) was obtained from Schircks Laboratoire (Jona, Switzerland). Thiopentone sodium was obtained from Rhône Merieux Ltd. (Harlow, Essex, UK). The PepG fragments were prepared by Dr. S.J. Foster (University of Sheffield). S. aureus lytic enzyme (SALE) was obtained from Dr. L. De Graaf (University of Utrecht, Utrecht, The Netherlands). Murine iNOS antibody was a gift from Dr. Claire Bryant (William Harvey Research Institute). All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK), and care was taken to prevent endotoxin contamination. For cell culture experiments, all solutions of agents were sterilized by filtration through a filter (pore size 0.22 µm).Statistical Analysis.
All data are presented as means ± SE of n observations. For the cell culture experiments, at least three independent experiments were performed in triplicate. In anesthetized rats, the pressor response to norepinephrine was calculated as the area under the response and expressed as mmHg·min. Statistical analysis was performed either by one-way analysis of variance for multiple comparison or by Student's t test as appropriate. If the analysis of variance yielded significance (P <0.05), Dunnett's test for multiple comparisons with one control mean or Bonferroni's test for multiple comparison on selected single means was performed (27). ![]() |
Results |
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Incubation of J774.2 macrophages
with PepG from S. aureus (1-30 µg/ml) in the presence of
IFN- (10 IU/ml) resulted in the accumulation of nitrite
after 24 h (Fig. 2 A). PepG alone (up to 100 µg/ml) did
not cause an increase in nitrite accumulation (Fig. 2 A; 2.0 ± 0.1 M nitrite for 100 µg/ml PepG). The enhanced formation of nitrite caused by PepG (10 µg/ml) in the presence of IFN-
(10 IU/ml) was prevented, in a concentration-dependent manner, by the NOS inhibitors NG-methyl-
L-arginine (L-NMMA; IC50 = 80 µM) or aminoethyl-isothiourea (AE-ITU; IC50 = 15 µM) (Fig. 2 B). Western
blot analysis revealed an increase in the expression of an
immunoreactive iNOS protein of ~130 kD in J774 macrophages challenged with PepG (10 µg/ml) and IFN-
(10 IU/ml) for 24 h (Fig. 2 C). In contrast, neither PepG nor
IFN-
alone caused the expression of iNOS protein. Additionally, the expression of iNOS protein in cells treated
with LTA (0.1 µg/ml) and PepG (10 µg/ml) was greater
than the level of iNOS expression caused by these components alone (Fig. 2 C).
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To further elucidate the structural elements of PepG that
are essential to induce iNOS activity in the presence of
IFN-, PepG from S. aureus was digested for 1 h with various enzymes before addition to macrophages. Preincubation with the N-acetylmuramidases mutanolysin or M1,
which hydrolyze the PepG backbone between NAM and
NAG (see Fig. 1), significantly attenuated the nitrite accumulation caused by S. aureus PepG in the presence of IFN-
(Fig. 3). Lysozyme, a muramidase which is unable to lyse
PepG derived from S. aureus, did not affect the increase in
nitrite caused by PepG plus IFN-
(Fig. 3). The endopeptidase lysostaphin, which breaks the pentaglycine interpeptide cross-bridge, markedly enhanced the formation of nitrite (Fig. 3). In contrast, SALE, which has both endopeptidase and amidase activity (i.e., hydrolyzes the bond NAM-
L-ala, which connects the sugar chain and stem peptide),
prevented the ability of PepG to synergize with IFN-
to
induce nitrite formation (Fig. 3). These effects were specific for PepG, as the induction of nitrite accumulation by
S. aureus LTA (1 µg/ml) was not affected by pretreatment
with these enzymes (results not shown).
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Hydrolysis of the PepG polymer affected the induction
of iNOS by PepG in the presence of IFN-, suggesting
that a small moiety within PepG is responsible for the synergism with IFN-
to cause induction of iNOS. To further
elucidate which fragment of PepG is involved, macrophages
were incubated with muropeptides, PepG-constituent amino
sugars, peptides, and related molecules. A concentration-dependent increase in nitrite formation by macrophages
was observed 24 h after the addition of either the NAG-NAM-L-ala-D-isoglutamine fragment (EC50 = 40 nM) or
the NAM-L-ala-D-isoglutamine fragment lacking the NAG
residue (EC50 = 100 nM) in the presence of IFN-
(Fig. 4
A). To investigate the specificity of these effects, two stereoisomers of NAM-L-ala-D-isoglutamine were tested: NAM-
L-ala-L-isoglutamine and NAM-D-ala-D-isoglutamine. The
maximal induction of nitrite formation was decreased significantly with NAM-L-ala-L-isoglutamine and nearly abrogated with NAM-D-ala-D-isoglutamine (Fig. 4 A). Other
fragments lacking the NAG-L-ala-D-isoglutamine moiety,
including NAM, NAG, stem peptide (L-ala-D-isoglutaminyl-L-lys-D-ala-D-ala), triglycine, and pentaglycine, did not
cause an increase in nitrite formation up to a concentration
of 100 µg/ml when given either alone or in combination
with IFN-
(results not shown). As with the PepG polymer, NAG-NAM-L-ala-D-isoglutamine or NAM-L-ala-
D-isoglutamine alone did not induce nitrite formation in
macrophages (2 ± 0.5 and 3 ± 1 µM, respectively, at 100 µg/ml).
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We have demonstrated previously that the S. aureus PepG polymer synergizes with LTA from the cell wall of S. aureus to induce iNOS in macrophages and in the anesthetized rat (18). In agreement with the present observations, the PepG fragment NAG-NAM-L-ala-D-isoglutamine or NAM-L-ala-D-isoglutamine synergized with LTA (0.1 µg/ ml) to induce an enhanced formation of nitrite in macrophages (Fig. 4 B). Again, this synergy was stereoselective, as the induction of nitrite formation was decreased for the stereoisomers of NAM-L-ala-D-isoglutamine (Fig. 4 B). None of the other fragments of PepG tested synergized with LTA to cause the formation of nitrite (results not shown).
Polymyxin B (an agent that binds and inactivates endotoxin) at a concentration sufficient to abolish the increase in
nitrite caused by LPS in macrophages (17) did not affect
the nitrite production caused by either PepG or its fragment NAG-NAM-L-ala-D-isoglutamine in the presence of
IFN- in the same cells (results not shown).
There are several studies reporting that
LTA (Sigma Chemical Co.) can induce iNOS activity in
macrophages or RASMC in culture and in vivo in anesthetized rats (15, 16, 28). This commercially available LTA is
extracted by the hot phenol-water procedure and is still
contaminated with various bacterial components, including polysaccharides, proteins, and nucleic acids. Two recent reports indicate that components other than purified LTA
contribute to (a) the release of IL-6 in a human astrocytoma cell line; (b) the release of IL-1, IL-6, and TNF-
from human monocytes; and (c) the induction of iNOS in
mononuclear cells (8, 29). Therefore, S. aureus LTA obtained from Sigma Chemical Co. was subjected to separation by hydrophobic interaction column chromatography,
and relevant fractions were obtained. As LTA contains
phosphates, detectable amounts of phosphate were observed in fractions 8-12 (corresponding to material not
bound by the column matrix) and in fractions 36-44 (Fig.
5). Only the latter fractions induced the accumulation of
nitrite in macrophages when given to these cells either
alone or in combination with the PepG fragment NAG-NAM-L-ala-D-isoglutamine. Most notably, the increase in
nitrite afforded by these fractions was not affected by polymyxin B (0.5 µg/ml; results not shown), demonstrating
that it was not due to the contamination of these fractions
with LPS.
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Treatment of RASMC for 48 h with S. aureus
LTA resulted in only a small increase in nitrite accumulation, whereas S. aureus PepG, IFN-, or NAG-NAM-
L-ala-D-isoglutamine did not induce a significant increase
(Fig. 6). LTA synergized with IFN-
in RASMC to induce a marked accumulation of nitrite. However, a combination of NAG-NAM-L-ala-D-isoglutamine or PepG with
LTA or IFN-
did not significantly enhance the release of
nitrite from these cells compared with treatment with LTA
or IFN-
alone (Fig. 6).
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Incubation of macrophages with whole heat-killed S. aureus (108 cells/ml) for 24 h resulted in a marked elevation of nitrite formation (Fig. 7). However, the induction of nitrite formation by the nonpathogenic bacterium B. subtilis (108 cells/ml) was only ~30% of that elicited by S. aureus (Fig. 7).
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We confirm that LTA from S. aureus elicits a concentration-dependent increase in nitrite formation from macrophages (Table 1). In contrast, LTA from B. subtilis (0.01-30 µg/ml) did not cause a significant increase in nitrite production from baseline within 24 h (Table 1).
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Although incubation of macrophages with PepG from S. aureus (up to 30 µg/ml, in the absence of IFN-) did not
result in an enhanced production of nitrite, PepG from B. subtilis elicited a small but significant increase in nitrite (at
the highest concentration tested; Table 1). The PepG from
S. aureus synergized only with LTA from S. aureus, not
with LTA from B. subtilis, to induce nitrite formation (Fig.
8 A). Similarly, coincubation of macrophages for 24 h with
PepG from B. subtilis and LTA from S. aureus markedly enhanced the nitrite formation compared with that elicited by either component alone, but no synergy was observed between PepG and LTA from B. subtilis (Fig. 8 B). Furthermore, NAG-NAM-L-ala-D-isoglutamine present in the
PepG monomer of both S. aureus and B. subtilis synergized
only with S. aureus LTA, not with (at 100-fold concentration) B. subtilis LTA, for the induction of iNOS protein
and nitrite formation in macrophages (Fig. 9).
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Treatment of rats with S. aureus LTA (3 mg/kg) and B. subtilis PepG (1 mg/kg) resulted in a significant decrease in blood pressure, from 125 ± 3 mmHg at time 0 to 84 ± 6 mmHg at 6 h (P <0.05 compared with sham-operated rats at the same time point [117 ± 3 mmHg]). Similarly, administration of other cell wall components also produced a delayed hypotension, but these were not significantly different from sham-operated animals (S. aureus LTA [3 mg/kg], 97 ± 6 mmHg; B. subtilis PepG [1 mg/kg], 82 ± 12 mmHg; B. subtilis LTA [3 mg/kg], 94 ± 7 mmHg; B. subtilis LTA [3 mg/kg] and B. subtilis PepG [1 mg/kg], 89 ± 7 mmHg; n = 5-7). Only in rats cotreated with S. aureus LTA and B. subtilis PepG was the delayed hypotension accompanied by a severe loss in the pressor response elicited by norepinephrine compared with sham-operated rats (Fig. 10 A). No significant change in the pressor response to norepinephrine was observed in rats receiving LTA and PepG from B. subtilis. There were no significant changes in the heart rate during the course of the experiment in any of the treatment groups (results not shown).
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Administration of S. aureus LTA (3 mg/kg) and B. subtilis PepG (1 mg/kg) resulted, within 6 h, in a significant elevation of serum levels of ALT (an indicator of hepatocellular injury), urea (results not shown) and creatinine (indicators of renal failure), and enhanced activity of iNOS in lung homogenates compared with sham-operated rats or rats receiving either component alone (Fig. 10, B-D). Rats receiving B. subtilis LTA (3 mg/kg) in combination with B. subtilis PepG did not show a significant rise in the serum levels of these biochemical markers of organ failure or in lung iNOS activity.
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Discussion |
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The cell wall component PepG derived from S. aureus
synergizes with IFN- to cause the induction of iNOS
protein and activity in J774.2 macrophages, as demonstrated by protein Western blot analysis and the accumulation of nitrite in the supernatant, which was attenuated by
the NOS inhibitors L-NMMA or AE-ITU. In the presence of IFN-
or LTA, NAG-NAM-L-ala-D-isoglutamine,
a small moiety within the PepG monomer, also caused an
increase in NO formation by these cells. In contrast, neither of the sugars NAG or NAM nor the stem peptide (L-ala-D-isoglutaminyl-L-lys-D-ala-D-ala) or the pentaglycine
bridge caused an increase in NO formation in J774.2 macrophages. Taken together, these results strongly suggest that
the NAG-NAM-L-ala-D-isoglutamine moiety accounts for
the induction of iNOS attributed to the whole PepG polymer. This conclusion is further supported by the following
findings. (a) Hydrolysis of the glycosidic bond between
NAG and NAM (sugar backbone of PepG) with two different muramidases (mutanolysin or M1) also reduced the
NO formation elicited by the whole PepG polymer (in the
presence of IFN-
) by approximately one third. We also
found that the NAM-L-ala-D-isoglutamine moiety was less potent than NAG-NAM-L-ala-D-isoglutamine to induce
this response. (b) Hydrolysis of the amide bond between
NAM and L-ala in PepG with SALE (30) completely abolished the NO formation caused by the whole PepG polymer (in the presence of IFN-
). Similarly, mutanolysin,
M1, or SALE have also been reported to attenuate the formation of TNF-
elicited by PepG from Staphylococcus epidermidis in human monocytes (21). It could be argued that
the observed effects of SALE are due to its ability to cleave
the pentaglycine bridge between PepG monomers rather
than its amidase activity. However, this is not the case, as
the hydrolysis of this glycine bridge by lysostaphin (31) enhanced (rather than decreased) the formation of NO. This
finding is not entirely surprising, as the cleavage of the glycine bridge by lysostaphin is likely to facilitate the interaction of the active moeity, NAG-NAM-L-ala-D-isoglutamine, with the macrophages. These results support our view that
NAM-L-ala-D-isoglutamine is the smallest possible fragment of PepG that is able to synergize with IFN-
to induce iNOS in macrophages, while an extra NAG residue results in a more potent induction.
We have reported previously that the PepG polymer
synergizes with another gram-positive cell wall component, LTA (18). Indeed, the identified active moieties
(NAG-)NAM-L-ala-D-isoglutamine also synergize with LTA
to cause induction of iNOS in macrophages. This synergy
with IFN- or LTA is stereoselective, as a decrease in activity is observed with the stereoenantiomers NAM-L-ala- L-isoglutamine and NAM-D-ala-D-isoglutamine. This conformational requirement suggests a distinct receptor-ligand
interaction. These results are in agreement with reports
demonstrating that NAM-L-ala-D-isoglutamine, also known
as adjuvant or muramyl peptide, is the smallest biologically active structure of bacterial PepGs (32, 33). Additionally, computer modeling studies have shown that a specific conformation of NAM-L-ala-D-isoglutamine is required to exert maximal biological activity (34).
Commercially available LTA is prepared by hot phenol-
water extraction of whole bacteria (35) and may still contain a variety of bacterial components coextracted during
the procedure. Further purification of this "crude" LTA
from S. aureus by hydrophobic interaction column chromatography shows that the increase in NO formation is
caused by those fractions containing phosphate (a major
component of the poly(glycerophosphate) backbone of
LTA) and eluted at 35-55% vol/vol propanol, the expected
hydrophobicity of LTA (13, 35). Similar results are found
for the synergy of these fractions with NAG-NAM-L-ala-
D-isoglutamine. Thus, the results strongly suggest that purified LTA induces the formation of NO by macrophages.
The LTA fractions 40 and 43 contain similar phosphate
concentrations; however, the more lipophilic fraction, 43, elicited a fivefold higher increase in NO formation than
fraction 40. This suggests that the activity of LTA to induce
iNOS increases with lipophilicity of the LTA fractions.
Similar results have been reported for two LTA fractions
isolated from Streptococcus faecalis (36). The more lipophilic
LTA fraction causes the release of TNF- from macrophages and regression of tumors in mice, whereas the more
hydrophilic is inactive. After column chromatography, fractions with significant concentrations of phosphate are also eluted at 15% vol/vol propanol, representing hydrophilic
fractions that do not contain LTA. Kusunoki et al. (22)
have shown that compound(s) in these fractions (and not in
lipophilic fractions containing purified LTA) elicit the release of IL-6 in human astrocytoma cells. However, in the
present study, the equivalent fractions do not induce the
formation of NO in murine macrophages. Therefore, it is
unlikely that components other than the purified LTA
contribute to the induction of iNOS in macrophages elicited by commercially available LTA extract.
S. aureus is a major cause of nosocomial infections and gram-positive septic shock (1), whereas B. subtilis is a nonpathogenic gram-positive bacterium (37, 38). We report here that macrophages challenged with whole heat-killed B. subtilis bacteria produce far less NO than macrophages activated with heat-killed S. aureus. Similarly, LTA from S. aureus is more potent than LTA from B. subtilis in enhancing the formation of NO in macrophages. It has been reported that LTA from S. aureus is also more potent in the activation of arachidonic acid metabolism in mouse macrophages than LTA from B. subtilis (39). The structure of LTA from S. aureus and B. subtilis is very similar, except that the glycosylation and alanine substitution of the polyglycerophosphate backbone is higher in S. aureus (13, 40, 41). This suggests that the lipophilic portion of LTA may be important for the biological activity. This notion is supported by findings that the immunodeterminants of LTA of various gram-positive bacteria are located mainly in their polyglycerophosphate moieties, including its glycosyl and alanyl substituents (42). Also, in the study by Takada et al. (36), the more lipophilic LTA fraction of S. faecalis with a high alanine content was more active than the hydrophilic LTA fraction with a low alanine content. In the present study, the more lipophilic fraction from LTA of S. aureus was also associated with higher activity. Thus, the degree of alanine substitution in LTA may be important for the induction of iNOS in macrophages, and may explain the lower activity of LTA from B. subtilis, but the exact role of alanine substitution remains to be further elucidated.
LTA from S. aureus synergizes with NAG-NAM-L-ala- D-isoglutamine, the active moiety of PepG present in the PepG of both S. aureus and B. subtilis (33, 43), to induce iNOS activity in macrophages. In contrast, LTA from B. subtilis does not synergize with this moiety to enhance the formation of NO. In support of this, PepG from either S. aureus or B. subtilis synergizes with LTA from S. aureus but not from B. subtilis to cause NO formation in these cells. These results further indicate that the structure of LTA determines whether or not a particular LTA is able to synergize with PepG to induce iNOS activity. The activity of PepG seems largely dependent on the NAG-NAM-L-ala- D-isoglutamine moiety, which is one of the most conserved parts of PepG from (pathogenic and nonpathogenic) gram-positive organisms (43). The diversity of PepG among gram-positive organisms is limited to the free combination of 11 different types of peptide side chains with two types of cross-linkage (44). Thus, our results suggest that the ability of gram-positive organisms to induce iNOS activity in macrophages, and possibly its pathogenicity, reside within the LTA component, while PepG serves to amplify a given biological response.
Surprisingly, in this study we found that neither NAG-NAM-L-ala-D-isoglutamine nor PepG from S. aureus synergizes with IFN- or LTA to induce NO formation in
RASMC. The reason for this is not clear, but may be that
these cells do not possess the "receptor" to which PepG
binds to activate these cells (45, 46). On the other hand, it
is possible that the PepG polymer has to be digested by
macrophages to release biologically active fragments, such as NAG-NAM-L-ala-D-isoglutamine, whereas smooth muscle cells are unable to digest PepG (47). Indeed, B. subtilis
PepG given to alveolar macrophages releases predominantly
NAG-NAM-L-ala-D-isoglutamine (48). But our finding that
direct exposure of smooth muscle cells to this fragment (in
the presence of IFN-
or LTA) does not result in an enhanced formation of NO would argue against this possibility. Thus, the reason underlying this lack of synergy in
smooth muscle cells remains to be established.
Previously, we have shown that the cell wall components LTA and PepG from S. aureus synergize in vivo to cause circulatory and multiple organ failure in anesthetized rats (18). We find here that LTA or PepG from B. subtilis when given either alone or in combination to anesthetized rats does not induce vascular hyporeactivity to norepinephrine, hepatic injury, or renal failure. The inability of the LTA and PepG-teichoic acid complex from B. subtilis to cause death in mice has been documented previously (37, 38). As in macrophages, PepG from B. subtilis and LTA from S. aureus synergized to cause vascular hyporeactivity to norepinephrine, liver injury, renal failure, and an enhanced iNOS activity in the lungs of rats (although either component alone was inactive). Thus, the importance of the LTA structure for synergy among the cell wall components extends from the in vitro (macrophages) to the in vivo (anesthetized rats) situation. In these experiments, the circulatory and multiple organ failure is associated with the induction of iNOS activity, suggesting a causative role for NO. Previous studies indicate that inhibition of NO synthesis prevents the circulatory failure and liver injury but not the renal failure elicited by the combined cell wall components from S. aureus (19). Thus, an enhanced formation of NO by iNOS accounts for the circulatory failure and contributes to the organ injury associated with gram-positive shock.
In summary, NAG-NAM-L-ala-D-isoglutamine is the
bioactive moiety in PepG responsible for the synergy of
PepG with IFN- or LTA to induce iNOS in macrophages. The finding that LTA from S. aureus but not B. subtilis synergizes with PepG from either bacterium to induce (a) NO formation in macrophages and (b) iNOS activity, shock, and multiple organ failure in anesthetized rats
suggests that the structure of LTA determines the pathogenicity of a particular bacterium, whereas PepG acts to amplify any response induced by LTA.
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Footnotes |
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Address correspondence to Chris Thiemermann, The William Harvey Research Institute, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. Phone: 44-171-982-6025; Fax: 44-171-251-1685; E-mail: c.thiemermann{at}mds.qmw.ac.uk
Received for publication 13 April 1998.
C. Thiemermann is a Senior Fellow of the British Heart Foundation (FS 016/96). S.J. Foster is supported by the Royal Society. This work was supported by a Biomed II grant of the European Commission.
Abbreviations used in this paper
AE-ITU, aminoethyl-isothiourea;
ALT, alanine aminotransferase;
D- or L-ala, D- or L-alanine;
iNOS, inducible NOS;
L-lys, L-lysine;
L-NMMA, NG-methyl-L-arginine;
LTA, lipoteichoic acid;
MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NAG, N-acetylglucosamine;
NAM, N-acetylmuramic acid;
NAG-NAM-L-ala-
D-isoglutamine, N-acetylglucosamine--[1
4]-N-acetylmuramyl-L-alanine-
D-isoglutamine;
NO, nitric oxide;
NOS, NO synthase;
PepG, peptidoglycan;
RASMC, rat aortic smooth muscle cell(s);
SALE, Staphylococcus aureus lytic
enzyme.
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