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INTRODUCTION |
Inflammation is a complex, nonspecific host defense response to
pathological situations such as infection. When localized, inflammation
is beneficial because it helps eradicate the invading organisms at the
infected site (1). In severe situations, however, the deregulation of
numerous host defense factors and soluble cellular mediators, such as
cytokines, may result in a dramatic cardiovascular collapse called
septic shock (2, 3). The cytokine tumor necrosis factor
(TNF)1 was shown to play a
critical role in triggering such an event (4, 5). Septic shock is a
classical response to severe infections due to Gram-negative bacteria,
in which the outer membrane lipopolysaccharide (LPS) is the principal
bacterial component triggering cytokine release from peripheral blood
mononuclear cells (PBMCs) and other responsive cells (6-9). To
stimulate cytokine release, LPS binds to the cell surface protein CD14
(10), a glycosylphosphatidylinositol-linked determinant that presumably
requires additional transmembrane elements such as the recently
described Tol receptor (11) to trigger cell response. In
vivo LPS also binds to lipopolysaccharide-binding protein, an
acute phase protein that catalyzes the binding of LPS to CD14 (12).
Septic shock is also a feature of severe Gram-positive infections
(13-16). In recent years, Gram-positive pathogens have become more
frequent than Gram-negative bacteria as a cause of nosocomial infections and shock (17, 18). This is particularly true in intensive
care unit patients and in neutropenic patients, two subgroups with
impaired host defenses that are especially prone to develop severe
sepsis. However, in contrast to their Gram-negative counterparts,
Gram-positive bacteria do not contain LPS. In certain cases, highly
pathogenic agents such as Staphylococcus aureus and
Streptococcus pyogenes produce toxins that may act as
superantigens and trigger an overwhelming nonspecific T-cell-mediated
inflammatory response (7, 13). With less pathogenic agents, such as
viridans group streptococci, triggering of inflammation and shock might be due to other cellular components, including the thick Gram-positive peptidoglycan. Gram-positive cell walls were shown to stimulate TNF
release and inflammation in several in vitro and in
vivo systems, including experimental meningitis, experimental
arthritis, and experimental septic shock (19-21).
To study the mechanism of peptidoglycan-induced inflammation, different
types of Gram-positive wall materials were utilized. On the one hand,
large molecules such as insoluble cell walls and soluble peptidoglycan
released from penicillin-treated staphylococci allowed the
demonstration that CD14 was also a receptor for Gram-positive wall
products (22, 23). On the other hand, the small molecule muramoyl
dipeptide was shown to be the minimal common peptidoglycan structure
carrying immunomodulatory activity (24). However, while very useful,
these compounds might not be ideal for solving the structure-activity
relationship between the wall degradation products occurring in the
nature and their cytokine-releasing capacity. First, molecules such as
soluble peptidoglycan are very large (Mr
125,000) (25, 26) and might be too complicated for refined analysis.
Second, muramoyl dipeptide is not a natural product of wall
degradation, and it has only limited cytokine-stimulatory power
in vitro (27).
To further explore this question, we attempted to isolate and
characterize small proinflammatory peptidoglycan fragments resulting from the digestion of pneumococcal walls with their native autolysins, thereby liberating products of digestion that are likely to be found
in vivo. Specifically, Streptococcus pneumoniae
contains a major autolytic enzyme (an
N-acetylmuramoyl-L-alanine amidase, which
hydrolyzes the bonds between the glycan chain and the stem peptides)
that is responsible both for wall solubilization and cell lysis during
the stationary growth phase or during
-lactam treatment (28-30).
The role of this autolytic enzyme in pneumococcal pathogenesis has been
demonstrated (31). In certain experiments, a second enzyme
(M1-muramidase) that hydrolyzes the glycan chain was used in parallel.
M1-muramidase is not formally a pneumococcal enzyme, but it has a
functional equivalent in these organisms (32). The resulting
soluble fragments were separated by reverse phase high pressure
chromatography (HPLC). Individual fractions were tested for their
TNF-releasing activity, and the molecular structure of single
components carrying inflammatory power was analyzed by mass
spectrometry (MS), amino acid and amino sugar analysis, and postsource
decay analysis.
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EXPERIMENTAL PROCEDURES |
Bacterial Strain and Growth Conditions--
The noncapsulated
lysis-prone positive S. pneumoniae strain R6 was used (33).
Bacteria were grown in the chemically defined liquid medium Cden (44).
Growth of the cultures was followed by their optical density at 620 nm
(A620) with a spectrophotometer (Sequoia-Turner,
Moutainville, CA). The cells were stored at
70 °C in Cden
containing 20% glycerol.
In certain experiments a label was introduced into the cell walls
by either of the following methods. (i) To label the glycan chains,
N-acetyl-D-[1-3H]glucosamine
(Amersham Pharmacia Biotech) was added to the culture to a final
concentration of 250 µCi/liter and 1.23 µg/liter. (ii) To label the
stem peptides, L-[4,5-3H]lysine (Amersham
Pharmacia Biotech) was added to a final concentration of 250 µCi/liter and 0.45 µg/liter. The cultures were allowed to grow for
4-5 generation times in the presence of the label before being
harvested as described below.
Preparation and Purification of Cell Walls--
Cell walls were
prepared as described previously (34). Briefly, one liter cultures
grown to the late logarithmic phase (A620 of
0.5) were rapidly cooled in an ice/ethanol bath and harvested by
centrifugation for 10 min at 16,000 × g and 4 °C.
The cells were resuspended in endotoxin-free water, and intrinsic
autolytic enzymes were inactivated by pouring the suspension dropwise
into boiling SDS (4%, final concentration). After 15 min of boiling, the SDS was removed by extensive washing with water, and the cells were
broken open by vortexing with an equal volume of acid-washed glass
beads with a 106-µm diameter (Sigma). The cells walls were pelleted
by centrifugation at 25,000 × g for 30 min at 4 °C;
resuspended in 0.1 M Tris-HCl buffer (pH 7.5) containing
0.05% sodium azide; and treated with DNase, RNase, and trypsin as
described (34). Peptides from protease digestion were extracted by
boiling for another 10 min with SDS (1% final concentration), and the
walls were incubated with 8 M lithium chloride and then 100 mM EDTA to remove material bound by ionic interactions. To
remove possible contaminating endotoxin, the insoluble preparation was
washed extensively with acetone followed by resuspension in
endotoxin-free water prior to drying by rotary evaporation. The absence
of contaminating endotoxin was confirmed by a negative result of the
Limulus amebocyte test (Chromogenix, Molndal, Sweden) with a
sensitivity of 4 pg/ml.
Digestion of Cell Walls by Autolytic Enzymes--
To hydrolyze
the bonds between the glycan chains and the stem peptides, the natural
pneumococcal autolysin
N-acetylmuramoyl-L-alanine amidase (referred to
as amidase in the following) was used. This enzyme was purified by
affinity chromatography (35) from Escherichia coli CM21
lysates, into which the pneumococcal amidase gene (lytA) had
been cloned (36). Purified pneumococcal walls were suspended in 50 mM MOPS buffer (pH 6.9) at a concentration of 1 mg/ml. The mixture was incubated with 100 units/ml (final concentration) of
amidase for 18 h with shaking at 37 °C. The digest was spun for
10 min at 15,000 × g, and the supernatant, containing
the solubilized walls, was collected. In certain experiments, the amount of radioactivity was counted to determine the extent of solubilization. The supernatant was then boiled for 10 min and centrifuged at 15,000 × g and 4 °C to denature and
remove the amidase. Eventually, the soluble walls were washed with
acetone and water, as described, to remove any contaminating endotoxin and subsequently dried by rotary evaporation and stored at
20 °C.
This enzymatic treatment resulted in >90% solubilization of the
purified material.
To hydrolyze the bonds between the N-acetylmuramoyl and
N-acetylglucosamine residues, mutanolysin from
Streptomyces globiosporus (Sigma; EC 3.2.1.17) was used. The
enzyme was added to a final concentration of 500 units/ml and 1 mg/ml
substrate in 0.25 mM potassium phosphate buffer (pH 6.5)
containing 10 mM magnesium chloride. The mixture was
incubated for 18 h with gentle shaking at 37 °C before boiling
and centrifugation to remove the enzyme as above. The supernatant was
subsequently washed with acetone, dried by rotary evaporation, and
stored at
20 °C as above.
In certain experiments, the glycan chains and stem peptides were
separated by differential precipitation in a mixture of
water/acetonitrile/propan-2-ol (in the ratio 50:25:25) containing 0.1%
trifluoroacetic acid. Typically, 600 µg of amidase-solubilized cell
walls were suspended in this solvent and kept on ice for 30 min.
Insoluble material containing the glycan-rich fraction was then
collected by centrifugation at 15,000 × g for 15 min
(4 °C). The supernatant contained the stem peptides. Solvents were
removed by rotary evaporation, and the peptides and glycan were
dissolved in water. The amount of material present in each fraction was
quantified by liquid scintillation counting.
Separation of Digested Peptidoglycan by HPLC and Identification
of Specific Fractions--
The HPLC system (Hitachi Instruments,
Ichige, Hitachinaka, Japan) consisted of the L-7200 autosampler, the
L-7100 gradient pump, with low pressure mixing, and the L-7400 UV
detector. Column temperature was maintained at 25 °C using a
pelcooler (LabSource, Reinach, Switzerland). The results were analyzed
using the D-7000 HPLC System Manager program (Hitachi).
Separation was performed by injection of a 100-µl sample, containing
100 µg of wall digest, into a C18 reverse phase column (SuperPac
Sephasil C18, 5 µm, 4 × 250-mm column, Amersham Pharmacia Biotech) protected with a guard cartridge (C18, 5 µm, 4 × 10 mm). The mixture was separated using a linear gradient of 0-15%
acetonitrile in 0.1% trifluoroacetic acid over 100 min with a flow
rate of 0.5 ml/min. Detection was at 210 nm. 1-min fractions were
collected. The solvent was removed by rotary evaporation. The amount of
material present in each fraction was too small to be measured either
by weight or by radioactive counts and was therefore calculated by the
percentage area method. Knowing the weight of material loaded, the
amount of material present in each fraction was deduced. The samples
were dissolved in 100 µl of water and stored at
20 °C.
The content of specific fractions was analyzed by the following means.
First their content in amino acids was determined using the OPAC
postcolumn derivatization method following hydrolysis of the samples
for 24 h in 6 M HCl at 110 °C (37). Second, their molecular mass was measured by matrix-assisted laser desorption ionization time of flight MS, using a Voyager-Elite TOF mass
spectrometer (PerSpective Biosystems Inc., Farmingham, MA) operating in
the positive ion mode and using an
-cyano-4-hydroxycinnamic acid matrix. The laser power was set to 1,600 units, and the accelerating voltage was set to 20,000 V. Postsource decay analysis was performed on
certain fractions.
N-Acetyl-D-glucosaminyl-(
1
4)-N-acetylmuramoyl-L-alanyl-D-isoglutamine (Calbiochem) was used as a standard (Mr
694.7).
Preparation, Stimulation of Human PBMCs or Whole Blood from
Rabbits, and Measurement of TNF-
Release--
Human PBMCs were
extracted from heparinized blood of healthy volunteers by
Ficoll-Hypaque (Seromed, Munich, Germany) density gradient
centrifugation as described by Heumann et al. (22). The
cells were suspended in RPMI 1640 medium (Life Technologies, Inc.) and
distributed into the wells of a flat bottomed 96-well tissue culture
plate at a concentration of 0.5 × 106 cells/well.
Each well contained a final volume of 200 µl, which comprised the
RPMI medium (140 µl), plasma from the donor (20 µl), and sample (20 µl). LPS from E. coli O111 (Sigma) in the concentration
range 0.01-100 ng/ml was used as a positive control. PBMCs incubated
with plasma or with medium alone were used as negative controls. The
plates were incubated at 37 °C in an atmosphere of 5%
CO2. After 8 h of incubation, samples (20 µl) of the
supernatants were taken, diluted 20-fold in RPMI medium, and stored at
80 °C for measurement of TNF concentrations.
For the whole blood assay, heparinized rabbit blood was diluted 4-fold
in RPMI 1640 medium supplemented with 10% fetal calf serum as
described (38). The diluted blood was distributed into tissue culture
plates (200 µl/well). Samples (20 µl) were added, and the plates
were incubated as above for 6 h. The plates were centrifuged for 5 min, and samples of the supernatant were diluted and
stored as described above for subsequent measurement of TNF concentrations.
WEHI clone 13 murine fibroblast cells (1 × 104/well)
were used for quantitation of TNF-
as described (39). Recombinant
mouse TNF-
were used as a standard. The sensitivity of the assay was 25 pg/ml.
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RESULTS |
TNF-stimulating Activity of LPS and Purified Pneumococcal
Walls--
Fig. 1 depicts a typical
titration experiment of the TNF-releasing activity of LPS and insoluble
pneumococcal walls exposed to human PBMCs in the presence of 10%
plasma. Both LPS and purified cell walls triggered TNF release from the
monocytes in a concentration-dependent fashion. However, it
required ~1000 times more pneumococcal walls than LPS to release the
same amounts of TNF from the target cells. This was in agreement with
previous observations reported by Timmerman et al. (40) with
insoluble staphylococcal peptidoglycan and Heumann et al.
(22) with pneumococcal peptidoglycan.

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Fig. 1.
TNF-releasing activity of LPS and
pneumococcal walls exposed to human PBMCs. Human PBMCs were
isolated as described and exposed for 8 h to LPS or purified
pneumoccal walls in the presence of 10% plasma. TNF production was
measured by its cytotoxic activity on murine fibroblasts. Recombinant
human TNF was used as a standard. The black bars
indicate the levels of TNF produced by stimulation with LPS from
E. coli O111, and the open bars
indicate the amounts of TNF produced by stimulation with purified
pneumoccal walls. Concentrations of TNF and LPS or cell wall stimulants
are indicated.
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To attempt to expose more active pneumococcal wall subcomponents that
might be embedded in the insoluble peptidoglycan, we first solubilized
the purified walls using the bacteria's own major autolysin amidase,
purified as described (35), and then sought inflammatory subcomponents
in the soluble material. Amidase solubilized >90% of the purified
walls. Nevertheless, the inflammatory power of this soluble mixture was
not greater than that of insoluble material. Similar observations were
made by Riesenfeld-Orn et al. (14). Indeed, when soluble and
insoluble walls were titrated for their TNF-releasing activity, as in
Fig. 1, they produced very similar stimulation profiles and had the
same stimulatory potency (see upper part of Table
I). This observation came in support of
the hypothesis that only part of the peptidoglycan might be
inflammatory and suggested that such specific inflammatory constituents
might now be present in the soluble mixture. Therefore, the wall
constituents were further separated by reverse phase HPLC, and
individual fractions were tested for their PBMC and/or whole
blood-stimulating activity.
Solubilization and HPLC Separation of Cell Walls--
Fig.
2 depicts the solubilization steps used
in these experiments. Purified insoluble pneumococcal walls (step 1 in
Fig. 2) digested with amidase (step 2 in Fig. 2) produced a wealth of soluble fragments comprising free stem peptides, separated from the
glycan backbone at the
N-acetylmuramoyl-L-alanine bonds, and glycan
chains decorated or not with teichoic acids and/or residual peptides
(34). However, although amidase solubilized >90% of the walls (step 2 in Fig. 2), the soluble fragments generated by this treatment could not
be properly resolved by the chromatography conditions used in these
experiments. The profile of this separation is shown in Fig.
3a.

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Fig. 2.
Steps used in the solubilization of the cell
walls and the separation of its components. The diagram
represents the principal wall components. The horizontal
lines indicate the glycan chains. The vertical
lines indicate the stem peptides and their interconnecting
bonds. The black circles represent the location
of glycan-attached teichoic acids. Insoluble cell walls were purified
from log phase cultures of S. pneumoniae (step 1). Purified
walls were digested with amidase (step 2), which hydrolyzed the bonds
between the glycan chain and the stem peptides. Note that amidase
hydrolyzes these bonds only in the presence of an adjacent teichoic
acid (black circle) (34). This digestion resulted
in soluble stem peptides with various degrees of cross-linkage and
glycan chains that still contained a few peptides. Amidase-digested
walls were then treated with muramidase (step 3A), which resulted in
the breakdown of the glycan chains. This preparation was submitted to
reverse phase HPLC separation (step 4A) and TNF stimulation assay (step
5A). In separate experiments, amidase-digested walls were also
separated into their peptide and glycan constituents by serial
precipitation (step 3B). Soluble peptides were then separated by HPLC
and analyzed for TNF-stimulating activity as described (steps 4A and
5A). On the other hand, the glycan-enriched fraction was submitted to
muramidase digestion (step 4B) and tested in the TNF-stimulation assay
(step 5B).
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Fig. 3.
Separation of soluble cell wall components by
HPLC. Equal amounts (100 µg) of the following pneumococcal wall
preparations were loaded onto a C18 Sephasil column: purified walls
digested with amidase alone (a), purified walls digested
with both amidase and muramidase (b), and stem peptides
prepared by sequential precipitation of amidase-digested walls
(c) (see Fig. 2). HPLC failed to resolve cell walls
solubilized by amidase alone (a). In contrast, the
amidase/muramidase, double-digested wall (b) and the
peptide-enriched fractions (c) could be resolved and gave
reproducible patterns that were very similar in both cases. The
insets show the putative composition of the unseparated
mixture.
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We reasoned that this might be due to some interference of the
undigested glycan backbone (step 2 in Fig. 2) with the C18 column. To
circumvent this problem, two strategies were adopted. In the first, the
amidase-solubilized walls were submitted to a second digestion with
muramidase (step 3A in Fig. 2), an enzyme that hydrolyzes the 1-4
-bonds between the N-acetylmuramoyl and N-acetylglucosamine residues of the glycan chain. The
profile of this second separation is depicted in Fig. 3b. In
contrast to Fig. 3a, Fig. 3b indicates that
double-digested wall material could now be resolved by HPLC. The
chromatogram was complex, indicating that the peptidoglycan contained a
variety of subcomponents. Nevertheless, this profile was perfectly
reproducible in terms of both interrun and intercell wall batch
resolution and over at least four independent experiments.
A second strategy (step 3B in Fig. 2) to encompass the interference of
the undigested glycan chains consisted of removing the glycans from the
amidase digest by serial precipitation in water/acetonitrile/propan-2-ol (as described under "Experimental Procedures") and then analyzing only the peptide-enriched fraction by
HPLC. Fig. 3c depicts the result of this separation. It can be seen that this chromatogram resembled very much that of Fig. 3b, indicating that the stem peptides of the cell walls were
the prevalent species resolved with both of these methods.
TNF-releasing Activity of Individual HPLC Fractions--
In a
first series of experiments, we determined the TNF-releasing activity
of 1-min fractions collected from the chromatogram presented in Fig.
3b. Individual fractions were exposed to human PBMCs as
described. Fig. 4 indicates that the
TNF-releasing activity was very low for material eluting before 30 min,
whereas it rose considerably later on, i.e. up to 100-fold
above background levels. Since the chromatograms resulting from the
separation of either double-digested or single amidase-digested
peptidoglycan were almost superimposable, we repeated the stimulation
experiment with 1-min fractions of both of these enzymatic digests. In
this second series of experiments, a new column was used, which
resulted in a slight increase in retention times. Fig.
5 presents these results. In these
experiments, the TNF assay was performed with whole blood from rabbits,
a system that gave similar results as human PBMCs but was much more
convenient for screening large numbers of fractions. It can be seen
that not only the chromatograms of double-digested or single
amidase-digested peptidoglycan were very similar, but their profiles of
TNF-release were comparable as well. This was consistent with the fact
that the peptide-enriched material contained active subcomponents
similar to those observed in the double-digested material.

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Fig. 4.
TNF triggering activity of 1-min fractions
collected from the separation shown in Fig. 3b.
Individual fractions were tested both for stimulation of PBMCs and
structural analysis based on MS and amino acid and amino sugar
analysis. Similar profiles were obtained irrespective of whether the
double digest or peptide-enriched fractions were tested (see Fig.
5).
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Fig. 5.
TNF released from rabbit blood following
stimulation by 1-min fractions collected from the separation of either
double-digested wall or single amidase-digested wall enriched for
peptides. The wall components were separated on a new column,
which resulted in a marginal increase in retention times compared with
Fig. 4. Fractions were analyzed by MS and tested for stimulation of TNF
release from rabbit blood. Similar profiles were obtained irrespective
of whether double-digested walls (a) or single
amidase-digested walls (b) enriched for peptides were
tested.
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Quantitation of TNF-releasing Activity in Separate HPLC
Fractions--
To more precisely evaluate the specific TNF-releasing
activity of the material present in individual HPLC fractions (Fig. 4),
the content of these fractions was quantified as follows. First, the
relative contribution of each peak to the whole chromatogram was
determined by comparing their areas under the curve to that of the
whole HPLC profile using the D-7000 HPLC System Manager software
(Hitachi). This permitted the calculation of the percentage of material
present in each fraction as compared with the total material loaded on
the column. Using this information, the concentration of material
present in each fraction was then calculated, and the specific
TNF-releasing activity of the fractions was determined.
Table I presents these results. It indicates an appraisal of the
relative stimulatory potency of the various materials tested in the
present experiments by denoting the minimum concentration of stimulants
necessary to release at least a 10-fold increase in the TNF levels
above background (defined as the minimal stimulatory concentrations
10×, or MSC10×). It can be seen that the most active
peaks eluted between 38 and 60 min. Additional material eluting until
105 min was also active but less well resolved. Together, these active
fractions of the chromatogram represented only 2% of the total
material. However, their specific (w/w) activity approached that of
LPS.
For instance, the fraction containing the inactive peak 5 (Fig. 4)
represented ~150 ng/ml wall material, but this concentration released
only 500 pg/ml TNF in the cytokine stimulation assay. In contrast, the
active fractions containing peaks 13 and 20 (Fig. 4) represented only
~27 and 23 ng/ml wall material, respectively, but these
concentrations triggered the release of >3000 and 1800 pg/ml TNF from
PBMCs. This indicated a difference of at least 20-30-fold in the
specific activity of these two types of material.
On the other hand, material eluting before 37 min was poorly
active (peaks 1-8 in Table I), and represented another 43% of the
loaded material. The rest (45%) of the loaded material eluted after
105 min and was essentially inactive. Taken together, these results
indicated that the most active fractions in the solubilized pneumococcal were found in only 2% of the material.
Molecular Characterization of Specific HPLC Fractions--
The
HPLC fractions corresponding to peaks 1-21 were further analyzed for
their molecular content by amino acid and amino sugar analysis and for
the molecular weights of their components by MS. All of the HPLC
fractions contained the basic pneumococcal wall amino acids alanine,
glutamine, and lysine. Some peaks also contained lower amounts of
serine, glycine, and aspartic acid, which are amino acids previously
reported to be present in the pneumococcal wall (41). No aminosugars
were found, confirming that the present experiments preferentially
analyzed cell wall stem peptides.
The MS analysis is presented in Fig.
6. It indicates that most fractions
contained more than one component. Moreover, the Mr of these components increased with the
retention time, from 600 to 950 in peaks 1-8 (retention time 15-37
min), to 950-1500 in peaks 9-21 (retention time 38-55 min). This MS
spectrum was similar when run with five consecutive HPLC separations,
three analyzing double-digested walls (step 3A in Fig. 2) and two
analyzing peptide-enriched materials (step 3B in Fig. 2).

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Fig. 6.
Molecular weights of components found in the
different HPLC fractions of the solubilized wall. HPLC fractions
were analyzed by MS. The molecular weights of their various components
were determined and plotted against their retention times. In this
experiment, fractions 5, 13, and 20 contained a single component that
could be unambiguously analyzed by postsource decay (Table II). The
dashed line shows the retention time cut-off
above which high TNF-stimulating activity was observed, as depicted in
Fig. 4. The many components found at the beginning of the chromatogram
indicate that this fraction contained the flow-through.
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On one occasion, three fractions (peaks 5, 13, and 20 in Fig. 4) gave a
single major ion by MS (Fig. 7), thus
allowing further characterization by postsource decay analysis. Table
II presents the molecular structures of
these components as tentatively deduced from (i) their content in amino
acids, (ii) their measured and calculated molecular weights, and (iii)
their postsource decay spectrum. Peak 5, which carried a very low
inflammatory activity (Table I and Fig. 4), consisted of a dipeptide
previously described in the pneumococcal wall (42). Peaks 13 and 20, which carried TNF-releasing activities almost equal to that of LPS
(Table I and Fig. 4), consisted of two tripeptides. One of them (peak
20) resembled a previously described structure (43), whereas the other
(peak 13) was new.

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Fig. 7.
Mass spectra of peaks 5, 13, and 20. Matrix-assisted laser desorption ionization time of flight mass
spectrometry was performed on HPLC fractions corresponding to peaks
1-21. Peaks 5, 13, and 20 were pure as indicated by the presence of a
single major ion. Their molecular weights are indicated. Postsource
decay analysis was performed on these ions. Their deduced structures
are presented in Table II.
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Table II
Molecular weight, amino acid composition, and proposed structures of
components found in peaks 5, 13, and
20
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TNF-releasing Activity of Glycan-enriched Fractions--
In the
present experiments, the double-digested walls (step 3A in Fig. 2) and
the peptide-enriched materials (step 3B in Fig. 2) gave very similar
HPLC and MS.
This indicated that the present chromatography conditions
preferentially resolved the soluble wall peptides and did not provide much information on the activity of the glycan moiety contained in the
original material. To address this question more specifically, the
glycan-enriched fraction released during amidase digestion was
precipitated as described (step 3B in Fig. 2) and further tested for
its TNF-releasing activity before and after digestion with muramidase.
It is noteworthy, however, that this fraction did not only contain the
glycan backbone but also some residual peptides that invariably escape
detachment by amidase (41). Table III
presents the type of material tested and the results of this
experiment. First, insoluble and amidase-digested walls had comparable
TNF-releasing activities as already described. Second, the
glycan-enriched fraction had an unaltered TNF-releasing activity as
compared with insoluble and amidase-digested walls. Third,
muramidase-treatment of this glycan-enriched material resulted in a
100-fold decrease in its specific TNF-releasing power. These results
indicated a role for the integrity of the glycan backbone to ensure
TNF-releasing activity.
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DISCUSSION |
The present results unraveled the existence of very active
proinflammatory stem peptides in the pneumococcal peptidoglycan by
digesting it with its natural autolytic enzyme amidase. Moreover, quantitative and structural analysis of these stem peptides indicated that a minimal degree of cross-linking between them was necessary to
confer TNF-releasing activity. Indeed, one dimeric form of branched
peptides (peak 5 with a Mr of 744 in Fig. 3,
b and c), which represented a large fraction on
the chromatogram, carried no activity. Moreover, material with a lower
molecular weight did not carry inflammatory power either. This
suggested that simpler structures such as monomeric stem peptides,
which also exist in the pneumococcal wall (44), were not inflammatory.
This was in agreement with Timmerman et al. (40), who did
not detect TNF-stimulating activity with the synthetic monomeric
peptide L-alanyl-D-isoglutaminyl-L-lysyl-D-alanyl-D-alanine
acetate. On the other hand, wall components with a molecular weight
greater than 1000 were highly proinflammatory. Several of them had
specific (w/w) activities
100-fold greater than that of the total
wall and almost equal to that of LPS. Two of them were structurally characterized and identified as branched tripeptides.
In the present experiments, the very active wall fragments released by
digestion with amidase or digestion with amidase plus muramidase
represented only ~2% of the total soluble material. However, this
2% of material was 10-100-fold more active than the 98% of the
remaining wall. Therefore, it was tempting to speculate that this 2%
fraction comprised the totality of the active wall subcomponents.
Although plausible, this hypothesis might greatly oversimplify the
reality. Indeed, the wall of Gram-negative bacteria is complex and may
contain different types of proinflammatory subcomponents. During
enzymatic digestion, it is possible that certain of these inflammatory
constituents were destroyed, while others (such as branched stem
peptides) were revealed by making them accessible to the target cells.
For instance, Dokter et al. (27) reported that the
disaccharide tetrapeptide from E. coli peptidoglycan was
almost as powerful as LPS as an inducer of interleukin-6 and -1
secretion by PBMCs. In the present experiments, we did not find such
structures in the pneumococcal wall. However, it is noteworthy that
putative pneumococcal disaccharide tetrapeptides could have been
destroyed during amidase digestion, because this enzyme hydrolyzes the
bonds between N-acetylmuramic acid and the stem peptide. In
this case, they would not have been revealed in the present
experiments. Alternatively, it is also possible that disaccharide
tetrapeptides of pneumococci were less active than those of E. coli. Indeed, differences exist between these two constituents of
the wall. In many Gram-positive bacteria, such as pneumococci and
staphylococci, the third amino acid in the stem peptide is an
L-lysine, whereas this residue is a diaminopimelic acid in
E. coli. Whether this subtle difference might affect the
proinflammatory activity of these molecules is as yet undetermined.
However, Kengatharan et al. (45) recently reported that the
closely related disaccharide pentapeptide of S. aureus was
poorly proinflammatory on its own. A synergism between lipoteichoic
acids and disaccharide pentapeptide was required for this
staphylococcal component to be active.
The proinflammatory potential of cross-linked peptidoglycan stem
peptides has never been tested before. Nonetheless, an indirect clue
for the importance of stem peptide cross-linkage may be drawn from
previous experiments, utilizing another soluble wall material that held
stem peptides close together. This material, so-called soluble
peptidoglycan, was released during penicillin treatment of
staphylococci (23) and thus consisted of disaccharide-pentapeptide polymers linked together via 1-4
-glycosidic bonds, but not
interpeptide bridges (due to the action of penicillin). Staphylococcal
soluble peptidoglycan approached/resembled somewhat the pneumococcal
glycan-enriched fraction schematized in Fig. 2 (step 3B). Both of them
carried intrinsic inflammatory activity. However, both of them lost
their inflammatory power after destruction of the glycan chain with muramidase, a process that also disconnected the stem peptides.
While these experiments indicated the importance of glycan chain
integrity to ensure a proinflammatory activity, they also might help
reconcile previous results with soluble peptidoglycan and the present
observations with cross-linked peptides under a common concept. It is
possible that in order to stimulate target cells, both soluble
peptidoglycan and cross-linked peptides must present at least three
stem peptides in close proximity. Below this level of polymerization,
peptidoglycan fragments are inactive. Above this level of
polymerization, they are active. This hypothesis does not exclude the
existence of other inflammatory components in the wall, and/or the
existence of components that might interact in synergism with lesser
active fragments, as described by Kengatharan et al. (45).
However, if true, this hypothesis would provide an important clue as to
the structure-activity relationship of some very active constituents of
the Gram-positive peptidoglycan. Further studies with synthetic
peptides or disaccharide peptide oligomers will be necessary to confirm
or disprove this model.
In conclusion, the observation that complex peptidoglycan branched
peptides carried high TNF-stimulating activity was novel. The extent of
the existence of these peptides occurring freely during infection
remains to be seen. Nonetheless, amidase-induced autolysis has been
implicated in the pathogenesis of pneumococci (31). The fact that
trimeric forms of these peptides were a minimal requirement for
activity might provide a clue as to the structural constraint necessary
for target cell stimulation. These types of cross-linked peptides are
likely to be conserved in the walls of many different Gram-positive
pathogens. Moreover, although some intrinsic variation may exist
between different bacteria, these cross-linked peptides might be
structurally close enough to obey a common stimulatory pathway of
target cells. Hence, they might represent a common target for potential
anti-inflammatory agents. The existence of such a common stimulatory
pathway and of freely circulating cross-linked peptides in infections
is currently under investigation.