| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité Medizinische Fakultät der Humboldt-Universität zu Berlin, Berlin, Germany; and
Laborgruppe Immunchemie, Forschungszentrum Borstel, Borstel, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
synthesis in human monocytes
increasing with culture time. This activity could be reduced
significantly by blocking the CD14-part of the LPS receptor using the
My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW
264.7, Treponema culture supernatants induced TNF-
secretion in a LPS binding protein (LBP)-dependent fashion. To enrich
for active compounds, supernatants were extracted with butanol, while
whole cells were extracted using a phenol/water method resulting in
recovery of material exhibiting a similar activity profile. An LPS-LBP
binding competition assay revealed an interaction of the treponeme
phenol/water extracts with LBP, while precipitation studies implied an
affinity to polymyxin B and endotoxin neutralizing protein. Macrophages
obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4
mutation were stimulated with treponeme extracts for NO release to
assess the role of TLRs in cell activation. Furthermore, NF-
B
translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was
studied. We found that phenol/water-extracts of the two strains use
TLRs differently with T. brennaborense-stimulating cells
in a TLR-4-dependent fashion, while T.
maltophilum-mediated activation apparently involved TLR-2.
These results indicate the presence of a novel class of glycolipids in
Treponema initiating inflammatory responses involving
LBP, CD14, and TLRs. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Treponemes are strictly anaerobic bacteria not easily maintained in culture. By molecular genetic analysis, several formerly "noncultivable" treponemes have been identified recently (14, 15, 16), some of which were associated with human periodontitis, a chronic inflammation of the periodontium causing severe costs to health care systems (17), or digital dermatitis, a disease commonly found in cattle (15). Two Treponema species were used in this study, Treponema brennaborense and Treponema maltophilum (15, 16, 18). Previous analyses of Treponema denticola, another putative periodontal pathogen, have shown that its cell wall contained a glycolipid chemically different from LPS (19). In this study, we focus on the interaction of treponeme glycolipids and host cells.
The host response to bacterial compounds is regulated and modulated by
certain serum proteins and cellular receptor molecules
(20, 21, 22). LBP and soluble CD14, both able to bind LPS, are
present in serum in high quantities (23, 24). LBP is an
acute-phase protein synthesized in the liver, the concentration of
which rises dramatically during systemic infection and the acute-phase
response (25, 26). It monomerizes LPS vesicles and
transports LPS to the CD14 part of the cellular LPS receptor enabling
inflammatory responses, such as TNF- synthesis. LPS effects can be
blocked by a range of inhibitors including polymyxin B, a polypeptide
known to bind lipid A, the active moiety of LPS (27). LPS
effects can also be blocked by the mAb My 4 directed against CD14
(21). In the last years, it has been shown convincingly
that members of the Toll-like receptor (TLR) family are involved in the
recognition of pathogens by a wide variety of host organisms
(28). In Drosophila, Toll has been shown to be
involved in antifungal responses (29), while a homologous
protein, 18-wheeler, induces antibacterial responses (30).
In vertebrates, strong evidence has been presented that TLR-4
recognizes LPS of Gram-negative bacteria (31, 32, 33), while
TLR-2 recognizes PG of Gram-positive bacteria, as well as lipoproteins
of mycobacteria or Borrelia (34, 35, 36, 37, 38, 39). Regarding
LTA, results for an involvement of TLR-2 or -4 have been controversial
(34, 35, 40).
Here we analyze the ability of two Treponema species isolated from a patient suffering from periodontitis and from a digital dermatitis lesion, respectively, to activate human monocytes, a murine macrophage cell line, macrophages obtained from C3H/HeJ mice, and Chinese hamster ovary (CHO) cells. First, we analyzed the involvement of the host LPS binding and receptor molecules LBP, CD14, as well as TLR-2 and -4. Second, a partial chemical purification was performed for structural analysis of the active compound. The results presented here should help to elucidate the role of spirochetes in chronic inflammatory reactions and to identify the mechanisms involved.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Frozen stocks of T. brennaborense and T. maltophilum cells (300 µl, each stored at -80°C) were inoculated in 3 ml of culture medium (OMIZ-Pat) as described previously (16). Bacteria were cultured under anaerobic conditions (Anaerogen, Oxoid, Germany) at 37°C for 3–4 days. The cultures were then transferred to a larger volume of OMIZ-Pat (20–100 ml) and further incubated for 1–2 days. Viability of treponemes and possible presence of contaminating bacteria were assessed by dark field microscopy (400-fold magnification, BH2-RFCA microscope, Olympus, Hamburg, Germany). Sterility controls of the medium were performed by incubating OMIZ-Pat medium under aerobic and anaerobic conditions at 37°C for 1 wk. The pH value of the culture medium was measured repeatedly. Cultures were stopped at pH 6.0 and centrifuged at 12,000 x g at 4°C for 20 min. The supernatant was passed through 0.2-µm sterile filters (Schleicher & Schuell, Dassel, Germany). For some studies, culture supernatants were heat-inactivated at 100°C for 20 min and passed again through 0.2-µm sterile filters. OMIZ-Pat medium (16), treated similarly, was used as control.
Extraction of culture supernatants and whole Treponema cells
We used a modification of a published protocol for the extraction of LPS from Gram-negative cell walls using n-butanol (41). Briefly, filtered and heat-inactivated culture supernatants were mixed with an equal volume of n-butanol and incubated at 4°C for 1 h on a 180° shaker. Subsequently, the mixture was centrifuged at 26,000 x g at 4°C for 1 h, and the upper butanol phase was recovered. These steps were repeated once. Combined butanol phases were recentrifuged and lyophilized. For stimulation experiments, lyophilized extracts were dissolved in RPMI 1640 medium (Life Technologies, Eggenstein, Germany) to the original volume of supernatants before extraction. For silver stain analysis, butanol extracts corresponding to 1 ml of culture supernatant were dissolved in 50 µl of distilled water. OMIZ-Pat medium was processed similarly and used as control. For an extraction of whole treponeme cells, aqueous suspensions of treponeme cells were digested with RNase (Sigma, Deisenhofen, Germany), DNase (Merck, Darmstadt, Germany), and proteinase K (Merck). The suspensions were dialyzed and extracted using a hot phenol/water extraction method (42) or, after drying, a phenol/chloroform/petroleum ether (PCP) method (43). In brief, the phenol/water extraction was performed by mixing the cell suspension with an equal volume of 90% phenol and stirring at 68°C for 10 min. After cooling on ice, the mixture was centrifuged at 3000 x g for 10 min at 0°C, and the upper phase was collected. This procedure was repeated twice, and combined phases were dialyzed and lyophilized. PCP extraction was conducted with a mixture of 90% PCP in a volume ratio of 2.5:2.5:4. PCP extraction resulted in yields of 0.075% for T. brennaborense and 0.036% for T. maltophilum according to wet weight. By using the phenol/water method, higher yields were obtained (T. brennaborense, 0.42%; T. maltophilum, 0.47%), therefore, the latter material was used for additional experiments.
Stimulation of human monocytes and the murine macrophage cell line RAW 264.7
Peripheral blood samples obtained from healthy volunteers were mixed with 50 U/ml of heparin as anticoagulant and diluted 1:2 with RPMI 1640. Then 30 ml of the diluted blood was carefully layered on top of 15 ml Lymphoprep (Nycomed, Oslo, Norway) and centrifuged at 600 x g without brake at 21°C for 15 min. The intermediate phase was recovered, washed twice with RPMI 1640, and recentrifuged at 600 x g at 21°C for 5 min. To separate platelets, cells were further spun at 100 x g at 21°C for 15 min. Remaining cells were diluted in RPMI 1640 containing 5% human AB serum (Sigma) to a final concentration of 1 x 106 cells/ml. Cells were transferred to 96-well cell-culture plates (100 µl/well) and incubated at 37°C for 1.5 h, followed by two washing steps with RPMI 1640 to remove nonadherent cells. Remaining monocytes were stimulated with culture supernatants, butanol extracts of Treponema isolates, extracts of whole treponeme cells, Escherichia coli 0111:B4 LPS (Sigma), or PMA (Sigma) in the presence or absence of 5% human AB serum in a total volume of 100 µl. For certain experiments, polymyxin B (Sigma) at a concentration of 5 µg/ml was added directly before stimulation, as indicated. For selected experiments, monoclonal anti-CD14 Ab My 4 (Coulter, Hamburg, Germany) was incubated with the cells at a concentration of 5 µg/ml at 37°C for 20 min before addition of stimuli to block CD14. After 4 h, supernatants were harvested and viability of cells was assessed via trypan blue staining. Additionally, 5 x 104 RAW 264.7 cells per well (kindly provided by T. Blankenstein, Max-Delbrück-Centrum, Berlin, Germany) were cultured overnight in 96-well tissue culture plates using RPMI 1640 supplemented with 10% FCS. After repeated washing with RPMI 1640, stimulation was performed in the presence or absence of 1 µg/ml recombinant murine LBP (rmLBP) in a total volume of 100 µl. RAW 264.7 supernatants were harvested after 4 h of incubation, and cells were stained with trypan blue, ensuring integrity of cells.
Preparation and stimulation of peritoneal elicited macrophages (PEM)
Peritoneal macrophages were isolated from C3H/HeJ or C3H/HeN mice (Charles River, Sulzbach, Germany), by thioglycollate elicidation. Female 7-wk-old mice were injected i.p. with 1.5 ml of 3% thioglycollate broth (Sifin, Berlin, Germany). After 3 days, mice were sacrificed and peritoneal macrophages were harvested by injection of 10 ml of ice-cold HBSS (Life Technologies) i.p. followed by aspiration. Cells were washed twice with RPMI 1640, and 2 x 105 cells were plated in 96-well tissue culture plates in RPMI 1640 containing 5% FCS. After 2 h, plates were washed twice with RPMI 1640 to remove nonadherent cells, and remaining cells were stimulated with treponeme phenol/water extracts or LPS for 24 h in RPMI 1640 containing 5% non-heat-inactivated FCS followed by NO detection as described below.
Quantitative detection of human and murine TNF- and NO
Nunc MaxiSorp ELISA plates (Nunc, Roskilde, Denmark) were coated
with 0.5 mg/ml of anti-human TNF (anti-hTNF) Ab (PharMingen,
Heidelberg, Germany) in 100 mM NaHCO3, pH 8.3, and blocked
with PBS containing 0.05% Tween 20 and 10% FCS. Cell supernatants and
rhTNF standard (R&D Systems, Wiesbaden, Germany) in PBS containing 10%
FCS were incubated at 4°C overnight. Bound hTNF was detected using a
biotinylated mouse anti-hTNF Ab (PharMingen) at a concentration of
0.5 mg/ml. Subsequently, 1 µg/ml streptavidin peroxidase conjugate
(Sigma) was added with ortho-phenylene-diphosphate (Sigma) as
substrate. The detection limit of this assay was 
10 pg/ml. For
quantitation of murine TNF-, MaxiSorp ELISA plates were coated with
3 µg/ml anti-murine TNF (anti-mTNF) Ab (PharMingen) in 100 mM
Na3PO4, pH 6.0. Samples and
rmTNF standard (R&D Systems) were incubated at room temperature for
3 h, followed by detection with a biotin-conjugated
anti-mTNF- Ab (PharMingen) and streptavidin-peroxidase with
ortho-phenylene-diphosphate as substrate. The detection limit was 15
pg/ml. All in vitro TNF- results were assessed statistically by the
Student’s t test, and the inhibitory effects of polymyxin B
and My 4 as well as the enhancing effects of LBP were highly
significant (p < 0.001).
NO2- accumulation in culture medium was
assessed according to a published protocol (44). In brief,
100 µl of Griess reagent (Sigma) was added to 100 µl of culture
medium in 96-well plates and measured in a microplate reader at 540 nm
with a standard of NaNO2 diluted in RPMI 1640.
Estimation of NF-B translocation
CHO cells transfected with human CD14 (CHO/CD14, generously
provided by L. Hamann, Forschungszentrum Borstel, Germany)
(45) were cultured overnight in six-well tissue culture
plates at 4 x 105 cells per well with
Ham’s nutrient medium F12 (PAA Laboratories, Linz, Austria)
supplemented with 10% FCS and 400 µg/ml hygromycin B (Calbiochem,
San Diego, CA). Before stimulation, cells were starved in FCS-free
Ham’s medium for 3 h and incubated with LPS or treponeme extracts
in the presence of 2% non-heat-inactivated FCS. After 1 h, cells
were washed with ice-cold PBS containing 1 mM
Na3VO4 and incubated in 150 µl of buffer
A (1 mM Na3VO4, 10 mM HEPES, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1 mM NaF). After 20
min, cells were harvested mechanically, transferred to 1.5-ml tubes,
mixed with 25 µl Nonidet P-40, and centrifuged at 13,000 x
g at 4°C for 1 min. Pellets were resuspended in 50 µl of
buffer B (400 mM NaCl, 1 mM
Na3VO4, 20 mM HEPES, 1 mM
EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 1 mM NaF), incubated for 30
min at 4°C, and spun at 13,000 x g at 4°C for 5
min. Supernatants containing nuclear proteins were collected, and
nuclear extracts were analyzed by EMSA as described previously
(46) using two synthetic oligonucleotides (Eurogentec,
Seraing, Belgium) containing the NF-B binding sequence of the murine
Ig light chain gene enhancer.
Limulus assay
Treponema culture supernatants, both native and
butanol extracted, as well as extracts of whole cells were assayed for
endotoxin contamination by using a chromogenic Limulus
amoebocyte lysate (LAL) assay (LPS, Sinntal-Oberzell, Germany). The
endotoxin content of the OMIZ-Pat culture medium was below 2 ng/ml
regardless of whether treponemes were cultured or the medium was
incubated without bacteria for control. Butanol extracts of the culture
supernatants contained endotoxin of 25 pg/ml. The phenol/water
extracts of T. maltophilum and T. brennaborense
exhibited a LAL activity of 16.3 pg/µg corresponding to 0.16
endotoxin units (EU) and 35.2 pg/µg corresponding to 0.35 EU,
respectively.
Electrophoresis and silver staining of treponeme extracts
Stacking gels (5%) and separating gels (15, 16, and 20%, respectively) were prepared without SDS. Prestained and unstained low molecular mass markers ranging from 3 to 43 kDa (Life Technologies), 30 µl of culture supernatants, 30 µl of butanol extracts, and 30 µl of LPS solutions derived from E. coli 0111:B4 or Salmonella minnesota Re 595 LPS (Sigma) were boiled in sample buffer (2 ml 1 M Tris, 4 ml 1 M DTT, 800 mg SDS, 40 mg bromophenol blue, and 4 g glycerol ad 10 ml H2O) for 5 min, loaded onto the gels, and submitted to electrophoresis. Gels were stained with the silver stain plus kit (Bio-Rad, Munich, Germany) according to the manufacturers instructions. In addition to the original protocol, gels were oxidized with 0.7% periodic acid after fixation (47). For some experiments, glycolipids were hydrolyzed and/or dephosphorylated as explained below.
Chemical analysis of the phenol/water-extracted cell wall fractions of T. maltophilum and T. brennaborense
Phosphate was determined according to Lowry (73), and 3-deoxy-D-manno-octulosonic acid (Kdo) was estimated by the thiobarbituric acid method (43). Amino acids were identified as their phenyl isothiocyanate derivatives by reversed-phase HPLC using a Waters PICO-TAG system (Waters, Eschborn, Germany) under conditions described previously (48). Amino sugars were analyzed using HPLC (49). Gas-liquid chromatography (GLC) and combined gas-liquid chromatography/mass spectrometry (GLC-MS) were applied for the analysis of neutral sugar alditol acetates (50) and fatty acid methyl esters liberated after strong methanolysis (2 M HCl/MeOH, 120°C, 24 h) and extraction with chloroform. GLC was performed on a model 3700 Varian gas chromatograph (Varian Associates, Palo Alto, CA), and GLC-MS was performed on a Hewlett Packard 5989A instrument equipped with a gas chromatograph (model 5890 Series II, Hewlett-Packard, Palo Alto, CA) operating under identical conditions as for GLC. For structural analysis of treponeme glycolipids, 3.8 mg of T. maltophilum and 4.9 mg of T. brennaborense phenol/water extracts were dephosphorylated in 100 µl HF (48% by volume) at 4°C for 24 h in a sealed Teflon tube. Samples were extensively dialyzed (cut-off at 10–14 kDa) against water and lyophilized. The yields obtained were 1.3 mg for T. maltophilum and 1.8 mg for T. brennaborense. Dephosphorylated glycolipids were peracetylated in 0.8 ml pyridine/acetic acid anhydride (5:3 by volume, 85°C, 30 min) and subjected to GLC-MS analysis. For analysis of the glycosyl part, peracetalyted glycolipids were further purified on a silica gel column (3.5 x 1.5 cm, Kieselgel 60, Merck, 230–400 mesh), eluted with a stepwise gradient of increasing amounts of ethanol in toluene (1–50% by volume). For GLC-MS analysis, fatty acids were released from the glycolipid by alkaline hydrolysis (50 µl 0.5 M NaOH, 1 h at 65°C), and the product was permethylated (51). GLC-MS analysis of the permethylated and deacylated glycolipids was performed using a gradient of 150°C (3 min) to 330°C at 10°/min.
Binding of phenol/water extracts to rmLBP, endotoxin neutralizing protein (ENP), and polymyxin B
Binding of phenol/water extracts to mLBP was investigated with a slightly modified competition assay published elsewhere (52). Briefly, MaxiSorp ELISA plates were coated with E. coli 0111:B4 LPS. Free protein binding sites were blocked by incubation with 10 mg/ml BSA in 150 mM NaCl, 50 mM HEPES, pH 7.4, at 37°C for 30 min. Washing and dilution steps were performed with blocking buffer containing 1 mg/ml BSA. E. coli 0111:B4 LPS and S. minnesota Re 595 LPS, and phenol/water extracts of T. brennaborense and T. maltophilum were assayed for their ability to bind to 100 ng/ml mLBP by inhibiting binding of LBP to LPS-coated plates. LPS-bound LBP was detected by a polyclonal rabbit-anti-mLBP Ab and incubated with goat anti-rabbit IgG-Ab, conjugated with HRP (Biogenes, Berlin, Germany). Ortho-phenylene-diphosphate was used as a substrate. E. coli 0111:B4 LPS and treponeme phenol/water extracts were precipitated by polymyxin B-coupled Agarose beads (Sigma) or Sepharose beads conjugated with ENP (Associates of Cape Cod, Falmouth, MA), respectively. LPS and extracts in a volume of 500 µl at a final concentration of 10 µg/ml were mixed with 50 µl of polymyxin B beads or with ENP beads, respectively, as recommended by the manufacturer. Samples were incubated at 4°C for 24 h using a 180° shaker. After centrifugation at 3000 x g at 4°C for 10 min, supernatants were collected and loaded onto 15% SDS-PAGE gels, followed by silver staining as described above. Control samples were treated accordingly, however, without addition of any beads. Murine LBP was expressed in a baculovirus system and purified as described (26).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in myeloid cells by treponeme culture
supernatants in the presence of serum, polymyxin B, mAb My 4, or rmLBP
Treponema culture supernatants induced TNF- in
freshly isolated human monocytes. This activity increased with culture
time reaching a maximum at day 3 (Fig. 1
). OMIZ-Pat culture medium alone,
incubated with monocytes for the same period of time, failed to induce
any detectable amounts of TNF- (data not shown). For the following
experiments, bacteria were cultured for 3 days. Cultures were monitored
by pH measurement (6.0) to guarantee similar growth conditions. Both
viability and motility of treponemes were assessed by dark field
microscopy. To compare the activity of the treponeme supernatants with
LPS, we performed experiments with monocytes in the presence and
absence of serum. Cytokine induction caused by treponeme culture
supernatants increased significantly in the presence of 5% human serum
(Fig. 2A). As compared with
T. brennaborense, serum-independent stimulation was
significantly stronger for T. maltophilum culture
supernatants (Fig. 2A). Both polymyxin B and the inhibitory
monoclonal anti-CD14 Ab My 4 were able to significantly reduce
cytokine levels induced by both treponeme cultures. However, the effect
was more pronounced for LPS (Fig. 2B). The cytokine-inducing
activity of T. brennaborense and T.
maltophilum culture supernatants was inhibited in the presence of
polymyxin B or My 4 mAb at least by 50%. In contrast, polymyxin B and
My 4 did not influence cytokine induction caused by PMA, a phorbol
ester causing cytokine induction by activating protein kinase C
directly without receptor interaction (53) (data not
shown). To investigate LBP effects on cytokine induction caused by
treponemes, purified rmLBP and the murine macrophage cell line RAW
264.7 were used. In RAW 264.7 cells, TNF--induction caused by both
treponeme culture supernatants was significantly increased by addition
of mLBP (Fig. 2C).
|
|
Based upon the notion that the stimulatory activity found within
the supernatants shared characteristics with LPS, we purified the
compounds from culture supernatants as well as from whole cells using
extraction methods commonly used for LPS. Supernatants were treated
with butanol, while whole cells were subjected to phenol/water or the
PCP extraction. Yields obtained from whole cells with the phenol/water
method were clearly higher; therefore, this method was used in the
following experiments. To identify possible LPS contamination during
the preparation, a mock extraction including all media and chemicals
used during the procedure, was performed. TNF- induction in human
monocytes caused by butanol extracted Treponema supernatants
was clearly reduced by both polymyxin B and My 4 (Fig. 3A). In RAW 264.7, an
LBP-dependent cytokine induction was observed (Fig. 3B). In
contrast to the culture supernatants, no TNF- was induced in the
absence of LBP. Similarly, phenol/water extracts of whole treponeme
cells revealed a serum-dependent cell-stimulating capacity in human
monocytes (data not shown). However, to achieve a TNF- release
equivalent to that caused by LPS, the concentrations of the extracts
had to be increased by 1000-fold. Addition of polymyxin B and mAb My 4
led to a significant decrease of cytokine levels (Fig. 3C).
The cytokine induction in RAW 264.7 cells was greatly increased by the
addition of rmLBP (Fig. 3D). In case of T.
maltophilum, it was LBP dependent. In all experiments, the mock
extracts did not cause cytokine induction.
|
The role of TLR-4 in treponeme-mediated cell stimulation was
analyzed using PEM derived from LPS hyporesponsive C3H/HeJ mice, a
strain bearing a dominant negative mutation in the gene encoding TLR-4
(31). We isolated PEM from C3H/HeJ mice, as well as from
the control C3H/HeN strain normally responsive to LPS. Cells were
stimulated with increasing amounts of LPS and treponeme phenol/water
extracts, followed by measurement of NO. LPS exhibited a significantly
stronger stimulatory activity toward C3H/HeN as compared with C3H/HeJ
PEM (Fig. 4). While the mock extract
failed to stimulate cells, phenol/water extracts derived from T.
brennaborense revealed a stimulation pattern comparable to LPS,
leading to a significantly weaker NO release by C3H/HeJ macrophages as
compared with C3H/HeN cells. In contrast, extracts derived from
T. maltophilum led to a comparable NO production in PEM of
both strains, suggesting a less important role of TLR-4.
|
B in CHO cells by treponeme phenol/water
extracts
To elucidate the role of TLR-2 in treponeme-mediated signaling, we
investigated CHO cells. These cells carry a mutation for TLR-2 leading
to a defective receptor expression (54). CHO cells
transfected with human CD14 inducing responsiveness to LPS (CHO/CD14)
were stimulated with treponeme phenol/water extracts as well as with
LPS. LPS induced a strong translocation of NF-B in CHO/CD14 cells as
shown in an EMSA (Fig. 5). T.
brennaborense phenol/water extracts induced a translocation of
NF-B at concentrations of 1 µg/ml comparable to the LPS effect. In
contrast, T. maltophilum-derived extracts, at 1
µg/ml, failed to induce NF-B translocation, indicating an
involvement of TLR-2.
|
To identify and further characterize the active components in pure
or butanol-extracted treponeme culture supernatants, the material was
analyzed on 15% polyacrylamide gels that were silver stained
subsequently. Visible bands appeared in the range of about 4 and 6 kDa
in butanol-extracted supernatants from T. maltophilum and
T. brennaborense, respectively (Fig. 6A). The low molecular
material reflected rough LPS derived from S. minnesota Re
595 consisting only of lipid A and the inner core region with a size of
2.5 kDa (lane 1). The relative concentration present
in the butanol extract of T. brennaborense revealed that
this material contained about twice as much of the low molecular
compound as the T. maltophilum extract. Digestion of both
butanol extracts with pronase failed to change the profile of bands
observed in SDS-PAGE, suggesting a nonproteinaceous nature of the
immunostimulatory compound (data not shown). Phenol/water extracts of
both strains were analyzed by silver-stained SDS-PAGE, revealing
striking differences (Fig. 6B). The material obtained from
T. brennaborense displayed a ladder-like pattern similar to
that of smooth LPS presumably containing numerous repeating
carbohydrate units. In contrast, T. maltophilum extracts
exhibited few repeating units of larger molecular size. For both
butanol-extracted culture supernatants and phenol/water extracts of
whole cells, the size of their smallest units was similar.
|
The results of a chemical analysis of phenol/water extracts from
T. maltophilum and T. brennaborense are
shown in Table I. Most of the components
in both fractions could be analyzed and quantified (56% (w/w) for
T. maltophilum and 71% for T. brennaborense). As
expected from SDS-PAGE analysis, the amount of total fatty acids in the
smaller glycolipid of T. maltophilum was significantly
higher (8.3%, w/w) as compared with the high molecular T.
brennaborense glycolipid (2.8%). LPS-characteristic
ß-hydroxylated fatty acids, as well as Kdo and heptose, were
completely lacking in both strains. In T. maltophilum,
galactosamine was identified to be the main sugar component (13%),
whereas in T. brennaborense, glucose was identified as the
dominating sugar (50.8%). In both preparations, a characteristically
high amount of phosphate could be identified (5–10%). Besides traces
of contaminating residual amino acids, alanine was the only amino acid
identified in T. maltophilum, whereas T.
brennaborense completely lacked amino acids.
|
, A and B). To cleave phosphate-interlinked sugar
chains, glycolipids were dephosphorylated by HF, resulting in material
of 1 kDa size, which also could not be stained after KOH treatment
(Fig. 7, A and B). This result suggests the
presence of diacylglycerol as lipid anchor in both glycolipids and
excludes sphingosine and steroids. For identification of the lipid
anchor in the glycolipid of T. maltophilum, extracts were
peracetylated after dephosphorylation with HF. GLC-MS analysis (CI
mode) of the peracetylated glycolipids from T. maltophilum
revealed one major set of peaks
(tR 17 min) expressing
pseudomolecular ions
([M+NH4]+) of
m/z = 572 and 586, respectively. Their molecular masses
(Mr = 554 and 568 kDa, respectively)
are consistent with monoacetylated diacyl-glycerol carrying two
tetradecanoic acid residues (14:0) and one pentadecanoic acid (15:0),
respectively (Fig. 7C). However, attempts to identify a
similar lipid anchor in T. brennaborense by using the same
approach were unsuccessful, most likely due to the small proportion of
the lipid in the whole molecule (see Table I
).
|
D). Both permethylated glycosylglycerides were
present in 1:9 proportion. The electron impact-mass spectrometry of
the major permethylated glycosylglyceride showed diagnostic fragments
derived from the reducing part of the molecule (m/z =
307, 557) as well as those from the nonreducing part
(m/z = 464, 668), thus allowing the sequence of the
trisaccharide to be assigned to Hex-HexNAc-Hex-Gro (Fig. 7D). Despite several attempts, the structure of the glycosyl
part of the putative glycolipid isolated from T.
brennaborense could not be revealed. Binding of treponeme phenol/water extracts to mLBP, ENP, and polymyxin B
To analyze the potential interaction of Treponema
glycolipids with LBP, we performed competition assays with rmLBP (Fig. 8A). These studies revealed
that the glycolipids are able to compete with LPS-LBP binding. Both
extracts displayed a comparable affinity, while LPS derived from
S. minnesota Re 595 and LPS from E. coli 0111:B4
exhibited a stronger activity. Next, we investigated possible
interactions of Treponema extracts by precipitation with
polymyxin B and beads coated with ENP, an endotoxin binding protein
used in the Limulus assay. These experiments indicate that
the glycolipids studied interact with both, polymyxin B, and ENP, as
precipitation led to a marked reduction of bands shown in SDS-PAGE
analysis (Fig. 8B). While for ENP no differences were
observed comparing LPS and the treponeme extracts, the effect was less
pronounced for polymyxin B, especially regarding T.
maltophilum interaction.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Immunostimulatory elements of bacteria interact with soluble and cell-bound receptor molecules of the host organism, a key element of the host’s repertoire to modulate an inflammatory reaction. As we have shown recently, the acute-phase response to a systemic infection leading to elevated levels of the hepatic acute-phase protein LBP can greatly modulate the hosts response to a systemic challenge with LPS (26). It is likely that LBP serves for toxic Treponema cell wall products as well as a modulator in vivo and may be able to reduce or enhance the inflammatory reaction. Recent results by others and us provide evidence that LBP interacts not only with LPS, but also with other bacterial products, i.e., LTA (7). Furthermore, our results showing an involvement of the CD14 part of the LPS receptor are in agreement with CD14 acting as a pattern recognition receptor (56, 57).
The immunostimulatory treponeme cell wall compounds described here are
apparently released by live bacteria or after cell death. We present
evidence that the compounds retained from the supernatants correspond
to the glycolipids extracted from whole cells regarding size and
biological characteristics. After cell death, bacteria release
immunostimulating particles such as LPS in Gram-negative bacteria,
causing a strong inflammatory response potentially leading to septic
shock and subsequent death of the host (58, 59). In
Gram-positive bacteria, elements like PG and LTA of the outer cell
wall, released after cell disintegration often following antibiotic
treatment, also stimulate cytokine release in host cells (3, 60). However, for spirochetes, the predominant inflammatory
active element of their cell wall has not yet been clearly identified.
For some spirochetes like T. hyodysenteriae and T.
innocens, the presence of LPS-like molecules has been described,
while for others it has been clearly ruled out (10).
Borrelia burgdorferi fails to contain LPS in the cell wall
(8, 9); however, it contains PG (61) and a
set of outer membrane proteins eliciting inflammatory responses in
immune cells (62, 63, 64). It is likely that these proteins
are identical with lipoproteins described to induce TNF--synthesis
in human monocytes (12, 65). T. pallidum
possesses a number of TNF--inducing membrane proteins, and for this
spirochete the presence of LPS was definitively ruled out after the
completion of the whole genome sequence (8, 11, 66). Our
observations provide evidence that treponemes contain a glycolipid-like
material within their membranes that is chemically different from LPS
while exhibiting comparable biological characteristics including, for
one of our isolates, involvement of TLR-4.
Analysis of the role of TLRs is important in light of the recent paradigm of Gram-negative bacteria using TLR-4 via LPS and other bacteria stimulating cells via TLR-2. Recently, two immunostimulatory fractions isolated from the cell walls of T. denticola have been compared, lipoproteins on one hand and lipooligosaccharides, comparable to the glycolipids isolated in our study, on the other hand (67). Both fractions were able to induce cytokines and NO in host cells of normal and C3H/HeJ mice, suggesting a TLR-4-independent activity, which is in line with our results for T. maltophilum. Lipoproteins isolated from Borrelia recently also have been found to stimulate host cells via TLR-2 similar to lipoproteins isolated from mycobacteria (36, 37, 39). According to our results, active cell wall compounds from genetically closely related spirochetes stimulate cells via different TLRs. However, chemical analysis revealed differences in the composition of the glycolipids isolated. Thus structural differences may explain the differential use of pattern recognition receptors. While the T. brennaborense glycolipid contained significantly more carbohydrates and revealed the presence of a high number of small "repeating units" in silver gel analysis, T. maltophilum glycolipids displayed a small number of larger "repeats." Recently, the TLR-2/TLR-4 paradigm was questioned by two other studies showing involvement of TLR-4 in non-LPS-mediated cell stimulation. Viable mycobacteria, in contrast to isolated lipoarabinomannan, stimulated CHO cells overexpressing both TLR-2 or TLR-4 (68). A recent study comparing the TLR-2 and the TLR-4 knockout mouse provided evidence that LTA from Gram-positive bacteria also stimulate macrophages via TLR-4 (40). Furthermore, this group compared different types of LPS leading to a different degree of use of members of the TLR family (69).
We describe here the molecules involved in the reaction pattern of myelo-monocytic host cells to contact with cell wall components of recently identified spirochetes. Certain features of this interaction, i.e., involvement of LBP, CD14, and the use of TLR-4 by T. brennaborense, as well as the inhibitory effect of polymyxin B, resemble the cell stimulation pattern induced by LPS of Gram-negative bacteria. However, for polymyxin B, it recently has been shown that it interacts with numerous structures including phospholipids (70). Furthermore, the Treponema glycolipids described here bound to ENP, a protein usually considered to bind specifically to LPS. However, it is known that agents other than LPS cross-react in the Limulus assay, potentially due to similar physical properties (71, 72). Because the glycolipids described in this study interact with a range of other LPS-binding structures, it is likely that the discrete LAL activity observed is caused by the extracted compounds themselves and not by contaminating LPS. Furthermore, precipitation studies using ENP revealed a specific affinity of the treponeme extracts to this protein.
Our chemical analysis suggests a glycolipid structure in T.
maltophilum and T. brennaborense differing
significantly from that of LPS. This is based on the absence of
structural components characteristic for LPS, such as heptose, Kdo, and
ß-hydroxy fatty acids. In contrast, Treponema
glycolipids displayed LTA-like elements such as sugar, high phosphate,
and alanine similar to that previously identified in T.
denticola (19). This similarity was further supported
by isolation and analysis of the dephosphorylated glycosyl part of the
repeating units, being a hexasaccharide in T.
maltophilum and a glucan in T. brennaborense (data not
shown). Moreover, in T. maltophilum we identified two
glycolipids composed of Hex3Gro and
Hex-HexN-Hex-Gro (Fig. 7D). GLC-MS analysis of the lipid
anchor revealed two monoacetylated diacylglycerols, the predominant one
containing two tetradecanoic acids (14:0) (Fig. 7C). Our
interpretation that T. brennaborense contains a glycolipid
of similar structure is based on results obtained from SDS-PAGE (Fig. 7, A and B) and from TLC analysis (data not
shown).
Taken together our chemical results indicate that T.
maltophilum and T. brennaborense both exhibit a
glycolipid consisting of a diacylglycerol-lipid anchor, a core region,
in the case of T. maltophilum consisting of three sugars,
and carbohydrate repeating units (Fig. 7E). As indicated by
silver stain analysis, T. maltophilum exhibits a low number
of large repeating units, each being composed of 20–30 sugars,
while T. brennaborense contains a high number of small
repeating units, each being composed of 5 sugars. Like in T.
denticola (19), these glycolipids share structural
characteristics with LTA and apparently represent the major membrane
component. The differences in chemical composition between the two
strains are significant considering the close genetic relatedness of
both strains (15) and may be the cause for the different
interactions with TLRs. A more detailed chemical analysis will be
performed in our laboratories to further support this
interpretation.
Our data complement the list of bacterial cell wall components recognized by TLRs and CD14, explaining results by others showing a CD14 involvement for spirochete-mediated host cell stimulation (13, 64). The differential use of TLRs by the treponeme glycolipids may help in understanding basic mechanisms of innate immunity caused by spirochetes as well as other microorganisms.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Department of Lipid Biochemistry, Merck Research Laboratories, Rahway, NJ 07065.
3 Address correspondence and reprint requests to Dr. Ralf R. Schumann, Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Dorotheenstrasse 96, D-10117 Berlin, Germany.
4 Abbreviations used in this paper: LTA, lipoteichoic acid; CHO, Chinese hamster ovary; ENP, endotoxin neutralizing protein; Kdo, 3-deoxy-D-manno-octulosonic acid; LAL, Limulus amoebocyte lysate; LBP, LPS binding protein; OMIZ-Pat, Treponema culture medium; PCP, phenol/chlorophorm/petroleum ether; PEM, peritoneal elicited macrophages; PG, peptidoglycan; TLR, Toll-like receptor; h, human; m, murine; GLC, gas-liquid chromatography; MS, mass spectrometry.
Received for publication June 2, 1999. Accepted for publication June 20, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B activation. Eur. J. Immunol. 30:211.[Medline]
, signal transduction. J. Immunol. 161:3001. in a murine model of shock due to Streptococcus pyogenes. Crit. Care Med. 21:1207.[Medline]
B. Infect. Immun. 64:3845.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  G. Nussbaum, S. Ben-Adi, T. Genzler, M. Sela, and G. Rosen Involvement of Toll-Like Receptors 2 and 4 in the Innate Immune Response to Treponema denticola and Its Outer Sheath Components Infect. Immun., September 1, 2009; 77(9): 3939 - 3947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sterzenbach, S. K. Lee, B. Brenneke, F. von Goetz, D. B. Schauer, J. G. Fox, S. Suerbaum, and C. Josenhans Inhibitory Effect of Enterohepatic Helicobacter hepaticus on Innate Immune Responses of Mouse Intestinal Epithelial Cells Infect. Immun., June 1, 2007; 75(6): 2717 - 2728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Alugupalli, S. Akira, E. Lien, and J. M. Leong MyD88- and Bruton's Tyrosine Kinase-Mediated Signals Are Essential for T Cell-Independent Pathogen-Specific IgM Responses J. Immunol., March 15, 2007; 178(6): 3740 - 3749. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Zhang, Z. Liu, and K. E. Meier Lysophosphatidic acid as a mediator for proinflammatory agonists in a human corneal epithelial cell line Am J Physiol Cell Physiol, November 1, 2006; 291(5): C1089 - C1098. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mueller, C. Stamme, C. Draing, T. Hartung, U. Seydel, and A. B. Schromm Cell Activation of Human Macrophages by Lipoteichoic Acid Is Strongly Attenuated by Lipopolysaccharide-binding Protein J. Biol. Chem., October 20, 2006; 281(42): 31448 - 31456. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-H. Lee, K.-K. Kim, I.-C. Rhyu, S. Koh, D.-S. Lee, and B.-K. Choi Phenol/water extract of Treponema socranskii subsp. socranskii as an antagonist of Toll-like receptor 4 signalling Microbiology, February 1, 2006; 152(2): 535 - 546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. W.J. Schroder and R. R. Schumann Non-LPS targets and actions of LPS binding protein (LBP) Innate Immunity, August 1, 2005; 11(4): 237 - 242. [Abstract] [PDF]
|
|
|
|
|
|
S.-H. Lee, K.-K. Kim, and B.-K. Choi Upregulation of Intercellular Adhesion Molecule 1 and Proinflammatory Cytokines by the Major Surface Proteins of Treponema maltophilum and Treponema lecithinolyticum, the Phylogenetic Group IV Oral Spirochetes Associated with Periodontitis and Endodontic Infections Infect. Immun., January 1, 2005; 73(1): 268 - 276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. W. J. Schroder, H. Heine, C. Alexander, M. Manukyan, J. Eckert, L. Hamann, U. B. Gobel, and R. R. Schumann Lipopolysaccharide Binding Protein Binds to Triacylated and Diacylated Lipopeptides and Mediates Innate Immune Responses J. Immunol., August 15, 2004; 173(4): 2683 - 2691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-W. Lee, C.-S. Chien, and C.-M. Yang Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells Am J Physiol Lung Cell Mol Physiol, May 1, 2004; 286(5): L921 - L930. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. O. Chung and B. A. Dale Innate Immune Response of Oral and Foreskin Keratinocytes: Utilization of Different Signaling Pathways by Various Bacterial Species Infect. Immun., January 1, 2004; 72(1): 352 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.E. LeClair Four Reasons to Consider a Novel Class of Innate Immune Molecules in the Oral Epithelium Journal of Dental Research, December 1, 2003; 82(12): 944 - 950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hashimoto, Y. Asai, and T. Ogawa Treponemal Phospholipids Inhibit Innate Immune Responses Induced by Pathogen-associated Molecular Patterns J. Biol. Chem., November 7, 2003; 278(45): 44205 - 44213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Van Amersfoort, T. J. C. Van Berkel, and J. Kuiper Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clin. Microbiol. Rev., July 1, 2003; 16(3): 379 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. W. J. Schroder, S. Morath, C. Alexander, L. Hamann, T. Hartung, U. Zahringer, U. B. Gobel, J. R. Weber, and R. R. Schumann Lipoteichoic Acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus Activates Immune Cells via Toll-like Receptor (TLR)-2, Lipopolysaccharide-binding Protein (LBP), and CD14, whereas TLR-4 and MD-2 Are Not Involved J. Biol. Chem., April 25, 2003; 278(18): 15587 - 15594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Liu, T. Matsuguchi, N. Tsuboi, T. Yajima, and Y. Yoshikai Differences in Expression of Toll-Like Receptors and Their Reactivities in Dendritic Cells in BALB/c and C57BL/6 Mice Infect. Immun., December 1, 2002; 70(12): 6638 - 6645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lorenz, D. D. Patel, T. Hartung, and D. A. Schwartz Toll-Like Receptor 4 (TLR4)-Deficient Murine Macrophage Cell Line as an In Vitro Assay System To Show TLR4-Independent Signaling of Bacteroides fragilis Lipopolysaccharide Infect. Immun., September 1, 2002; 70(9): 4892 - 4896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Applequist, R. P. A. Wallin, and H.-G. Ljunggren Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines Int. Immunol., September 1, 2002; 14(9): 1065 - 1074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Vasselon and P. A. Detmers Toll Receptors: a Central Element in Innate Immune Responses Infect. Immun., March 1, 2002; 70(3): 1033 - 1041. [Full Text] [PDF]
|
|
|
|
 |
|
 P. I. Song, T. A. Abraham, Y. Park, A. S. Zivony, B. Harten, H. F. Edelhauser, S. L. Ward, C. A. Armstrong, and J. C. Ansel The Expression of Functional LPS Receptor Proteins CD14 And Toll-Like Receptor 4 in Human Corneal Cells Invest. Ophthalmol. Vis. Sci., November 1, 2001; 42(12): 2867 - 2877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fan, R. D. Ye, and A. B. Malik Transcriptional mechanisms of acute lung injury Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1037 - L1050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B W Jones, K A Heldwein, T K Means, J J Saukkonen, and M J Fenton Differential roles of Toll-like receptors in the elicitation of proinflammatory responses by macrophages Ann Rheum Dis, November 1, 2001; 60(90003): iii6 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. W. Jones, T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle, and M. J. Fenton Different Toll-like receptor agonists induce distinct macrophage responses J. Leukoc. Biol., June 1, 2001; 69(6): 1036 - 1044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. W. J. Schroder, D. Pfeil, B. Opitz, K. S. Michelsen, J. Amberger, U. Zahringer, U. B. Gobel, and R. R. Schumann Activation of Mitogen-activated Protein Kinases p42/44, p38, and Stress-activated Protein Kinases in Myelo-monocytic Cells by Treponema Lipoteichoic Acid J. Biol. Chem., March 23, 2001; 276(13): 9713 - 9719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Opitz, N. W. J. Schroder, I. Spreitzer, K. S. Michelsen, C. J. Kirschning, W. Hallatschek, U. Zahringer, T. Hartung, U. B. Gobel, and R. R. Schumann Toll-like Receptor-2 Mediates Treponema Glycolipid and Lipoteichoic Acid-induced NF-kappa B Translocation J. Biol. Chem., June 15, 2001; 276(25): 22041 - 22047. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
