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*
Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;
Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
Eijkman Winkler Institute of Medical and Clinical Microbiology, University of Utrecht, Utrecht, The Netherlands;
Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;
¶ Boston University School of Medicine, Boston, MA 02118;
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Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway;
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Department of Pediatrics, University of California at San Diego, La Jolla, CA 92093; and
**
Department of Pediatrics, Free University, Berlin, Germany.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, the secretion of TNF-
, and the
formation of NO in mouse macrophages. Macrophages from mice with a
targeted disruption of MyD88 failed to secrete TNF- in response to
both heat-inactivated whole bacteria and GBS-F, suggesting that
Toll-like receptors (TLRs) are involved in different aspects of GBS
recognition. Immune cell activation by whole bacteria differed
profoundly from that by secreted GBS-F. Whole GBS activated macrophages
independently of TLR2 and TLR6, whereas a response to the secreted
GBS-F was not observed in macrophages from TLR2-deficient animals. In
addition to TLR2, TLR6 and CD14 expression were essential for GBS-F
responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved.
Heat lability distinguished GBS-F from peptidoglycan and lipoproteins.
GBS mutants deficient in capsular polysaccharide or 
-hemolysin had
GBS-F activity comparable to that of wild-type streptococci. We
suggest that CD14 and TLR2 and TLR6 function as coreceptors for
secreted microbial products derived from GBS and that cell wall
components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or
6. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expeditious elimination of microorganisms like GBS that invade from mucosal surfaces is a primary goal of the innate immune system. APCs recognize traces of microbial components and subsequently orchestrate the antibacterial defense. The crucial sensory function is assigned to pattern recognition receptors that distinguish self from conserved microbial structures shared by different pathogens (4, 5). Mammalian Toll-like receptors (TLRs) lately have been identified as type I transmembrane signaling receptors with pattern recognition capabilities (6). Recent observations attributing the dependence of mammalian immune responses to LPS, the major cell wall component of Gram-negative bacteria, to the expression of functional TLR4 (7, 8, 9, 10) serve as a paradigm for our understanding of the biology of TLRs. Previously enigmatic observations that the inability to respond to LPS correlated to reduced resistance to Gram-negative bacterial infections can now be understood in this context.
Although Gram-positive and Gram-negative bacterial sepsis are due to distinct microorganisms, the similarities in the physiological consequences of invasive infection with the bacteria are extraordinary. There is extensive evidence that the immediate immune response to Gram-negative bacterial invasion is mediated by LPS, but no surface equivalent of LPS in Gram-positive bacteria has ever been identified. GBS subcellular cell wall components include its capsular polysaccharide, peptidoglycan, and lipoteichoic acid and have all been reported to have proinflammatory activities in vitro (11, 12). TLR2 has been identified as a receptor that is central to the innate immune response to several whole Gram-positive bacteria, as well as a receptor for peptidoglycan and lipoteichoic acid (13, 14, 15, 16). Although it is an attractive hypothesis that capsular polysaccharide, peptidoglycan, and lipoteichoic acid are responsible for the immune activity associated with GBS infection, none of these bacterial products is a potent inducer of cytokine production. Moreover, we failed to observe a role for TLR2 in the innate immune response to killed GBS, despite its content of peptidoglycan and lipoteichoic acid (17). This suggests that the ability of peptidoglycan and lipoteichoic acid to activate cells via TLR2 and the ability of whole organisms to activate the innate immune system are not causally related.
To further delineate the capabilities of different TLRs to discriminate microbial products, we hypothesized that GBS would be recognized by both known and novel Toll receptors. We specifically examined the role of the TLR adapter molecule MyD88 and the receptors TLR1, 2, 4, and 6 using the companion approaches of cellular transfection with cDNA constructs (gain of function) and the examination of macrophages from genetically deficient animals (loss of function). We found that GBS engagement of TLR2 and TLR6 is essential for cells to respond to the presence of bacteria, but that this recognition is due to a novel secreted (or shed) bacterial product. We hypothesize that this secreted factor might prove to be a toxin that can systemically activate immune cells in vivo and contribute to the sepsis syndrome in newborn infants. In contrast to the effects of this soluble product, GBS cell walls appear to engage TLRs different from TLR2 and TLR6, although the precise bacterial ligand, as well as the TLR that is activated, have not yet been determined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBS, Ham’s F-12 medium, RPMI 1640, and trypsin-versene mixture (trypsin-EDTA) were from BioWhittaker (Walkersville, MD). Low endotoxin FBS was from Summit Biotechnology (Greeley, CO). Ciprofloxacin was a gift from Miles Pharmaceuticals (West Haven, CT). Hygromycin B was purchased from Calbiochem (San Diego, CA), puromycin and polymyxin B were purchased from Sigma-Aldrich (St. Louis, MO), and G418 was from Life Technologies (Gaithersburg, MD). Protein-free LPS from Escherichia coli K 235 was a gift from S. Vogel (Uniformed Services University of the Health Sciences, Bethesda, MD), immunoaffinity purified native OspA from Borrelia burgdorferi was a gift from J. Radolf (University of Connecticut Health Center, Farmington, CT), and soluble peptidoglycan from Staphylococcus aureus was donated by R. Dziarski (Indiana University, Gary, IN). Microcentrifuge filters were purchased from Sigma- Aldrich. The LPS antagonist B1287 was a gift from the Eisai Research Institute (Andover, MA).
Cell lines
The Chinese hamster ovary (CHO)/CD14.ELAM.tac reporter cell line
(clone 3E10) is stably transfected with human CD14 and expresses
inducible membrane CD25 (tac Ag) under the transcriptional control of
the NF-B-dependent human E-selectin promoter (18). The
CHO/CD14/huTLR2.Elam.tac cells were constructed by stable
cotransfection of CHO/CD14.ELAM.tac with the cDNA for human TLR2
(huTLR2), together with the neor-containing
plasmid pcDNA3 (Invitrogen, San Diego, CA) as described
(14). CHO cell lines were grown in Ham’s F-12 medium
containing 10% FBS and 10 µg/ml ciprofloxacin. CHO/CD14.Elam.tac
were cultured with 400 U/ml hygromycin B; CHO/CD14/huTLR2.Elam.tac were
cultured with hygromycin plus 0.5 mg/ml G418. The LPS-resistant
nonresponder CHO/CD14 cell line, clone 7.19 (as described in Ref.
18) was transfected with huTLR2 (7.19/TLR2). Clone 7.19
carries a point mutation resulting in a C95Y amino acid exchange for
the TLR4 coreceptor, MD-2, that renders it unresponsive to LPS
(19). The 7.19/TLR2.Elam.tac was cultured with hygromycin
and G418.
Bacterial strains, the generation of bacterial supernatant, and inactivated GBS
The -hemolytic GBS type III strains COH1and M781, initially
isolated from newborn infants with sepsis, have been previously
described (20, 21). Unless otherwise stated, experiments
were performed with strain COH1. The nonhemolytic mutant COH1-20, an
isogenic derivative of strain COH1, was generated by a single insertion
of Tn916
E into its chromosome (22). All strains were
grown overnight on blood agar plates (REMEL, Lenexa, KS). Bacterial
colonies were removed from the plates in stationary phase, washed three
times with PBS, and then used to inoculate chemically defined medium
(23) prepared from endotoxin-free water or, where
indicated, RPMI 1640 with 10% FBS to
ABS650 = 0.03 and grown to midlog phase
(ABS650 = 0.27–0.30). For the culture of strain
COH1-20, the medium was supplemented with chloramphenicol (5 µg/ml)
and the nonhemolytic phenotype was confirmed in each experiment (blood
agar). Subsequently, cultures were clarified of bacteria by
centrifugation and were filtered (0.2-µm pore diameter). For some
experiments, the GBS supernatant was size fractionated by spin filters
using molecular mass cutoff at 30, 100, and 300 kDa.
Heat-inactivated GBS was prepared from midlog phase cultures, washed
three times with PBS, resuspended at a density of 3 x
1010 CFU/ml, and heat-inactivated (30 min,
80°C). Cell-free supernatants and heat-inactivated bacteria were
stored at -80°C for further use. Endotoxin was not detectable in the
above preparations using a Limulus assay with a sensitivity
of <0.6 EU/ml (BioWhittaker). Quantitative protein
determination in GBS supernatants was performed using the Bio-Rad
Protein Assay per the manufacturer’s protocol (Bio-Rad, Hercules,
CA).
Purification of the GBS supernatant by anion exchange column chromatography
Filtered GBS supernatant was concentrated 20-fold using a PREP/SCALE Cartridge (Millipore, Bedford, MA) diluted 1/3 in 10 mmol Tris/EDTA (pH 8.0) and loaded on a MonoQ anion exchange column (Pharmacia, Peapack, NJ) using fast protein liquid chromatography (Äkta) and Unicorn 3.00 software (both from Amersham Pharmacia Biotech, Uppsala, Sweden). Bound proteins were eluted in a total volume of 80 ml with a linear NaCl gradient up to 1 M. Fractions were screened for soluble factor released by GBS (GBS-F) activity with 7.19/TLR2 cells for induction of CD25 expression.
Transient transfection
Transient transfection was performed using the SuperFect reagent
(Qiagen, Valencia, CA) according to the manufacturer’s protocol. HEK
293 cells were plated at a density of 0.5 x
106 cells/well in six-well dishes and incubated
for 18 h. Plasmid DNA was added to 100 µl of serum-free DMEM per
well. For each transfection, a total of 2.5 µg of plasmid DNA per
well, consisting of 1 µg of a previously described reporter plasmid
(pELAM-luc) (10), together with 1 µg of huTLR2 in pcDNA3
and 0.5 µg of human MD2 in the mammalian expression plasmid pEFBOS
(24) were used. The next day, the cells were stimulated
for 5 h and cytoplasmic extracts were prepared as described by the
manufacturer (Promega, Madison, WI). The response to stimulation was
measured by assessing luciferase activity using luciferase substrate
(Promega) and a Monolight 3010 luminometer (BD PharMingen, San Diego,
CA), per the manufacturer’s protocol. Depicted in Fig. 6
are the mean
values ± SD expressed as fold induction above background. Each
point was assayed in triplicate; one representative of three
experiments is shown.
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MyD88-/- (25),
TLR2-/-, TLR2+/-
(16, 26), and TLR6-/-
(27) mice were engineered as described. The generation of
the TLR1-/- mice will be described in an independent
manuscript. MyD88-/-,
TLR2-/-, TLR2+/-, and
TLR6-/- mice, Chinese hamsters (Cytogen,
Boston, MA), Golden Syrian hamsters (Charles River Breeding
Laboratories, Wilmington, MA), C57BL/6J, C3H/HeOuJ, and C3H/HeJ mice
(The Jackson Laboratory, Bar Harbor, ME) were injected i.p. with 2.5 ml
(mice and Chinese hamsters) or 10 ml (Syrian hamsters) of 3%
thioglycolate (REMEL). After 3 days, peritoneal exudate cells were
harvested by lavage with RPMI 1640 medium containing 10% FBS and 10
µg/ml ciprofloxacin. The cells were washed with medium, counted in a
hemocytometer, and plated at a density of 1 x
106 cells/well in six-well dishes (for NF-B
assays) or 1 x 105 cells/well in 96-well
dishes (for the determination of cytokine release and NO). After 24–72
h, nonadherent cells were removed by washing with medium and adherent
cells were stimulated.
Isolation of PBMCs and measurement of TNF-
Human PBMCs were isolated by gradient centrifugation of
heparinized blood from healthy donors on Histopaque 1077
(Sigma-Aldrich) according to the manufacturer’s protocol. The cells
were resuspended in RPMI 1640 medium containing 10% FBS and plated at
a density of 2 x 106/ml in a 96-well dish.
For blocking experiments, PBMCs were preincubated with 10 µg/ml of
the human CD14 mAb 3C10 (American Type Culture Collection, Manassas,
VA) or the control mAb 6H8, which recognizes a widely distributed
180-kDa glycoprotein (T. A. Espevik and B. Naume, unpublished
observations), for 30 min at room temperature before further
stimulation. After addition of the indicated preparations, incubation
proceeded for an additional 16 h at 37°C and 5%
CO2. Supernatants were collected and stored
at -80°C until assayed for TNF- concentrations with a commercial
ELISA for human TNF- (R&D Systems, Minneapolis, MN).
Measurement of proinflammatory activity of peritoneal macrophages
Nuclear translocation of NF-B was determined as follows.
Peritoneal exudate macrophages (PEMs; 1 x
106/well in six-well dishes) were incubated for
2 h as indicated in the figures. For coculture experiments,
macrophages (1 x 106/well) were seeded in
the lower compartment and midlog GBS in the upper compartment of a
Transwell dish (Costar, Cambridge, MA), in which compartments are
separated by a 0.4-µm membrane. Macrophages/bacteria were cultured in
a total of 3 ml of RPMI 1640 plus 10% FBS/well for 16 h. Nuclear
extracts were subsequently isolated and analyzed for binding to a
32P-labeled NF-B-specific oligonucleotide by
EMSA, exactly as described (28). Depicted in Figs. 1 and 3 is one representative experiment of three. For determination of
TNF- and NO, RAW 267.4 cells or peritoneal exudate cells (1 x
106 cells/ml in 96-well dishes) were incubated
for 16 h at 37°C in a 5% humidified CO2
environment. Supernatants were processed directly for the determination
of released TNF- by ELISA (R&D Systems) and NO by Griess reaction
(naphtylethylenediamine, sulfanilamide, and sodium nitrite were all
from Sigma-Aldrich), per the manufacturers’ protocols. Depicted
are the means ± SD of triplicates from one representative
experiment of three.
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Cells were plated at a density of 7 x 104/well in 24-well dishes. On the next day, the cells were stimulated as indicated in Ham’s F-12 medium containing 10% FBS (total volume of 0.5 ml/well) and incubated for 18 h. Subsequently, the cells were harvested with trypsin-EDTA, spun down by centrifugation at 800 x g for 5 min, and labeled with FITC-labeled anti-human CD25 mAb (BD Biosciences, Mountain View, CA) in PBS/1% FBS for 30 min on ice, as described (18). The cells were analyzed by flow cytometry using a FACScan microfluorometer. Data were analyzed with CellQuest software (BD Biosciences). Depicted are representative results from three or more experiments.
Sequencing of TLR2 from Syrian hamsters
Genomic DNA was extracted from the livers of Chinese and Syrian hamsters. Extirpated tissue was homogenized in Trireagent, and DNA was separated from RNA by a 1-bromo-3-chloropropane (Molecular Research Center, Cincinnati, OH) gradient. A 2-µl portion of the resulting DNA was used as a template in a 25-µl PCR as described (29). The PCR was conducted in an automatic thermal cycler (Thermo Hybaid, Franklin, MA) using primers for hamster TLR2 (5'-ATCTCATTTCGTTTTCTTTG and 3'-GATCTTCATGACCAGTAACAC) that flank the previously described mutation (30). The resulting product was extracted from a 2% agarose gel using a QIAEX II gel extraction kit (Qiagen) and sequenced at the Boston University Core Facility (Boston, MA) using an ABI 373A automated sequencer (Applied Biosystems, Foster City, CA). The result was confirmed in an independent PCR experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address the question of whether the proinflammatory effect of
GBS was restricted to a physical interaction between bacteria and host
cells, murine macrophages (RAW 267.4 and peritoneal macrophages) were
stimulated with heat-inactivated GBS, with live GBS (harvested during
logarithmic growth) separated from the macrophages by a 0.4-µm
membrane, or with filtered supernatants of GBS harvested during log
growth. Heat-inactivated whole GBS induced secretion of TNF- and NO
in a dose-dependent manner (Fig. 1
, A and D). Interestingly, coculture of live GBS
with macrophages in a Transwell dish system that prevents direct
interaction between bacteria and macrophages induced nuclear
translocation of NF-B (Fig. 1B). This effect could be
reproduced by using cell-free bacterial supernatants of GBS that
induced the secretion of both NO and TNF- at concentrations as low
as 0.5% (v/v) (bacterial supernatant/total volume; Fig. 1, C and D). This finding strongly suggested that
GBS secrete one or more proinflammatory factors. The activation
observed was not due to the production of -hemolysin, because the
nonhemolytic GBS strain COH1-20 induced the translocation of NF-B
and formation of TNF- similarly, in comparison with the
-hemolytic parental strain COH1 (Fig. 1, B and
C). The proinflammatory effect of both bacterial
preparations was designated GBS-F. GBS-F was not significantly
inhibited by addition of the TLR4 antagonist B1287, a lipid A analog
that completely blocked activation by LPS (Fig. 1C).
Supernatants of GBSIII serotype M781 induced a quantitatively similar
proinflammatory response in RAW 267.4 cells when compared with
strain COH1 (data not shown). Thus, GBS release a proinflammatory
factor that is not -hemolysin.
The secretion of TNF- in response to GBS and GBS-F was abrogated
in peritoneal macrophages from mice with a targeted deletion of the TLR
adapter protein MyD88
The adapter protein MyD88 has been identified as a signal
transduction molecule linking TLRs to NF-B and/or protein kinase
activation. To test whether MyD88 was involved in the cellular response
to heat-inactivated GBS and GBS-F, PEMs from normal C57BL/6J mice and
MyD88-/- mice were exposed to either whole
heat-inactivated GBS or soluble GBS-F. Whereas whole GBS and GBS-F
induced secretion of TNF- in normal PEMs, both GBS and GBS-F failed
to induce significant TNF- secretion by
MyD88-/- macrophages, suggesting that one or
multiple TLRs are necessary for activation by whole bacteria as well as
soluble GBS-F (Fig. 2). Macrophages from
MyD88-/- and C57BL/6J mice did not differ in
their ability to internalize whole streptococcal organisms (data not
shown).
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The finding that responses to GBS-F and GBS were MyD88 dependent
prompted an effort to define which Toll-like receptors were involved in
GBS-induced activation. Our laboratory has previously reported that
macrophages from Chinese hamsters exhibit a frameshift mutation at bp
1758 in the TLR2 gene that results in a premature stop codon in the
predicted extracellular portion of TLR2. The resultant TLR2 transcript
fails to produce functional TLR2 (30). We functionally
characterized TLR2 of elicited peritoneal exudative macrophages from
Golden Syrian hamsters. Macrophages from Golden Syrian hamsters, unlike
Chinese hamster cells, responded to TLR2 ligands including soluble
peptidoglycan from S. aureus and nOspA, a lipoprotein from
B. burgdorferi, as indicated by the inducible translocation
of NF-B (data not shown). Sequence analysis of PCR products that
were amplified using flanking primers and genomic DNA from hamster
liver as template revealed a normal sequence for Syrian hamster TLR2.
The results contrast to the previously reported frameshift mutation in
Chinese hamster DNA (30).
Macrophages from Chinese and Golden Syrian hamsters were exposed to
whole GBS and soluble GBS-F. Macrophages from Golden Syrian hamsters,
but not from the TLR2-negative Chinese hamsters, translocated NF-B
in response to GBS-F. Both types of macrophages responded similarly to
whole, heat-inactivated GBS as well as to LPS (Fig. 3A). We next tested peritoneal
macrophages from C3H/HeJ mice, an LPS hyporesponder strain that
expresses a dominant-negative TLR4 (8). Peritoneal
macrophages from C3H/HeJ and normal C3H/HeOuJ mice responded equally to
GBS and GBS-F (Fig. 3B). These data expanded our previous
findings that macrophages from C3H/HeJ and normal C3H/HeOuJ mice are
equally activated by lipoteichoic acid and protein-free GBS cell walls
(31). Thus, expression of TLR2, but not TLR4, appeared to
be essential for responses to GBS-F. However, expression of neither
TLR2 nor TLR4 was critical for NF-B translocation in response to
whole, heat-inactivated streptococcal organisms.
We confirmed the TLR2 dependence of the soluble GBS-F, but not whole,
heat-inactivated GBS, using peritoneal macrophages from
TLR2-/- mice by assessing the induced secretion
of TNF- as a marker of proinflammatory activity (Fig. 4, A and C). TLR2
expression was essential for nuclear translocation of transcription
factors and secretion of proinflammatory cytokines in response to
GBS-F, but not to whole, heat-killed GBS. The lack of a significant
TLR2 engagement by whole GBS appeared not to be simply due to heat
treatment of GBS, in that ethanol-inactivated and washed GBS (70%
ethanol, 45 min) activated TLR2-/- and
TLR2+/- peritoneal macrophages similarly (data
not shown).
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, B and C). These
data are consistent with the concept that TLR2, TLR6, and MyD88 are
portions of a single receptor complex that recognizes GBS-F.
To complement these studies in macrophages from genetically engineered
animals, TLR2 was tested as a signaling molecule in heterologously
transfected cell lines. GBS-F failed to induce translocation of NF-B
in CD14-transfected CHO cells (which are null for TLR2)
(30). In contrast, these cells responded to concentrations
as low as 10 pg/ml LPS (data not shown). However, stable transfection
into a CHO/CD14 background of huTLR2, but not huTLR4, conferred
responsiveness to GBS-F (Fig. 5). We PCR
amplified and partially sequenced TLR6 from a CHO-K1 cDNA library (data
not shown) by using cross-species primers derived from the known
sequences of mice and humans. Hence, CHO-K1 cells transcribe endogenous
TLR6 that may serve as part of a signaling complex if coexpressed with
exogenous TLR2.
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CD14 is a 55-kDa glycosylphosphatidylinositol-linked protein that
functions as a coreceptor for numerous bacterial products, including
LPS, peptidoglycan, and bacterial lipoprotein. Hence, we tested whether
signaling in response to GBS-F was inhibited by the CD14 mAb 3C10. We
found a significant reduction of the GBS-F-induced TNF secretion from
human PBMCs by preincubation with 3C10, but not with the control, mAb
6H8 (Fig. 6, A).
Another potential coreceptor both for TLR2 and TLR4 in response to
peptidoglycan and LPS is the secreted protein MD2 (33, 34). We investigated the role of MD2 in GBS-F-induced signaling
using two experimental approaches. First, we tested an LPS nonresponder
cell line, clone 7.19 (18), which we have recently
determined to contain a mutation in MD-2 (19). This cell
line was further engineered to express huTLR2 and is referred to as
7.19/TLR2. The 7.19/TLR2 cell line was found to be activated by GBS-F,
but not by LPS (Fig. 6, B). Thus, a mutation in MD2 that
abrogated LPS-induced signaling did not significantly interfere with
NF-B translocation in response to soluble GBS-F. Second, we tested
whether expression in HEK 293 cells of MD2 together with TLR2 would
amplify the response to GBS-F when compared with expression of TLR2
alone. Whereas cotransfection of MD2 and TLR4 dramatically enhanced the
response to LPS when compared with TLR4 alone (data not shown), MD2
expression did not significantly affect the response of HEK 293 cells
to GBS-F (Fig. 6, C).
GBS-F is heat labile with a molecular mass that exceeds 30 kDa
Certain cell wall components of Gram-positive bacteria, including
peptidoglycan and lipoproteins, have been demonstrated to activate
cells in a TLR2-restricted manner. To exclude these cell wall
components as immunologically active substances in GBS supernatant and
to further investigate the nature of GBS-F, we subjected the filtered
GBS supernatant to heat treatment (100°C, 15 min). Heat treatment
abrogated the response of CHO/CD14-TLR2 to GBS-F, but not to soluble
staphylococcal peptidoglycan or the lipoprotein nOsp A from B.
burgdorferi (Fig. 7, upper
panel, inset).
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5 µg/ml total protein. Biological activity was
generally observed at a 1/100 dilution of such cultures, suggesting
that GBS-F is capable of activating cells at concentrations as low as
50 ng/ml or less. Furthermore, we subjected the supernatant to spin
filters that separate fractions of different molecular mass. Fractions
>30 kDa, but not those <30 kDa, activated the CHO/CD14-TLR2 cells,
and most of the activity was observed at a molecular mass cutoff of
<100 kDa (Fig. 7, lower panel). Similarly, these fractions
were far more potent than other fractions with respect to activation of
RAW 267.4 cells tested in a TNF- release assay (data not shown).
Anion exchange chromatography also suggested that GBS-F is negatively
charged, although the active column fractions consisted of more than a
single band on an SDS-PAGE gel (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The currently accepted paradigm regards TLR2 as an essential receptor for many eubacterial cell wall components, including lipoproteins and peptidoglycan. Bacterial species as diverse as mycobacteria, spirochetes, mycoplasma, S. aureus, and Streptococcus pneumoniae have all been shown to mediate cellular activation via TLR2 (14, 39, 43). In contrast to expectations, we found here that the deletion of TLR2 did not significantly alter the cellular response to whole GBS as compared with normal cells, suggesting a lesser role of peptidoglycan in streptococcal pathogenesis than might otherwise have been predicted. An alternative hypothesis that is currently the subject of investigation is that peptidoglycan from GBS activates TLRs that are different from those engaged by peptidoglycan of S. aureus (14). The requirement for MyD88 expression, but not TLR2, suggests that GBS is recognized by other TLRs. As documented here, using macrophages from mutant mice carrying targeted deletions or spontaneous mutations, these TLRs are not TLR1, TLR4, or TLR6.
In addition to narrowing our understanding of TLR use by the innate
immune system in response to GBS, the present study has identified the
existence of a novel proinflammatory factor from GBS. This factor,
GBS-F, is secreted or shed in bacterial culture, and this activity is
already present in early log phase of bacterial growth. GBS-F appears
to have considerable inflammatory potency as GBS supernatant, with a
total protein content as low as 5 µg/ml activated macrophages
and TLR2-transfected CHO cells in dilutions of up to 1/100. The total
bacterial mass removed from the supernatant that produced this activity
was only 180 µg/ml (dry weight per volume). GBS-F engages both TLR2
and TLR6 and hence is distinctly different from whole GBS, whose cell
wall constituents induce signaling in the absence of TLR2 or TLR6
expression. The complexity of GBS recognition by cells of the innate
immune system is summarized in Fig. 8.
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-hemolysin
from GBS has been assigned a proinflammatory function
(48). None of these known molecules is likely to be the
GBS-F described herein. GBS-F clearly differs from -hemolysin in
that GBS-F activity was produced similarly by -hemolytic and
nonhemolytic GBS strains. Similarly, GBS-F is not a capsular
polysaccharide, in that GBS-F activity was detected from mutant GBS
deficient in the production of capsular polysaccharide (data not
shown). GBS-F can be discriminated from the cell wall components
lipoteichoic acid, peptidoglycan, and bacterial lipoproteins by its
heat lability and its release starting at early log phase, before cell
wall lysis is observed. In addition, no proinflammatory lipoproteins
from Gram-positive bacteria have been identified yet. When GBS was
compared with a pneumolysin-deficient strain of S.
pneumoniae, a bacterial species that exhibits extensive autolysis,
only GBS released a proinflammatory factor (data not shown). These
results challenge the current dogma that TLR2 is a critical mediator
for cell wall components common to all bacteria (36).
Instead, they support the argument that species-specific molecules from
different genera of Gram-positive bacteria are selectively recognized
by this important receptor. We conclude that GBS-F is a novel proinflammatory factor secreted by GBS. GBS-F, but not whole bacteria, engages TLR2 and TLR6 as essential receptors. Currently we do not know whether GBS-F is secreted and immunologically relevant in vivo. However, it is tempting to speculate that the spillage of GBS-F from the lung or other sites of infection into the blood stream drives systemic hyperinflammation in GBS sepsis. We believe that the discovery of GBS-F will prove to lend significant insight into the mechanisms by which the invasion of human tissue by GBS results in sepsis, excessive morbidity, and even death.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Douglas T. Golenbock, Department of Medicine, University of Massachusetts Medical School, NRB Floor 3, 364 Plantation Street, Worcester, MA 01605. E-mail address: douglas.golenbock{at}umasssmed.edu
3 Abbreviations used in this paper: GBS, group B streptococcus; TLR, Toll-like receptor; CHO, Chinese hamster ovary; huTLR2, human TLR2; GBS-F, soluble factor released by GBS; PEM, peritoneal exudate macrophage.
Received for publication June 4, 2001. Accepted for publication October 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by type III group B streptococci. Infect. Immun. 64:5042.[Abstract]
, signal transduction. J. Immunol. 161:3001.-hemolysin expression is associated with injury of lung epithelial cells. Infect. Immun. 64:3818.[Abstract]
B induced by lipopolysaccharide does not require tyrosine kinase activity. J. Biol. Chem. 269:22253.B activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls. J. Immunol. 160:4535.B through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J. Biol. Chem. 274:7611.-hemolytic streptococcal disease. Proc. Natl. Acad. Sci. USA 84:51.-hemolysin induces nitric oxide production in murine macrophages. J. Infect. Dis. 182:150.[Medline]This article has been cited by other articles:
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  S. Kenzel, S. Santos-Sierra, S. D. Deshmukh, I. Moeller, B. Ergin, K. A. Fitzgerald, E. Lien, S. Akira, D. T. Golenbock, and P. Henneke Role of p38 and Early Growth Response Factor 1 in the Macrophage Response to Group B Streptococcus Infect. Immun., June 1, 2009; 77(6): 2474 - 2481. [Abstract] [Full Text] [PDF]
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M. Puliti, S. Uematsu, S. Akira, F. Bistoni, and L. Tissi Toll-Like Receptor 2 Deficiency Is Associated with Enhanced Severity of Group B Streptococcal Disease Infect. Immun., April 1, 2009; 77(4): 1524 - 1531. [Abstract] [Full Text] [PDF]
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 S. Weiss, H. Levy, M. Fisher, D. Kobiler, and Z. Altboum Involvement of TLR2 in innate response to Bacillus anthracis infection Innate Immunity, February 1, 2009; 15(1): 43 - 51. [Abstract] [PDF]
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T. G. Loof, O. Goldmann, and E. Medina Immune Recognition of Streptococcus pyogenes by Dendritic Cells Infect. Immun., June 1, 2008; 76(6): 2785 - 2792. [Abstract] [Full Text] [PDF]
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 P. Henneke, S. Dramsi, G. Mancuso, K. Chraibi, E. Pellegrini, C. Theilacker, J. Hubner, S. Santos-Sierra, G. Teti, D. T. Golenbock, et al. Lipoproteins Are Critical TLR2 Activating Toxins in Group B Streptococcal Sepsis J. Immunol., May 1, 2008; 180(9): 6149 - 6158. [Abstract] [Full Text] [PDF]
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S. Lehnardt, J. Wennekamp, D. Freyer, C. Liedtke, C. Krueger, R. Nitsch, I. Bechmann, J. R. Weber, and P. Henneke TLR2 and Caspase-8 Are Essential for Group B Streptococcus-Induced Apoptosis in Microglia J. Immunol., November 1, 2007; 179(9): 6134 - 6143. [Abstract] [Full Text] [PDF]
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 R. Graveline, M. Segura, D. Radzioch, and M. Gottschalk TLR2-dependent recognition of Streptococcus suis is modulated by the presence of capsular polysaccharide which modifies macrophage responsiveness Int. Immunol., April 1, 2007; 19(4): 375 - 389. [Abstract] [Full Text] [PDF]
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 S. Liang, M. Wang, R. I. Tapping, V. Stepensky, H. F. Nawar, M. Triantafilou, K. Triantafilou, T. D. Connell, and G. Hajishengallis Ganglioside GD1a Is an Essential Coreceptor for Toll-like Receptor 2 Signaling in Response to the B subunit of Type IIb Enterotoxin J. Biol. Chem., March 9, 2007; 282(10): 7532 - 7542. [Abstract] [Full Text] [PDF]
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G. Mancuso, A. Midiri, C. Biondo, C. Beninati, S. Zummo, R. Galbo, F. Tomasello, M. Gambuzza, G. Macri, A. Ruggeri, et al. Type I IFN Signaling Is Crucial for Host Resistance against Different Species of Pathogenic Bacteria J. Immunol., March 1, 2007; 178(5): 3126 - 3133. [Abstract] [Full Text] [PDF]
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 G. Elson, I. Dunn-Siegrist, B. Daubeuf, and J. Pugin Contribution of Toll-like receptors to the innate immune response to Gram-negative and Gram-positive bacteria Blood, February 15, 2007; 109(4): 1574 - 1583. [Abstract] [Full Text] [PDF]
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G. Hajishengallis, M. Wang, E. Harokopakis, M. Triantafilou, and K. Triantafilou Porphyromonas gingivalis Fimbriae Proactively Modulate {beta}2 Integrin Adhesive Activity and Promote Binding to and Internalization by Macrophages. Infect. Immun., October 1, 2006; 74(10): 5658 - 5666. [Abstract] [Full Text] [PDF]
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S. Santos-Sierra, D. T. Golenbock, and P. Henneke Toll-like receptor-dependent discrimination of streptococci Innate Immunity, October 1, 2006; 12(5): 307 - 312. [Abstract] [PDF]
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S. Lehnardt, P. Henneke, E. Lien, D. L. Kasper, J. J. Volpe, I. Bechmann, R. Nitsch, J. R. Weber, D. T. Golenbock, and T. Vartanian A Mechanism for Neurodegeneration Induced by Group B Streptococci through Activation of the TLR2/MyD88 Pathway in Microglia J. Immunol., July 1, 2006; 177(1): 583 - 592. [Abstract] [Full Text] [PDF]
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P. Henneke and R. Berner Interaction of neonatal phagocytes with group B streptococcus: recognition and response. Infect. Immun., June 1, 2006; 74(6): 3085 - 3095. [Full Text] [PDF]
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S. Kenzel, G. Mancuso, R. Malley, G. Teti, D. T. Golenbock, and P. Henneke c-Jun Kinase Is a Critical Signaling Molecule in a Neonatal Model of Group B Streptococcal Sepsis. J. Immunol., March 1, 2006; 176(5): 3181 - 3188. [Abstract] [Full Text] [PDF]
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 F.C. Gibson III, H. Yumoto, Y. Takahashi, H.-H. Chou, and C.A. Genco Innate Immune Signaling and Porphyromonas gingivalis-accelerated Atherosclerosis Journal of Dental Research, February 1, 2006; 85(2): 106 - 121. [Abstract] [Full Text] [PDF]
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K. O. Omueti, J. M. Beyer, C. M. Johnson, E. A. Lyle, and R. I. Tapping Domain Exchange between Human Toll-like Receptors 1 and 6 Reveals a Region Required for Lipopeptide Discrimination J. Biol. Chem., November 4, 2005; 280(44): 36616 - 36625. [Abstract] [Full Text] [PDF]
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T. Kielian, A. Haney, P. M. Mayes, S. Garg, and N. Esen Toll-Like Receptor 2 Modulates the Proinflammatory Milieu in Staphylococcus aureus-Induced Brain Abscess Infect. Immun., November 1, 2005; 73(11): 7428 - 7435. [Abstract] [Full Text] [PDF]
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M. A. Hughes, C. S. Green, L. Lowchyj, G. M. Lee, V. K. Grippe, M. F. Smith Jr., L.-Y. Huang, E. T. Harvill, and T. J. Merkel MyD88-Dependent Signaling Contributes to Protection following Bacillus anthracis Spore Challenge of Mice: Implications for Toll-Like Receptor Signaling Infect. Immun., November 1, 2005; 73(11): 7535 - 7540. [Abstract] [Full Text] [PDF]
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 H. Fan, B. Zingarelli, O. M. Peck, G. Teti, G. E. Tempel, P. V. Halushka, and J. A. Cook Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric G{alpha}i proteins Am J Physiol Cell Physiol, August 1, 2005; 289(2): C293 - C301. [Abstract] [Full Text] [PDF]
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M. B. Zeisel, V. A. Druet, J. Sibilia, J.-P. Klein, V. Quesniaux, and D. Wachsmann Cross Talk between MyD88 and Focal Adhesion Kinase Pathways J. Immunol., June 1, 2005; 174(11): 7393 - 7397. [Abstract] [Full Text] [PDF]
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P. Henneke, S. Morath, S. Uematsu, S. Weichert, M. Pfitzenmaier, O. Takeuchi, A. Muller, C. Poyart, S. Akira, R. Berner, et al. Role of Lipoteichoic Acid in the Phagocyte Response to Group B Streptococcus J. Immunol., May 15, 2005; 174(10): 6449 - 6455. [Abstract] [Full Text] [PDF]
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A. L. Kau, S. M. Martin, W. Lyon, E. Hayes, M. G. Caparon, and S. J. Hultgren Enterococcus faecalis Tropism for the Kidneys in the Urinary Tract of C57BL/6J Mice Infect. Immun., April 1, 2005; 73(4): 2461 - 2468. [Abstract] [Full Text] [PDF]
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S. Totemeyer, P. Kaiser, D. J. Maskell, and C. E. Bryant Sublethal Infection of C57BL/6 Mice with Salmonella enterica Serovar Typhimurium Leads to an Increase in Levels of Toll-Like Receptor 1 (TLR1), TLR2, and TLR9 mRNA as Well as a Decrease in Levels of TLR6 mRNA in Infected Organs Infect. Immun., March 1, 2005; 73(3): 1873 - 1878. [Abstract] [Full Text] [PDF]
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U. Hasan, C. Chaffois, C. Gaillard, V. Saulnier, E. Merck, S. Tancredi, C. Guiet, F. Briere, J. Vlach, S. Lebecque, et al. Human TLR10 Is a Functional Receptor, Expressed by B Cells and Plasmacytoid Dendritic Cells, Which Activates Gene Transcription through MyD88 J. Immunol., March 1, 2005; 174(5): 2942 - 2950. [Abstract] [Full Text] [PDF]
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 B. Opitz, S. Forster, A. C. Hocke, M. Maass, B. Schmeck, S. Hippenstiel, N. Suttorp, and M. Krull Nod1-Mediated Endothelial Cell Activation by Chlamydophila pneumoniae Circ. Res., February 18, 2005; 96(3): 319 - 326. [Abstract] [Full Text] [PDF]
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G. Mancuso, A. Midiri, C. Beninati, C. Biondo, R. Galbo, S. Akira, P. Henneke, D. Golenbock, and G. Teti Dual Role of TLR2 and Myeloid Differentiation Factor 88 in a Mouse Model of Invasive Group B Streptococcal Disease J. Immunol., May 15, 2004; 172(10): 6324 - 6329. [Abstract] [Full Text] [PDF]
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S. Knapp, C. W. Wieland, C. van 't Veer, O. Takeuchi, S. Akira, S. Florquin, and T. van der Poll Toll-Like Receptor 2 Plays a Role in the Early Inflammatory Response to Murine Pneumococcal Pneumonia but Does Not Contribute to Antibacterial Defense J. Immunol., March 1, 2004; 172(5): 3132 - 3138. [Abstract] [Full Text] [PDF]
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M. A. Campos, G. M. S. Rosinha, I. C. Almeida, X. S. Salgueiro, B. W. Jarvis, G. A. Splitter, N. Qureshi, O. Bruna-Romero, R. T. Gazzinelli, and S. C. Oliveira Role of Toll-Like Receptor 4 in Induction of Cell-Mediated Immunity and Resistance to Brucella abortus Infection in Mice Infect. Immun., January 1, 2004; 72(1): 176 - 186. [Abstract] [Full Text] [PDF]
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O. Levy, R. M. Jean-Jacques, C. Cywes, R. B. Sisson, K. A. Zarember, P. J. Godowski, J. L. Christianson, H.-K. Guttormsen, M. C. Carroll, A. Nicholson-Weller, et al. Critical Role of the Complement System in Group B Streptococcus-Induced Tumor Necrosis Factor Alpha Release Infect. Immun., November 1, 2003; 71(11): 6344 - 6353. [Abstract] [Full Text] [PDF]
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 B. Beutler, K. Hoebe, X. Du, and R. J. Ulevitch How we detect microbes and respond to them: the Toll-like receptors and their transducers J. Leukoc. Biol., October 1, 2003; 74(4): 479 - 485. [Abstract] [Full Text] [PDF]
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 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]
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S. S. Mambula, K. Sau, P. Henneke, D. T. Golenbock, and S. M. Levitz Toll-like Receptor (TLR) Signaling in Response to Aspergillus fumigatus J. Biol. Chem., October 11, 2002; 277(42): 39320 - 39326. [Abstract] [Full Text] [PDF]
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P. Henneke, O. Takeuchi, R. Malley, E. Lien, R. R. Ingalls, M. W. Freeman, T. Mayadas, V. Nizet, S. Akira, D. L. Kasper, et al. Cellular Activation, Phagocytosis, and Bactericidal Activity Against Group B Streptococcus Involve Parallel Myeloid Differentiation Factor 88-Dependent and Independent Signaling Pathways J. Immunol., October 1, 2002; 169(7): 3970 - 3977. [Abstract] [Full Text] [PDF]
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G. Mancuso, A. Midiri, C. Beninati, G. Piraino, A. Valenti, G. Nicocia, D. Teti, J. Cook, and G. Teti Mitogen-Activated Protein Kinases and NF-{kappa}B Are Involved in TNF-{alpha} Responses to Group B Streptococci J. Immunol., August 1, 2002; 169(3): 1401 - 1409. [Abstract] [Full Text] [PDF]
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)
or with TLR2 plus human MD2 and the ELAM.luc reporter plasmid, (
).
After 24 h, cells were stimulated over 5 h with GBS-F, and
luciferase activity was determined as outlined in Materials and
Methods. ND, Not detectable.
