Veterinary Microbiology 156 (2012) 147–156

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

The role of toll-like receptors in the pathogenesis of Streptococcus suis

Han Zheng a, Xia Luo a, Mariela Segura b, Hui Sun a, Changyun Ye a,
Marcelo Gottschalk b, Jianguo Xu a,*
a
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for
Disease Control and Prevention, Changping, Beijing, China
b
Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de médecine vétérinaire, Université de Montréal, Québec, Canada

A R T I C L E I N F O A B S T R A C T

Article history: Streptococcus suis is an important agent of swine and human meningitis. Sequence type
Received 16 May 2011 (ST) 7 emerged in China and was responsible for the human epidemic caused by S. suis in
Received in revised form 8 October 2011 2005. The virulence of S. suis ST7 is greater than the wild type pathogenic S. suis, ST1;
Accepted 12 October 2011 however, the mechanisms for this increased pathogenicity are unknown. The aim of this
study was to determine the role of different toll-like receptors (TLRs) involved in
Keywords: regulating the host response to the S. suis infection and to speculate on differing
Streptococcus suis sequence type 7 mechanisms used by ST7 strains to induce disease. Here we compared two ST7 strains
Streptococcal toxic shock-like syndrome
isolated in the 2005 Sichuan outbreak to two ST1 strains. Our data show TLR2, 6 and 9 are
Toll-like receptors
involved in the recognition of heat-killed S. suis independent of the ST type. We found the
Cytokines
TLR-dependent cytokine production differed between the two types of strains using whole
cell lysate proteins. TLR6 played a greater role in cytokine production induced by the
whole cell lysate proteins from the ST7 strain than in that induced by the ST1 strain lysates.
The data suggest that mechanisms of inflammation induced by S. suis strains differ where
this will be useful in designing efficient strategies in combating streptococcal toxic shock-
like syndrome caused by the S. suis ST7 strains.
ß 2011 Elsevier B.V. All rights reserved.

1. Introduction previously reported (Ye et al., 2006). Using multi-locus

Streptococcus suis (S. suis) serotype 2 is an important
swine pathogen causing large economic losses to the pork
industry (Reams et al., 1994). The infection is a zoonotic
disease causing primarily meningitis in humans involved
in slaughtering and processing pork-derived products;
occasionally patients present other infections, e.g. endo-
carditis, arthritis, and pneumonia (Gottschalk et al., 2007).
A large outbreak of 215 human cases emerged in 2005 in
Sichuan Province, China with 61 cases presenting a
streptococcal toxic shock-like syndrome (STSLS) not
* Corresponding author at: State Key Laboratory for Infectious Disease
Prevention and Control, National Institute for Communicable Disease
Control and Prevention, Chinese Center for Disease Control and
Prevention, Changbai Road 155, Changping, Beijing 102206, China.
Tel.: +86 1058900748.
E-mail address: xujianguo@icdc.cn (J. Xu).
sequence typing (MLST), S. suis sequence type (ST) 7 was
recognized as the causative agent for the Sichuan outbreak
(King et al., 2002; Ye et al., 2006).
In a previous study, the pro-inflammatory cytokine
levels in patients was shown to be elevated, a significant
contributing factor to STSLS caused by the ST7 strains (Ye
et al., 2009). An animal study showed the increased
virulence of S. suis ST7 was associated with an increased
ability to stimulate the host immune system to produce
unusually high levels of serum pro-inflammatory cyto-
kines that may be responsible for the shock syndrome
(Zheng et al., 2008). Toll-like receptors (TLRs) are receptors
that play a key role in the innate immune response. When
those receptors are activated, a signaling cascade is
initiated that results in the release of pro-inflammatory
cytokines. We hypothesized that TLR1, 2, 6 and 9 may be
involved in the response to S. suis due to their PAMP
specificities. To investigate the role of these TLRs in

0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2011.10.014
148 H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156
regulating the host response to S. suis infection, and to
speculate on the differing mechanisms used by ST1 and
ST7 strains to induce disease, heat-killed bacterial
suspensions, whole cell lysate proteins, and DNA of S. suis
ST1 and ST7 were used as stimulators to determine the role
of the TRLs in the cytokine cascade responsible for STSLS.
2. Materials and methods
2.1. Bacterial strains and reagents
The strains used in this study were S. suis GZ1 and
31533, typed as ST1; and strains SC49 and SC84, typed as
ST7. Strain GZ1 was isolated from a meningitis patient
from another area of China in 2005. Strain 31533 was
isolated in the 1990s from a diseased pig in France and has
been widely used in previous studies (Dominguez-Punaro
et al., 2007, 2010; Segura et al., 2002, 2006). SC84 was
isolated from a patient with STSLS and meningitis during
the Sichuan outbreak in 2005 and SC49 was isolated from a
diseased pig whose owner was the host of SC84. SC84 and
GZ1 were selected because their entire genomes were
sequenced by our laboratory (Genebank: NC 012924 and
CP 000837). S. suis strains were grown overnight on
Columbia blood base agar plates (Detgerm Microbiological
Science, Guangzhou, China) at 37 8C, and isolated colonies
were used to inoculate 10 ml Todd-Hewitt broth (THB,
Oxoid Ltd., London, UK). This culture was incubated for 8 h
at 37 8C with agitation (100 rpm). Working cultures were
prepared by transferring 300 ml of the 8 h cultures into
30 ml of THB and incubating stationary for 15 h at 37 8C
with 5% CO2. Bacteria were washed twice in phosphate-
buffered saline (PBS, pH 7.4, Invitrogen, Carlsbad, CA). The
pellet was re-suspended in PBS. Serial dilutions of the
suspension were plated onto blood base agar plates to
determine the CFU per ml.
Heat killed bacteria were obtained according to
previously described method (Zheng et al., 2008). To
stimulate with purified TLR ligands, we used Pam3CSK4,
Pam2CSK4 and ODN2006 (all from InvivoGen, San Diego,
CA). For inhibition of TLR1, TLR2, TLR6 and TLR9, we used
mouse monoclonal anti-TLR1 (clone GD2.F4, isotype:
mouse IgG1), mouse monoclonal anti-TLR2 (clone TL2.1,
isotype: mouse IgG2a), mouse monoclonal anti-TLR6
(clone C5C8, isotype: mouse IgG1), and ODNTTAGGG,
respectively (all from InvivoGen). In order to exclude non-
specific inhibition, the isotype controls of corresponding
neutralizing antibody (SouthernBiotec, Birmingham, AL)
were used.
2.2. Cell culture
Human peripheral blood was obtained from healthy
donors at the Red Cross of China, Beijing Branch. Peripheral
blood mononuclear cells (PBMCs) were isolated by
centrifugation (400  g for 30 min) using Ficoll-Paque
(GE Healthcare Biosciences, Uppsala, Sweden). PBMCs
were harvested; washed three times with RPMI medium
1640 (Invitrogen); and then re-suspended in white blood
cell medium (GenMed Scientifics, Shanghai, China). Viable
cells were counted using light microscopy with trypan blue
exclusion. For experiments, PBMCs were seeded into 96-
well plates (Becton Dickinson, Texarkana, TX) at a density
of 1.0  105 per well or 24-well plates (Becton Dickinson)
at a density of 1.0  106 per well. 293/hTLR9 and 293/Null
cells (all from InvivoGen) were cultured according to the
manufacturer’s instructions. For experiments, transfected
cells were seeded in 24-well plates (Becton Dickinson) at a
density of 1.0  106 per well.
2.3. Preparation of whole bacterial cell lysate proteins
Bacteria (2  1012) were collected and washed four
times with PBS using centrifugation at 4 8C. The final pellet
was re-suspended in lysis buffer: 8 M urea, 4% (wt/vol) 3-
[(3-cholamidopropyl)dimethylammonio]-1-propanesul-
fonate (Bio-Rad, Hercules, CA), 1% dithiothreitol (DTT,
Promega, Madison, WI), and 1% (wt/vol) Bio-Lyte 3/10
supplemented with 5 mg/ml lysozyme (Sigma, St. Louis,
MO) incubated at 37 8C for 30 min. Half of a tablet of an
EDTA-free protease inhibitor cocktail tablet (Roche,
Rotkreuz, Switzerland) was added. Bacterial cells were
then disrupted using six 2 min-bursts with a sonicator
(Sonics 130 W Ultrasonic Processor), cooling on ice
between bursts until the suspension was clarified. The
suspension was incubated at room temperature for 1 h
with 125 mg/ml RNase A and 10 U/ml DNase (Promega).
Cellular debris was removed using centrifugation at
40,000  g for 1 h at 15 8C. The supernatant was incubated
on ice 1 h in the presence of 10% trichloroacetic acid (TCA,
Fluka-Sigma, St. Louis, MO) and then pelleted using
centrifugation at 4 8C. Pellets of TCA-precipitated proteins
were washed three times in cold acetone. Finally, pellets
were dried in a Marin Christ (Osterode, Germany) and
solubilized in lysis buffer. The solution was retained as the
whole cell lysate protein preparation. The yield of whole
cell lysate proteins extracted from 2  1012 bacteria was
50 mg as determined by the Quick Start Bradford Protein
Assay (Bio-Rad). When used, whole cell lysate proteins
were diluted to 5 mg/ml (equivalent to the whole cell
lysate protein concentration from 2  108 heat-killed
bacteria per ml) in white blood cell medium. To rule out
toxicity of lysis buffer diluted in white blood cell medium
for the PBMCs, PBMCs were cultured with dilute lysis
buffer. Dilute lysis buffer was not toxic for human PBMCs
(data not shown).
2.4. DNA extracted from test strains
Chromosomal DNA of all tested isolates was extracted
using the TIANamp Bacteria DNA Kit (Tiangen Biotech,
Beijing, China) according to the manufacturer’s direc-
tions. DNA samples were analyzed using agarose gel
electrophoresis and quantified with a GeneQuant spec-
trophotometer ND-1000 (NanoDrop, Wilmington, DE).
Purity of the nucleic acid samples was measured using a
spectrophotometer. The ratio of the A260/A280 repre-
sented the purity, where between 1.80 and 2.0 was pure.
All DNA was stored at 20 8C in distilled H2O. The
working concentration of bacterial DNA was 10 mg/ml;
equivalent to a DNA concentration from 2  108 heat-
killed bacteria per ml.
 H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156 149

2.5. In vitro cytokine production and TLR blocking
We previously found live bacterial strains were toxic for
human PBMC using incubation times as short as 4 h (Ye
et al., 2009). Thus, the high levels of cytotoxicity with live
bacteria rendered long incubation times impossible.
Therefore, heat killed (56 8C for 60 min) S. suis (equivalent
to 2  108 organisms/ml) were added to the PBMC and
incubated for 3, 6, 9, 12, and 24 h in 96-well flat-bottom
plates. The supernatant was collected to measure cytokine
concentrations using the Bio-Plex Pro Human Cytokine
Reagent Kit (Bio-Rad). Cytokine levels were analyzed with
the Bio-Plex ManagerTM 6.0 software.
Blocking experiments for TLRs were performed using
blocking antibodies and/or peptides (TLR9) according to
previous publications (Mogensen et al., 2006; Schroder
et al., 2003) and protocols provided by the manufacturer.
Briefly, cells (1  106 cells/ml) were incubated with 10 mg/
ml of each antibody or 40 mg/ml (5 mM) peptide for 1 h
prior to incubation at 37 8C with killed bacteria
(2  108 organisms/ml) or bacterial DNA (10 mg/ml). Cells
(1  106 cells/ml) were incubated with 1 mg/ml of each
antibody or 40 mg/ml peptide for 1 h prior to incubation at
37 8C with whole cell lysate proteins (5 mg/ml) or pure
TLRs ligands (0.1 mg/ml Pam3CSK4, 2 mg/ml Pam2CSK4,
and 10 mg/ml ODN2006). To exclude non-specific blocking
of antibodies, heat-killed or whole cell lysate proteins of S.
suis (SC84 and GZ1) were treated with the isotype controls
with corresponding antibodies (10 mg/ml). The super-
natant was collected at 24 h post-incubation to measure IL-
6 and TNF-a concentrations using the Bio-Plex Pro Human
[(Fig._1)TD$IG]
Cytokine Reagent Kit (Bio-Rad). Cytokine levels were
analyzed with the Bio-Plex ManagerTM 6.0 software.
2.6. NF-kB p65 activity
PBMCs (1  106/ml) were incubated with whole cell
lysate proteins of S. suis (5 mg/ml), DNA (10 mg/ml), or the
corresponding TLR ligands (0.1 mg/ml Pam3CSK4, 2 mg/ml
Pam2CSK4, and 10 mg/ml ODN2006) for 25 min in 24-well
flat-bottom plates. 293/hTLR9 or 293/Null cells (1  106/
ml) were incubated with S. suis DNA (10 mg/ml) or
ODN2006 (10 mg/ml) for 25 min in 24-well flat-bottom
plates.
Lysates derived from cell culture were prepared using a
cell lysis kit (Bio-Rad) according to the protocol provided
by the manufacturer. Protein concentrations were deter-
mined using the Quick Start Bradford Protein Assay (Bio-
Rad). The protein concentration of all samples was
adjusted to 300 mg/ml. Phosphoprotein assays for NF-kB
p65 were determined using a phosphoprotein detection
reagent kit (Bio-Rad) according to the manufacturer’s
directions. The values were presented as fluorescence
intensity (MFI) using Bio-Plex ManagerTM 6.0 software.
2.7. Statistics
Cytokine values and NF-kB p65 activity were expressed
as the means  standard deviations. Statistical analyses
were performed using the Student unpaired t test. Differ-
ences between the groups were considered significant at a P
value of <0.05.

Fig. 1. Kinetics of cytokines produced by PBMCs in response to heat-killed S. suis (panels A and B) and whole cell lysate proteins of S. suis (panels C and D).
PBMC (1  106/ml) were cultured with heat killed ST7 or ST1 S. suis (2  108/ml) and whole cell lysate proteins of ST7 or ST1 S. suis (5 mg/ml). Experiments
were repeated three times with similar results. Heat-killed S. suis and whole cell lysate proteins of S. suis were prepared independently for each experiment.
*Significant, P < 0.05, the cytokine value of the ST7 group compared to the ST1 group.
150 H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156

3. Results ST7 strains at 3, 6, and 9 h post-incubation than when

3.1. Kinetics of cytokines secreted by PBMCs in response to
heat-killed S. suis and whole cell lysate proteins of S. suis
Stimulation with heat-killed S. suis, whole cell lysate
proteins and genomic DNA of S. suis induced time-
dependent IL-6 and TNF-a production. PBMCs produced
significantly higher levels of cytokines when stimulated by
heat-killed ST7 strains at 3, 6, and 9 h post-incubation than
when stimulated by heat-killed ST1 strains. After 12 h of
incubation, no differences in cytokine levels were observed
between the two ST groups (Fig. 1A and B). Similar results
were obtained using whole cell lysate proteins of S. suis as
stimulators. PBMCs produced significantly higher levels of
TNF-a when stimulated by whole cell lysate proteins of
[(Fig._2)TD$IG]
stimulated by whole cell lysate proteins of ST1 strains.
Differences in IL-6 levels between ST1 and ST7 were only
observed at 6 and 9 h post-incubation (Fig. 1C and D). To
appropriately evaluate the blocking efficacy of different
inhibitors, 24 h incubation was used in the following
experiments.
3.2. Evaluation of the blocking efficacy of different inhibitors
The efficacy of TLR inhibitors was evaluated using
corresponding TLR ligands to stimulate PBMCs. Our data
indicate cytokine production induced by TLR ligands was
significantly inhibited, although their production was not
completely abrogated (Fig. 2). To exclude non-specific

Fig. 2. The production of cytokines by PBMCs in response to TLR ligands was inhibited by corresponding blocking antibodies or peptide (TLR9). PBMC
(1  106/ml) were pretreated with blocking antibodies (1 mg/ml) or TLR9 peptide (40 mg/ml) prior to addition of the TLR ligands (panels A and B: Pam3CSK4
at 0.1 mg/ml; panels C and D: Pam2CSK4 at 2 mg/ml; and panels E and F: ODN2006 at 10 mg/ml). The experiment was repeated three times with similar
results. *Significant, P < 0.05, the cytokine value of the unblocked TLR group compared to the blocked TLR group.
[(Fig._3)TD$IG] H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156 151

Fig. 3. PBMCs produced cytokines in response to heat-killed S. suis using different TLRs. PBMCs (1  106/ml) were pretreated with blocking antibodies
(10 mg/ml) or TLR9 peptide (40 mg/ml) prior to addition of heat killed S. suis (2  108/ml) (panels A–D). Blocking antibodies (10 mg/ml) or TLR9 peptide
(40 mg/ml) alone did not stimulate production of cytokines (panels E and F). The experiment was repeated three times with similar results. Heat-killed S.
suis used in the study were prepared independently for each experiment. *Significant, P < 0.05, the cytokine value of the unblocked TLR group compared to
the blocked TLR group.

blocking by antibodies, heat-killed or whole cell lysate
proteins of S. suis (SC84 and GZ1) were treated with the
isotype controls of corresponding antibodies. Isotype
controls did not affect the production of cytokines induced
using the stimulators (results not shown).
3.3. Roles of TLR1, TLR2, TLR6 and TLR9 on cytokine
production in PBMCs stimulated with heat-killed S. suis ST1
and ST7
No significant effect on cytokine levels was observed
when PBMCs were pre-treated with anti-TLR1 antibody
and then stimulated with heat-killed S. suis ST1 or ST7;
even when using the antibody at concentrations as high as
10 mg/ml. In contrast, pretreatment of PBMCs with anti-
TLR2 antibody significantly inhibited the production of
both cytokines induced by either heat-killed S. suis ST1 or
ST7. The production of IL-6 was not affected by pretreat-
ment of PBMCs with anti-TLR6 antibody; however, the
production of TNF-a was significantly affected. Similar
results were obtained when PBMCs were pre-treated with
anti-TLR9 peptide (Fig. 3).
3.4. Differential involvement of TLRs in cytokine production
induced by S. suis ST7 and ST1 whole cell lysate proteins
The effects of TLR1, TLR2, TLR6 and TLR9 on the
induction of cytokine production by whole cell lysate
proteins of S. suis ST1 and ST7 were investigated. The
secretion of IL-6 and TNF-a by PBMCs in response to S. suis
ST7 whole cell lysate proteins may be partially TLR2 and
TLR6 dependent; and secretion of IL-6 and TNF-a by
PBMCs in response to S. suis ST1 whole cell lysate proteins
was in-part TLR2 dependent. TLR1 and TLR9 were not
involved in the recognition of S. suis whole cell lysate
proteins by the innate immune system (Fig. 4).
[(Fig._4)TD$IG]
152 H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156

Fig. 4. The dependence of TLRs was different between ST7 and ST1 in the production of cytokines by PBMCs in response to whole cell lysate proteins. PBMCs
(1  106/ml) were untreated or pretreated with blocking antibodies (1 mg/ml) or TLR9 peptide (40 mg/ml) prior to the addition of the whole cell lysate
proteins (5 mg/ml) (panels A–D). Blocking antibodies (1 mg/ml) alone did not stimulate the production of cytokines (panels E and F). The experiment was
repeated three times with similar results. Whole cell lysate proteins were prepared independently. *Significant, P < 0.05, the cytokine value of the
unblocked TLR group compared to the blocked TLR group.

3.5. Effect of TLR antagonists on NF-kB activation by whole 3.6. Stimulation of cytokine expression by bacterial DNA of S.
cell lysate proteins of S. suis suis ST7 and ST1 strains is through TLR9

Transcription factor NF-kB is a key regulator of genes
involved in inflammation. Our data showed an increase in
intra-nuclear NF-kB after stimulation with whole cell
lysate proteins from S. suis ST7. This was partially, but
significantly, inhibited by pretreatment with anti-TLR2
and anti-TLR6 antibodies; whereas the activation of NF-kB
by stimulation with whole cell lysate proteins of S. suis ST1
was affected by pre-treatment with anti-TLR2 antibody.
These data indicate NF-kB may play an important role in
the induction of cytokine secretion via TLR6 stimulated by
whole cell lysate proteins of S. suis ST7. Inhibition of TLR1
and TLR9 did not affect the activation of NF-kB stimulated
with whole cell lysate proteins of either ST1 or ST7 strains
(Fig. 5).
Since TLR9 was involved in the recognition of heat-
killed S. suis by the innate immune system, we determined
if the DNA of S. suis ST1 and ST7 had immuno-stimulation.
TLR9 becomes activated within the lysosomal compart-
ment by endocytosed DNA (Hacker et al., 1998; Takeshita
et al., 2001). Conversely, it has been suggested that CpG
ODN can bind to the plasma membrane and does not need
to be internalized to trigger the TLR9 response (Liang et al.,
1996). Here, we did not use transfection reagents to
increase the delivery of DNA or of the TLR9 peptide
inhibitor. DNA stimulated the release of cytokines in a
dose-dependent manner with stimulatory effects at a
concentration of 10 mg/ml (equivalent to the DNA con-
centration of 2  108 heat killed bacteria per ml). Our data
[(Fig._5)TD$IG] H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156 153

Fig. 5. Whole cell lysate proteins of S. suis ST1 and ST7 led to NF-kB activation through different TLR dependent signaling pathways (panels A and B).
Corresponding TLR ligands used as positive controls are shown in panels C and D. PBMCs (1  106/ml) were pretreated or untreated with blocking antibodies
(1 mg/ml) prior to addition of whole cell lysate proteins (5 mg/ml) or corresponding TLR ligands (0.1 mg/ml Pam3CSK4 and 2 mg/ml Pam2CSK4). The
experiment was repeated three times with similar results. *Significant, P < 0.05, the MFI of the unblocked TLR group compared to the blocked TLR group.
§Significant, P < 0.05 the MFI of the blank compared to the unblocked or blocked group.

indicate DNA from S. suis possessed the capacity to induce
the production of cytokines. We did not observe differ-
ences in cytokine levels between ST1 and ST7 at any
incubation time (Fig. 6A and B). The induction by S. suis
DNA of IL-6 and TNF-a production by PBMCs was
dependent on TLR9; pre-treatment of PBMCs with a
TLR9 inhibitor significantly attenuated the production of
these cytokines and the activation of NF-kB in DNA-
stimulated PBMCs (Fig. 6C–E). Further evidence for TLR9
involvement in sensing genomic DNA of S. suis was shown
when the genomic DNA of S. suis was used to stimulate
293/hTLR9 and 293/Null cells. After 25 min incubation,
nuclear extracts were purified, and NF-kB activity was
measured. As seen in Fig. 7, DNA of S. suis was able to
activate cells through TLR9.
4. Discussion
In this report, we identify the receptors involved in
innate immune recognition of S. suis by PBMCs. To
determine the contribution of TLRs, we tested the
requirements of different types of TLRs for inducing the
production of cytokines by PBMCs after exposure to heat-
killed S. suis, whole cell lysate proteins, or bacterial DNA.
TLR2 was included because it has been previously shown
to play an important role in the inflammatory response
caused by S. suis as well as other Gram-positive Strepto-
coccus (Dominguez-Punaro et al., 2010; Echchannaoui
et al., 2002; Graveline et al., 2007; Schroder et al., 2003;
Zheng et al., 2011). TLR1 and TLR6 were chosen because
they interact with TLR2 to form receptor clusters (hetero-
dimers) in response to different microbial ligands (Trian-
tafilou et al., 2006). Finally, TLR9 may be engaged in
response to bacterial CpG DNA, and thus may also be
involved in S. suis recognition.
When we used a heat-killed bacterial suspension as a
stimulator, ST7 induced the production of higher levels of
cytokines at early stages of incubation with PBMCs than did
ST1. The production of IL-6 was TLR2-dependent and the
production of TNF-a was TLR2-, TLR6-, and TLR9-depen-
dent. Different pathways may be involved in S. suis-induced
production of IL-6 and TNF-a, as previously shown using
heat-killed S. suis stimulated mouse macrophages (Segura
et al., 1999). Nevertheless, the amplification-loop of IL-6
production mediated by other cell released cytokines may
also contribute to the differences observed. Faulkner et al.
(Faulkner et al., 2005) found that an early burst of TNF-a is
responsible for the lethality during toxic shock. This higher
burst of TNF-a may be responsible for the STSLS observed in
the Sichuan outbreak. Understanding the mechanism of
TNF-a production will increase our knowledge of the S. suis-
induced inflammatory response. In this study, blocking of
TLR6 and TLR9 efficiently inhibited the production of TNF-a
induced by heat-killed S. suis. This provides potential
methods to mitigate the inflammatory cascade induced
by S. suis.
Our results demonstrate that DNA from S. suis is a
strong stimulus for TLR9 activation and may play a role
during the induction of the overwhelming inflammatory
response. Based on our data, DNA represents a microbial
component that may trigger the pathogenesis of S. suis.
Mogensen et al. (2006) also found TLR9 mediated cellular
recognition of pneumococci in vitro. This is the first report
showing a role of TLR9 in S. suis interactions with host cells.
Our data shows that the host uses multiple TLRs to
recognize S. suis and initiate the inflammatory response. As
[(Fig._6)TD$IG]
154 H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156

Fig. 6. Kinetics of cytokines produced by PBMCs in response to DNA of S. suis (panels A and B). PBMC (1  106/ml) were cultured with DNA of ST7 or ST1 S. suis
(10 mg/ml). The production of cytokines by PBMCs in response to the DNA of S. suis was through TLR9 (panels C and D) and the DNA of S. suis ST1 and ST7 led
to NF-kB activation through TLR9 dependent signaling pathways (panel E). PBMC (1  106/ml) were pretreated or untreated with the blocking peptide of
TLR9 (40 mg/ml) prior to addition of bacterial DNA (10 mg/ml). ODN 2006 was used as the positive control shown in panel F. The experiment was repeated
three times with similar results. Bacterial DNA used in the study was prepared independently. *Significant, P < 0.05, the cytokine value of the unblocked
TLR9 group compared to the blocked TLR9 group.

PBMCs express TLRs in cell type-specific patterns, activa-
tion of multiple TLRs allows for stimulation of more cell
types. For instance, human TLR9 is expressed abundantly
on plasmacytoid DC (pDC) and B cells but not on
macrophages, whereas the two former cell types do not
express TLR2, which is expressed on macrophages. TLR6 is
expressed on most cell types in PBMCs, e.g. PDC, B cells, NK
cells, T cells, and monocytes (Hornung et al., 2002;
Mogensen et al., 2006). Therefore, in the case of S. suis,
TLR2, TLR6, and TLR9 activate different cell types and
together mount the inflammatory response. Engagement
of more than one TLR may also be a means for the host to
avoid immune evasion by the infectious agent.
Further, the involvement of different TLRs in the
induction of TNF-a and IL-6 production was different
when comparing ST1 and ST7 whole cell lysates. The whole
cell lysate proteins of S. suis ST1 and ST7 both stimulated
cytokines through TLR2; but TLR6 contributed more to the
production of cytokines induced by whole cell lysate
proteins of S. suis ST7 strains than by those of ST1. These
data are in agreement with previously published studies
with experimental mice suggesting both S. suis ST1 and ST7
induced high levels of serum cytokines that may be
responsible for the shock syndrome; but they did so using
possibly different components. Heat sensitive components
of ST7 played a central role during induction of the
overwhelming inflammatory response (Zheng et al., 2008).
Similarly, in a recent published work, Schreur et al.
(Schreur et al., 2010) compared the activation of human
TLRs by S. suis serotype 2 and 9 strains. They showed S. suis
lipoproteins activate TLR2/6 but not TLR1/2, and the
activation levels of the hTLR2/6 complex were higher for
serotype 9 strains compared to serotype 2 strains
suggesting intrinsic differences in cell wall composition
between the serotypes. Thus, we hypothesize that different
bacterial components, especially differences in lipopro-
teins of S. suis ST7, may play a distinct role compared to
those derived from ST1 in the pathogenesis of the disease.
Differences in TLR6 engagement between ST1 and ST7
strains were not observed when using heat-killed S. suis,
probably because lipoteichoic acid (LTA) from heat-killed
S. suis is a very potent and well-known stimulator of TLR2/
6. As such, the differential effects of proteins could be
overcompensated by LTA. The components of S. suis ST7
whole cell lysate proteins, especially the diacyl lipopep-
tides that are recognized by TLR6 remain to be studied.
[(Fig._7)TD$IG] H. Zheng et al. / Veterinary Microbiology 156 (2012) 147–156 155

Fig. 7. NF-kB activation by the DNA of S. suis ST1 and ST7 in 293/hTLR9 cells. 293/hTLR9 (panel A) and 293/null (panel B) (1  106/ml) were incubated with
bacterial DNA (10 mg/ml) or ODN 2006 (10 mg/ml). The experiment was repeated three times with similar results. Bacterial DNA used in the study was
prepared independently.

After recognition of a pathogen-associated molecule,
TLRs associated with MyD88 and/or other signaling
molecules induce a cascade of signaling events leading
to NF-kB activation and subsequent transcription of genes
encoding pro-inflammatory cytokines such as IL-6 and
TNF-a (Takeda and Akira, 2002). Our data show blocking of
TLR2 led to decreased levels of NF-kB p65 activation when
PBMCs were stimulated using whole cell lysate proteins of
both S. suis ST1 and ST7. The increased activation of NF-kB
p65 in PBMCs stimulated by whole cell lysate proteins of S.
suis ST7 was partially, but significantly, blocked when
PBMCs were pre-treated with anti-TLR6 antibody. Based
on our data, activation of NF-kB may be involved in the
production of IL-6 and TNF-a by whole cell lysate proteins
of S. suis.
In contrast to TLR6, TLR1 may not be involved in the
recognition of S. suis. This is in agreement with Schreur
et al. (2010). Hajjar et al. (2001) reported TLR1 and TLR6
had opposite effects on the TLR2-mediated response to the
phenol-soluble modulin of Staphylococcus epidermidis.
These divergent effects appear to result from differences
in their extra-cellular domains that may reflect differences
in the interaction between these receptors and TLR2 in
ligand binding.
In conclusion, here we show data describing the role of
TLRs in host recognition of S. suis as well as the potential
contribution of whole cell lysate proteins in S. suis ST7 and
S. suis ST1 pathogenesis. S. suis is primarily recognized via
TLR2/TLR6 and TLR9; this interaction results in the release
of pro-inflammatory mediators. Likely, the presence of
lipoproteins plays an important role by interacting with
TLR6 and increasing the activity of NF-kB, and thus
contributes to the pathogenesis of STSLS caused by the ST7
strains. DNA represents a new stimulatory microbial
component that needs further research to better under-
stand its relationship to the virulence of S. suis strains for
both humans and pigs.
Acknowledgements
This work was supported by grants (50153-3) from the
State Key Laboratory for Infectious Disease Prevention and
Control (to HZ.) and grants (2009ZX10004-108) from the
Ministry of Science and Technology, PR China. Funding was
provided by MDEIE, Collaboration Chine-Quebec to M.G.
and M.S. and China-Quebec Collaboration Grant to J. Xu
(2008FA31830 and 2008DFA31830).
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