RESEARCH LETTER

Regulation of sagA , siaA and scpC by SilCR, a putative signaling

peptide of Streptococcus pyogenes
Kowthar Y. Salim1, Joyce C. de Azavedo2, Darrin J. Bast2 & Dennis G. Cvitkovitch1
1
Department of Microbiology, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada; and 2Department of Laboratory Medicine and
Pathobiology, University of Toronto, Toronto, ON, Canada

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Correspondence: Dennis G. Cvitkovitch,
Department of Microbiology, Faculty of
Dentistry, University of Toronto, Rm. 449A,
124 Edward Street, Toronto, ON, Canada
M5G 1G6. Tel.: 1416 979 4917; fax: 1416
979 4936; e-mail:
d.cvitkovitch@dentistry.utoronto.ca
Received 12 May 2008; accepted 29 August
2008.
First published online 30 October 2008.
DOI:10.1111/j.1574-6968.2008.01375.x
Abstract
SilCR, a 17 amino acid putative signaling peptide, was proposed to modulate gene
expression in Streptococcus pyogenes. We showed that SilCR added exogenously to
an M1 serotype strain lacking the sil locus upregulates the in vitro expression of
sagA, siaA, and scpC, genes associated with S. pyogenes pathogenesis. Interestingly,
only sagA and siaA were upregulated by SilCR in vivo, whereas the expression of
scpC remained unaltered. A previous report indicated that exogenously added
SilCR protects mice to some degree from developing necrotic lesions caused by an
invasive strain of S. pyogenes. In contrast to this report, we found that SilCR did
not reduce lesion formation in a subcutaneous murine model of S. pyogenes
infection but rather appeared to delay wound healing.

Editor: Robert Burne

Keywords
group A Streptococcus ; cell signaling; gene
regulation; virulence.

and was reported to be involved in DNA transfer and

Introduction
Streptococcus pyogenes causes a variety of infections in
humans, ranging in severity from pharyngitis to necrotizing
fasciitis (Cunningham, 2000). Recently, SilCR, a putative
peptide possessing features characteristic of signaling pep-
tides in gram-positive bacteria involved in quorum sensing,
was identified in S. pyogenes (Miller & Bassler, 2001;
Hidalgo-Grass et al., 2002, 2004). The silCR gene encodes a
putative 41 amino acid propeptide with a characteristic
glycine–glycine sequence motif, which allows cleavage of
the propeptide into a 17 amino acid mature peptide
(Hidalgo-Grass et al., 2002, 2004). Encoded on the sil locus
are silA/B, a putative two-component signal transduction
system (TCSTS), silD/E, a putative ATP-binding cassette
(ABC) transporter, and silC a small ORF preceded by a
TACGAATA combox promoter sequence (Morrison & Lee,
2000). The silCR gene was found to overlap silC and
transcribed on the reverse strand (Hidalgo-Grass et al.,
2002; Eran et al., 2007).
The sil locus in S. pyogenes is homologous to the quorum-
sensing competence regulons of Streptococcus pneumoniae
invasive disease (Hidalgo-Grass et al., 2002). Significantly
smaller necrotic lesions were observed in mice injected
subcutaneously with an invasive isolate of S. pyogenes in the
presence of exogenously added SilCR compared with mice
injected with the invasive isolate alone (Hidalgo-Grass et al.,
2004). Furthermore, analysis of genome databases of various
M serotypes of S. pyogenes indicated that either silCR or
other genes in the sil operon were mutated or absent in the
genomes of invasive isolates (Hidalgo-Grass et al., 2002).
Hence, Hidalgo-Grass and colleagues proposed that SilCR
might have a therapeutic effect on the invasive potential of
S. pyogenes by modulating gene expression (Hidalgo-Grass
et al., 2006), whereby its absence increases pathogenicity and
its presence, either as a natural product of the strains or as
an added pharmacological agent, attenuates virulence (Hi-
dalgo-Grass et al., 2004). To gain further insight into a
potential mechanism for the proposed therapeutic effect of
SilCR, we assessed the influence of this peptide on the
expression of three genes, sagA, siaA, and scpC, all of which
are known to play a role in necrotic lesion formation in
animal models of invasive S. pyogenes infections.

FEMS Microbiol Lett 289 (2008) 119–125

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
120 K.Y. Salim et al.

The sagA gene encodes streptolysin S (SLS), an oxygen- growth. Bacterial pellets were snap-frozen in liquid nitrogen
stable, broad-spectrum cytolysin responsible for necrotic and stored at  70 1C until mRNA extraction. All gene
lesion formation in mice injected subcutaneously with expression analyses were conducted in duplicate cultures.
S. pyogenes (Betschel et al., 1998; Nizet et al., 2000; Fontaine
et al., 2003; Engleberg et al., 2004; Datta et al., 2005). SagA- Infection of mice
deficient mutants of S. pyogenes were unable to form lesions
Four-week-old, immunocompetent, female, crl:SKH1
in mice and had reduced ability to spread systemically
(hrhr) hairless mice (Charles River Laboratories, MA) were
(Betschel et al., 1998; Nizet et al., 2000; Datta et al., 2005).
utilized (Betschel et al., 1998). For preparation of the
SiaA, also known as HtsA, constitutes a component of an
inoculum, an overnight culture of MGAS166 at a dilution
ABC transporter system that is part of a 10-gene operon
of 1/20 was inoculated into fresh TH medium and grown at
involved in iron acquisition (Lei et al., 2002, 2003; Bates
37 1C until the culture reached the mid-exponential growth
et al., 2003; Liu & Lei, 2005). Unlike the sagA-deficient
phase (OD600 nm = 0.4). Subsequently, the culture was cen-

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mutants, an siaB-deficient mutant produced more severe
trifuged at 39 410 g at 4 1C for 10 min, the pellet was washed
necrotic lesions relative to the wild-type parent strain in a
once in 1  phosphate-buffered saline (PBS), and resus-
zebrafish model of invasive infections (Montanez, 2005). In
pended in 1  PBS to the desired inoculum.
addition to the aforementioned genes, scpC (or spyCEP),
For in vivo gene expression analysis, groups of four
which is a serine protease of S. pyogenes, was also shown to
mice each were inoculated with either 106 CFU of
contribute to necrotic lesion formation. This protease pre-
MGAS16615.0 mg of SilCR or MGAS166 alone. SilCR was
vents recruitment and activation of neutrophils by degrad-
added to the inoculum at a concentration of 50 mg mL1 and
ing chemokines, such as IL-8, GCP-2, and Gro-a (Hidalgo-
left at room temperature for 20–30 min before infection. At
Grass et al., 2006; Sumby et al., 2008). Consequently, it was
24 and 48 h postinoculation, necrotic lesions were excised
proposed to contribute to the evasion of the host’s immune
from two mice each, snap-frozen in liquid nitrogen, and
system by S. pyogenes. However, unlike sagA and siaA, the
stored at  70 1C until mRNA extraction.
effect of scpC on necrotic lesion formation appears to be
To determine the effect of SilCR on the pathogenicity of
strain and/or serotype dependent. One study showed that a
S. pyogenes, groups of 18 mice were inoculated with
mutant of scpC in an M1 serotype strain increased lesion size
MGAS166 in the presence (5.0 mg) or absence of exogen-
relative to its wild-type parent strain (Sumby et al., 2008),
ously added SilCR as described above. Four mice at 2 and 5
whereas another showed the opposite effect in an M14
days postinoculation, 3 at 7 days postinoculation, and the
serotype strain (Hidalgo-Grass et al., 2006).
remaining 7 at 12 days postinoculation were sacrificed,
In our study, SilCR was synthesized as a mature 17 amino
lesions were photographed with a Powershot G6 camera
acid custom peptide, and its effect on in vitro and in vivo
(Canon, Mississauga, Canada), and images were analyzed
gene expression in an invasive M1 serotype strain of
with the OPENLAB 4.0 imaging program (Improvision,
S. pyogenes was investigated. Furthermore, the proposed
Coventry, UK) to determine lesion surface area. The lesions
therapeutic effect of SilCR was examined in a subcutaneous
were then excised, ground in a disposable tissue grinder
murine model of invasive disease by comparing the viru-
(Kendall, MA), serially diluted in sterile 1  PBS, and plated
lence of the invasive S. pyogenes isolate either in the absence
on Columbia Blood Agar to determine bacterial counts. The
or in the presence of exogenously added SilCR.
mice were also monitored for weight loss/gain. All experi-
mental procedures were conducted in accordance with the
Materials and methods principles of the Animal Care Committee of Mount Sinai
Hospital, Toronto, Canada.
Bacterial strains and in vitro growth conditions
Total RNA isolation and real-time reverse
Streptococcus pyogenes MGAS166 (M1 serotype) was used as
transcriptase (RT)-PCR analysis
the test strain for in vitro and in vivo experiments. SilCR was
custom synthesized as a 17 amino acid (DIFKLVIDHISM- Mouse tissue biopsies, macerated with a disposable tissue
KARKK) peptide (Mimotopes, Vic.). For in vitro analysis of grinder (Kendall), and frozen in vitro-grown bacterial pellets
gene expression, overnight cultures of MGAS166 grown in were processed for mRNA extraction as described previously
Todd–Hewitt (TH) broth (Difco Laboratories, MI) were (Salim et al., 2005, 2007). Gene expression analysis included
centrifuged at 39 410 g at 4 1C for 10 min, washed twice with the generation of standard curves for each target gene
fresh TH broth, and used to inoculate fresh TH broth with including the DNA gyrase A gene (gyrA), which was used as
(0.05, 0.5, or 5.0 mg mL1) or without SilCR at a dilution of an internal standard for normalizing gene expression as
1/20. Cultures were incubated at 37 1C and bacteria were discussed previously (Salim et al., 2005, 2007). Briefly, each
harvested at early-, mid-, and late-exponential phases of independently derived in vitro and in vivo sample was

c 2008 Federation of European Microbiological Societies FEMS Microbiol Lett 289 (2008) 119–125
Published by Blackwell Publishing Ltd. All rights reserved
SilCR regulates sagA, siaA and scpC expression 121

analyzed in triplicate using real-time RT-PCR to ensure little
variability within each sample. Primers and their optimal
annealing temperatures utilized for real-time RT-PCR ana-
lysis are listed in Table 1. Relative expression was determined
by obtaining the ratio of normalized target gene expression
under the desired conditions.
Statistical analyses
The data were compared utilizing an unpaired Student’s
t-test or a Wilcoxon–Mann–Whitney test, and a P-value
o 0.05 was considered significant.
S. pyogenes, MGAS166. Southern analysis using a probe
specific for the silCR gene confirmed that MGAS166 does
not encode silCR by comparison against a silCR-encoding
M18 isolate (data not shown). Furthermore, any residual
signaling molecule(s) from the overnight cultures of
MGAS166 were removed previously by washing. Thus, the
effect(s) of altered gene expression or pathogenicity of
MGAS166 is a direct consequence of the exogenous addition
of SilCR. To ensure that the addition of SilCR did not alter
the growth kinetics of MGAS166, we confirmed that con-
centrations ranging from 0.1 to 5.0 mg mL1 had no signifi-
cant effect on growth (data not shown).

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The addition of SilCR to MGAS166 resulted in the
Results and discussion upregulation of sagA, siaA, and scpC relative to growth in
TH medium alone (Fig. 1), indicating that SilCR affects the
In this study, SilCR was synthesized as a mature 17 amino expression of these loci. SilCR influenced sagA and siaA in a
acid peptide to investigate its effects on the gene expression concentration-dependent manner (Fig. 1a and b); however,
and pathogenicity of an invasive M1 serotype strain of this effect was not observed on the expression of scpC (Fig.
1c). There was no significant difference in scpC expression
Table 1. Primers and optimal annealing temperature used for the real- when SilCR concentrations ranged from 0.05 to 5.0 mg mL1.
time RT-PCR analysis of sagA, siaA, scpC, and gyrA Conversely, sagA was significantly upregulated in the pre-
Optimal annealing Forward primer:
sence of 0.5 mg mL1 relative to 0.05 and 5.0 mg mL1 of
Gene temperature ( 1C) Reverse primer: SilCR at the mid- and the late-exponential growth phase.
Similarly, at the late-exponential growth phase, siaA was
sagA 50 5 0 -AGGAGGTAAACCTTATGTTA-3 0
5 0 -TACCACCTTGAGAATTACCA-3 0 significantly upregulated in the presence of 0.5 mg mL1
siaA 65 5 0 -CAGCAGAGAATTGTAGCCACTTCG-3 0 relative to 0.05 and 5.0 mg mL1 of SilCR. This concentra-
5 0 -CCCACACGCTTAACAGCATCATAG-3 0 tion-dependent effect on gene expression, whereby a signal-
scpC 55 5 0 -GCACGAACTATCATTACGCCTTG-3 0 ing peptide stimulates gene expression at low concentrations
5 0 -CTTTTGCTGTTGCTGAGGTCG-3 0 and inhibits it at higher concentrations, has been observed
gyrA 65 5 0 -AGCGAGACAGATGTCATTGCTCAG-3 0
with the competence-stimulating factor of Bacillus subtilis
5 0 -CCAGTCAAACGACGCAAACG-3 0
(Lazazzera et al., 1997). It is not clear why varying the

(a) sagA expression in various SilCR concentrations (b) siaA expression in various SilCR concentrations
(µg mL–1) relative to TH medium alone (µg mL–1) relative to TH medium alone
187
Relative expression

40 156
Relative expression

30 125
5.00 5.00
20 94
0.50 0.50
63
10 0.05 0.05
32
0 1
Early-exponential Mid-exponential Late-exponential Early-exponential Mid-exponential Late-exponential
–10 –30
Growth phase Growth phase
(c) scpC expression in various SilCR concentrations
(µg mL–1) relative to TH medium alone
21
Relative expression

16
5.00
11
0.50
6
0.05
1
Early-exponential Mid-exponential Late-exponential
–4
Growth phase

Fig. 1. Expression of sagA (a), siaA (b), and scpC (c) grown in various concentrations of SilCR relative to growth in TH medium alone as determined
using real-time RT-PCR analysis.

FEMS Microbiol Lett 289 (2008) 119–125

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
122
concentrations of SilCR did not affect scpC in a manner
similar to that of sagA and siaA. These data, however,
suggest different modes of regulation and warrant further
investigation.
In addition to a concentration-dependent effect, we also
observed a density-dependent effect on sagA expression,
which was at its highest during the late-exponential growth
phase (Fig. 1a). This is concordant with previous data from
our laboratory that showed that sagA is involved in density-
dependent regulation (Salim et al., 2007). The 120–150-fold
increase in siaA expression in the early-exponential growth
phase (Fig. 1b) could only be accounted for by the exogen-
ous addition of SilCR because the experiments in this study
were not conducted under iron-restricted conditions. Lim-
ited iron concentrations are known to upregulate siaA
(Bates et al., 2003; Lei et al., 2003; Salim et al., 2007);
therefore, we conclude that SilCR is either directly or
indirectly involved in regulating iron acquisition or mod-
ulating physiologic homeostasis in S. pyogenes.
Interestingly, scpC was upregulated c. 16-fold in the
presence SilCR at the early-exponential growth phase (Fig.
1c). These data are in contrast to those reported previously
that indicated that in an M14 serotype strain, the exogenous
addition of SilCR resulted in the downregulation of scpC in
vitro via the TCSTS silA/B (Hidalgo-Grass et al., 2006; Eran
et al., 2007). We, however, used an M1 serotype strain that is
proposed to lack the entire sil locus (Hidalgo-Grass et al.,
2002, 2004). We confirmed that the strain used in our study
lacked not only silCR by Southern analysis but also silA and
silB by PCR using specific primers (data not shown). These
data suggest that although the sil operon TCSTS is absent in
MGAS166, one or more of the TCSTSs identified in
S. pyogenes (Ferretti et al., 2001; Beres et al., 2002, Bates
et al., 2003; Smoot et al., 2002; Nakagawa et al., 2003; Green
et al., 2005; Sumby et al., 2005) could be detecting and
responding to this peptide. Clearly, additional work will be
required to determine the TCSTS in M1 serotype strains that
allows for modulation of gene expression in the absence of
silA/B. We speculate that this TCSTS could be CovRS
because a recent report indicated that the expression of scpC
(a) Gene expression in vivo at 24 and 48 h relative to the
highest level of expression in vitro
16
Relative expression
Relative expression
11
siaA
6 sagA
scpC
1 1
24 48
–4
Time postinoculation (h)
Fig. 2. (a) In vivo expression of sagA, siaA, and scpC in the absence of SilCR at 24 and 48 h postinoculation relative to the highest level of expression in
vitro also in the absence of SilCR. (b) In vivo gene expression of sagA, siaA, and scpC at 24 and 48 h postinoculation with MGAS16615.0 mg SilCR
relative to MGAS166 alone.


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K.Y. Salim et al.
was negatively regulated by this system in an M1 serotype
strain (Sumby et al., 2008).
Our in vitro data showed that the addition of SilCR to
cultures increased the expression of genes involved in the
pathogenicity of S. pyogenes. These observations were not
congruent with the reported protective or therapeutic effect
of SilCR against invasive S. pyogenes infections (Hidalgo-
Grass et al., 2004). We investigated gene expression in vivo in
a subcutaneous murine model of invasive disease in the
presence or absence of exogenously added SilCR and also
tested the proposed therapeutic effect of SilCR. In the
absence of SilCR, sagA, siaA, and scpC were upregulated at
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24 and 48 h postinoculation in vivo relative to the highest
level of gene expression in vitro (Fig. 2a), indicating the
importance of these genes in this infection model. Interest-
ingly, the addition of SilCR to the inoculum resulted in
further upregulation of sagA and siaA relative to the
MGAS166 inoculum alone (Fig. 2b). Conversely, there was
no significant difference in the expression of scpC in vivo
regardless of whether SilCR was present or absent from the
inoculum (Fig. 2b). Furthermore, unlike Hidalgo-Grass
et al. (2004), who found a therapeutic effect of SilCR on
lesion formation at concentrations as low as 3.0 mg of
peptide, we did not observe such a beneficial effect even at
a dose of 5.0 mg of peptide.
Mice inoculated with MGAS166 either in the presence or
in the absence of exogenously added SilCR lost weight
significantly at days 1 and 2 postinoculation relative to day
0 (Fig. 3a). This suggests that SilCR did not have a
therapeutic effect on weight loss, which is a quantitative
measure of virulence in this murine model. A comparison of
lesion surface area on the same day between mice inoculated
with MGAS166 in the presence or absence of SilCR indi-
cated that there was no significant difference in lesion size
between these mice until 12 days postinoculation. At this
point, the mice inoculated with MGAS166 in the presence of
SilCR exhibited significantly larger lesions than those inocu-
lated with MGAS166 alone (Fig. 4a and b), indicating that
SilCR was not therapeutic during tissue necrosis but sig-
nificantly attenuated lesion healing. Determination of
(b) Gene expression in vivo at 24 and 48 h in MGAS166 +
5.0 µg SilCR relative to MGAS166 alone
3
2.5
siaA
2
sagA
1.5
scpC
24 48
0.5
Time postinoculation (h)
FEMS Microbiol Lett 289 (2008) 119–125
SilCR regulates sagA, siaA and scpC expression 123

(a) Weight change (b) Average log10(CFU mL–1) of bacteria

Average log10 (CFU mL–1)

3 10
2 * *
Median weight change

1 * * * 8
*
0 6
(g)

–1 4
–2
2
–3 0 1 2 3
–4 0
–5 2 5 7 12
Time postinoculation (days) Time postinoculation (days)

MGAS166 MGAS166 + SilCR MGAS166 MGAS166 + SilCR

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Fig. 3. (a) Median weight loss/gain ( range) of mice inoculated subcutaneously with MGAS166 in the presence or absence of SilCR. Statistical
significance (P o 0.05) utilizing a Wilcoxon–Mann–Whitney test determined for each group of mice inoculated with MGAS166 in the presence or
absence of SilCR at days 1, 2, and 3 postinoculation relative to day 0 indicated by the asterisks (). (b) Average log10 (CFU mL1) of bacteria recovered
from the inoculation site of groups of mice each inoculated with either MGAS166 alone or MGAS16615.0 mg SilCR. Statistical significance (P o 0.05) in
recovered bacterial counts between mice inoculated with MGAS166 in the presence or absence of SilCR determined by an unpaired Student’s t-test
indicated by the asterisks ().

(a) MGAS166 alone (b) MGAS166 + 5.0 µg SilCR

25 25
Lesion area (mm 2)
Lesion area (mm 2)

20 20

15 15

10 10

5 * 5
*
0 0
2 3 4 5 6 7 9 12 2 3 4 5 6 7 9 12
Time postinoculation (days) Time postinoculation (days)

Fig. 4. Boxplots of lesion surface area (mm2) per group of mice inoculated with MGAS166 in the absence (a) or presence (b) of 5.0 mg SilCR at 2, 3, 4, 5,
6, 7, 9, and 12 days postinoculation. Statistical significance (P o 0.05) based on a Wilcoxon–Mann–Whitney test determined for each group of mice on
the same day inoculated with MGAS166 alone relative to MGAS16615.0 mg SilCR indicated by the asterisks ().

bacterial counts further supports these observations because
at 12 days postinoculation significantly more MGAS166
were eradicated from the inoculation site compared with
MGAS16615.0 mg SilCR (Fig. 3b). Thus, SilCR appeared to
either directly or indirectly interfere with the ability of the
host to eradicate the bacteria and allow for healing of
necrotic tissue. The differences in our results and those of
Hidalgo-Grass et al. (2004) could be explained by the
different mouse strains and/or M serotype strains utilized
in both studies, although we propose that the latter is more
likely. Evidence to support the latter explanation was
presented in a recent report that showed that an isogenic
mutant of scpC in an M1 serotype strain caused significantly
larger skin lesions in mice compared with its wild-type
parental strain (Sumby et al., 2008).
Clearly, the delayed effect on wound healing observed in
this study was not a result of a change in the expression of
scpC, because there was no significant difference in expres-
sion levels in vivo in the absence or presence of SilCR (Fig.
2b). Whether the delayed effect on wound healing was due
to the increased expression of SLS is uncertain. The in-
creased expression of sagA in the presence of SilCR did not
significantly affect lesion formation as would be expected
because sagA contributes to lesion formation in the sub-
cutaneous murine infection model. However, increased SLS
production that did not correlate with increased virulence
has been recorded in a previous study (Lyon & Caparon,
2004). Interestingly, the upregulation of SiaA in an siaB-
deficient mutant resulted in increased virulence of S. pyo-
genes in a zebrafish model of invasive disease, which was
proposed to be a result of the accumulation of surface-
bound heme by SiaA (Montanez, 2005). Because heme itself
was shown to exhibit proinflammatory properties (Wagener
et al., 2003), the virulent phenotype observed with the siaB-
deficient mutant was attributed to the accumulation of
heme bound to SiaA on the bacterial surface (Montanez,

FEMS Microbiol Lett 289 (2008) 119–125

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
124
2005). It would be interesting to determine whether the
delayed response of wound healing in the presence of SilCR
observed in this study was due to an increased inflammatory
response resulting from bacterially associated heme accu-
mulation or the increased expression of sagA or other
virulence factor(s).
In conclusion, we have shown that exogenously added
SilCR upregulates sagA, siaA, and scpC expression in vitro.
However, only sagA and siaA were upregulated in vivo by
SilCR and the expression of scpC remained unaltered.
Furthermore, this study shows that SilCR does not appear
to have a therapeutic effect on the invasive potential of
S. pyogenes. Additional work will be required to determine
what effect SilCR might have on the expression of other
virulence factors of S. pyogenes and why the effect of SilCR
on the invasive potential of S. pyogenes varies depending on
the strain and/or the serotype background.
Acknowledgements
This work was supported by an Operating Grant from
Connaught Laboratories to D.G.C. and Infrastructure grants
from The Canadian Foundation for Innovation and Ontario
Innovative Trust. Additional support was provided by a
CIHR Strategic Training Fellowship in Cell Signaling in
Mucosal Inflammation and Pain (STP-53877).
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