SIC, a Secreted Protein of Streptococcus pyogenes
That Inactivates Antibacterial Peptides*
Inga-Maria
Frick
§,
Per
Åkesson¶,
Magnus
Rasmussen
,
Artur
Schmidtchen¶, and
Lars
Björck
From the
Section for Molecular Pathogenesis,
Department of Cell and Molecular Biology, Lund University, and
the ¶ Department of Medical Microbiology, Dermatology, and
Infection, Lund University Hospital, S-221 85 Lund,
Sweden
Received for publication, August 30, 2002, and in revised form, February 25, 2003
 |
ABSTRACT |
Some isolates of the significant human pathogen
Streptococcus pyogenes, including virulent strains of the
M1 serotype, secrete protein SIC. This molecule, secreted in large
quantities, interferes with complement function. As a result of natural
selection, SIC shows a high degree of variation. Here we provide a
plausible explanation for this variation and the fact that strains of
the M1 serotype are the most frequent cause of severe invasive S. pyogenes infections. Thus, protein SIC was found to inactivate human neutrophil
-defensin and LL-37, two major antibacterial peptides involved in bacterial clearance. This inactivation protected S. pyogenes against the antibacterial effect of the
peptides. Moreover, SIC isolated from S. pyogenes of the M1
serotype was more powerful in this respect than SIC variants from
strains of M serotypes 12 and 55, serotypes rarely connected with
invasive infections.
 |
INTRODUCTION |
Streptococcus pyogenes is one of the most
common and important human bacterial pathogens. It causes relatively
mild infections such as pharyngitis (strep throat) and impetigo but
also serious clinical conditions like rheumatic fever,
poststreptococcal glomerulonephritis, necrotizing fasciitis,
septicemia, and a toxic-shock syndrome (1, 2). Increases in the number
of life-threatening systemic S. pyogenes infections have
been reported world-wide since the late 1980s and have attracted
considerable attention and concern (3, 4). Based on the highly
polymorphic M protein, a surface protein of S. pyogenes (for
references see Ref. 5), isolates are divided into more than 100 serological subtypes, and systemic infections are most frequently
caused by organisms of the M1 serotype (6).
Protein SIC was originally isolated from the growth medium of an M1
strain (7). All strains of the M1 serotype secrete SIC and so do M57
organisms, whereas strains of 53 other serotypes were found to lack the
sic gene (7). Subsequent work has identified distantly
related sic variants also in M12 and M55 strains (8). SIC
stands for streptococcal inhibitor of
complement, as the protein incorporates into the membrane
attack complex of complement and inhibits complement-mediated
lysis of sensitized erythrocytes (7). This inhibition of membrane
attack complex was recently shown to be the result of SIC preventing
uptake of C567 onto cell membranes (9). A remarkable property of
SIC was reported by Stockbauer et al. (10). They found that
the sequences of a large number of sic genes from different
strains of the M1 serotype showed a unique degree of variation, which
is in striking contrast to the lack of M1 protein variation. Moreover,
in a mouse model of infection, Hoe et al. (11) discovered
that SIC variants arise rapidly on mucosal surfaces by natural
selection. They also reported that the inhibition of
complement-mediated lysis by SIC was not affected in the new SIC
variants arising from natural selection, suggesting that complement
inhibition is not the only function of SIC. Complement belongs to the
innate immune system, and antibacterial peptides represent another
important part of this defense system. These peptides, originally
described in silk worms (12), play important roles in the clearance of
bacteria at biological boundaries susceptible for infection (for
references see reviews in Refs. 13-16), and the starting point for
this investigation was the hypothesis that SIC, secreted in substantial
amounts by S. pyogenes (7), could interfere with the
activity of antibacterial peptides. Such a mechanism could help explain
the variation of SIC and the high frequency of S. pyogenes
infections caused by strains of the M1 serotype.
 |
EXPERIMENTAL PROCEDURES |
Bacterial Strains and Purification of Protein SIC--
The
S. pyogenes strains AP1 (40/58) of serotype M1 and AP12
(1/71) of serotype M12 were from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. The S. pyogenes strain W38 (GT
71-154) of serotype M55 was from the late Dr. L. W. Wannamaker, and
the S. pyogenes strains U15 and U17 of serotype M1 were
kindly provided by Dr. Stig Holm, Umeå University, Umeå, Sweden.
Bacteria were grown in Todd-Hewitt broth
(TH1; Difco) at 37 °C.
Protein SIC was purified from the S. pyogenes strains AP1,
U15, U17, AP12, and W38 as described (7) by precipitation of the
culture medium with 30% ammonium sulfate, followed by ion-exchange chromatography on Mono Q (Amersham Biosciences). Fractions
containing protein SIC were combined, dialyzed against 2 mM
NH4HCO3 and freeze-dried. For the antimicrobial
assay (see below) protein SIC was dissolved in 10 mM
Tris-HCl, pH 7.5, containing 5 mM glucose.
DNA Sequencing--
PCR was used to amplify the sic
genes from the S. pyogenes strains U15, U17, AP12, and W38.
For serotype M1 strains (U15 and U17), primers were constructed
corresponding to the nucleotide sequence starting 72 base pairs
upstream (ACCTTTACTAATAATCGTCTTTGTTTTATAATGA) and 179 base pairs
downstream (ATCTTTCTCGGACTCAGATAGTCCATAGC) of the coding sequence for
sic in the AP1 strain (7). For serotype M12 (AP12)
and M55 (W38) strains, a forward primer (CATTAACGAAATAATTTATTAAGGAGAG) corresponding to a region upstream of the coding sequence of
sic from AP1 and a reverse primer
(CCAATGATAGTCACCAGCAATTCAGG) corresponding to a region downstream of
the coding sequence of the distantly related sic from M12
and 55 strains (8). The PCR products were purified with a High Pure PCR
purification kit (Roche Applied Science) and used as templates in
sequencing reactions using an ABI PRISM®
BigDyeTM dideoxy terminator kit (BigDye terminator version
3.0 cycle sequencing Ready Reaction, Applied Biosystems), according to
the manufacturer's instructions.
Proteins, Peptides, Antibodies, and
radiolabeling--
Recombinant M1 protein was prepared as described
previously (17),
-defensin (HNP-1), ACYCRIPACIAGERRYGTCIYQGRLWAFCC
(Mr 3442) was purchased from Sigma, and
LL-37, LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (Mr
4492) was synthesized by Innovagen AB, Lund, Sweden. The peptide GCP28,
GCPRDIPTNSPELEETLTHTITKLNAEN, based on a sequence in human kininogen
has been described previously (18) and was a kind gift from Dr. Heiko
Herwald, Lund University, Lund, Sweden. Mouse monoclonal
-defensin
antibodies were from Bachem AG, and rabbit polyclonal LL-37 antibodies
were from Innovagen AB. Antiserum against protein SIC was raised in
rabbits. Protein SIC was labeled with 125I using the
chloramine T method (19).
Generation of a sic Mutant in S. pyogenes and Northern
Blotting--
To inactivate the gene encoding protein SIC, AP1
bacteria were subjected to an allelic replacement mutagenesis strategy.
A fragment of the up-stream sph and of the down-stream
IS1562 was amplified from AP1 by PCR as described (20) using
synthetic oligonucleotides hybridizing with nucleotides 486-512 and
1559-1533 in (21) and 516-539 and 1614-1641 in (22). The
oligonucleotides had sites for restriction enzymes that were used to
clone the sph product into multiple cloning site I of
plasmid pFW13 (23) and the IS1562 product into multiple
cloning site II to generate pFW13sic
. 20 µg of
pFW13sic
was electroporated as described (24)
into AP1 bacteria. Recombinants were selected on plates containing 150 µg/ml kanamycin. One transformant (SIC
) was obtained,
and growth media from this strain was incubated with 6%
trichloroacetic acid for 30 min on ice followed by
centrifugation at 15,000 × g (4 °C for 20 min).
Precipitated material was analyzed for SIC content using polyclonal
anti-SIC antibodies in Western blot experiments.
Total RNA from the AP1 and SIC
strains was isolated at
early logarithmic (A620 0.3), late
logarithmic (A620 0.6), or early stationary phase
(A620 0.8) as described previously (20). Northern blotting
was performed as described (20) using a probe generated with primers
hybridizing to sic (1-28, 912-889 in Ref. 7).
Animal Experiments--
Female NMRI mice weighing ~25 g
were injected subcutaneously with 104 mutant or wild-type
bacteria in 100 µl of PBS followed by 900 µl of air. Mice were
observed for 10 days. Statistical analysis of survival time was
performed with the Wilcoxin rank sum test.
Antimicrobial Assay--
AP1 bacteria were grown to mid-log
phase in TH broth, washed, and diluted in 10 mM Tris-HCl,
pH 7.5, containing 5 mM glucose. 50 µl of bacteria
(2 × 106 colony forming units (cfu)/ml) were
incubated together with
-defensin or LL-37 at various concentrations
for 2 h at 37 °C. In subsequent experiments, bacteria were
incubated with
-defensin or LL-37 at a concentration of 448 nM together with different concentrations of protein SIC or
M1 protein and the reactions were carried out for 2 h
(
-defensin) or 1 h (LL-37). To quantitate the bactericidal activity serial dilutions of the incubation mixtures were plated on TH
agar, incubated overnight at 37 °C, and the number of cfus were determined.
Bacterial Growth Assay--
AP1 and SIC
bacteria
were grown to stationary phase in TH broth. 200 µl of TH was
inoculated with 5 µl of the bacterial suspension in 96-well plates
(Falcon) at 37 °C. At early logarithmic phase various amounts of
LL-37 was added, and growth was followed by measuring the absorbance at
490 nm (using a BioRad 550 microplate reader). The amount of protein
SIC in the growth medium was estimated by ELISA (see below).
Slot-binding, SDS-PAGE, and Western Blot Analysis--
Peptides
were applied to polyvinylidene difluoride (PVDF) membranes (Immobilon,
Millipore) using a Milliblot-D system (Millipore). Membranes were
blocked in TBS (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl) containing 3% bovine serum albumin, incubated with
125I-labeled protein SIC for 3 h, and washed with TBS
containing 0.05% Tween 20. Autoradiography was carried out using Kodak
x-Omat AR films and regular intensifying screens. SDS-PAGE was
performed as described by Laemmli (25) using a polyacrylamide
concentration of 10% or 12% and 3.3% cross-linking. Samples were
boiled for 3 min in sample buffer containing 2% SDS and 5%
2-mercaptoethanol. Gels were stained with Coomassie Blue or subjected
to Western blot analysis. Separated proteins were transferred to PVDF
membranes using a trans-blot semidry transfer cell (Bio-Rad). Membranes were blocked with PBS containing 0.05% Tween 20 (PBST) and 5% dry
milk powder (blocking buffer), incubated with antibodies against protein SIC (1:1000) in blocking buffer for 30 min at 37 °C, washed with PBST, and incubated with a horseradish peroxidase-conjugated antibody against rabbit IgG (1:3000) for 30 min at 37 °C. The membranes were washed, and detection of bound antibodies was performed using the chemiluminescence method.
ELISA--
Indirect ELISA was performed by coating microtiter
plates (Maxisorb, NUNC) overnight with protein SIC or M1 protein
at a concentration of 2.9 nM. The plates were washed in
PBST, blocked in PBST containing 2% bovine serum albumin for 30 min
and incubated with 58 nM
-defensin or LL-37 for 1 h. Bound antibacterial peptides were detected with specific antibodies
against
-defensin (1:2000) or LL-37 (1:5000), and binding was
visualized by a peroxidase-conjugated secondary antibody against mouse
or rabbit IgG (1:3000). All incubations were performed at 37 °C for
1 h followed by a washing step. Substrate solution, 0.1% (w/v)
diammonium-2,2-azino-bis-(3-ethyl-2,3-dihydrobenzthiazoline)-6-sulfonate, 0.012% (v/v) H2O2 in 100 mM
NaH2PO4, pH 4.5, was added, and the change in
absorbance at 405 nm was determined after 5 min. To determine the
concentration of SIC in growth medium plates coated with AP1 or
SIC
growth medium were incubated with antibodies against
protein SIC (1:1000) followed by a secondary antibody against rabbit
IgG (1:3000). Visualization of binding was detected as above, and absorbance was determined after 15 min.
 |
RESULTS AND DISCUSSION |
Two major and well characterized human antibacterial peptides,
-defensin (HNP-1) and LL-37, were used in this investigation. These
peptides have broad antibacterial activity against both Gram-positive
and Gram-negative bacteria.
-defensin (HNP-1) is found in the
azurophilic granules of human neutrophils, but analogues to neutrophil
-defensins are produced also by intestinal Paneth cells (26, 27).
LL-37 is produced by neutrophils and epithelial cells.
-defensin and
LL-37 are both found in extracellular fluids, including wound fluid,
and the two peptides act synergistically on target bacteria (28). In
S. pyogenes, the sic gene is part of the
so-called mga regulon, and like the other genes of this regulon sic is expressed at an early growth phase (7),
suggesting that SIC will be secreted as soon as S. pyogenes
bacteria carrying the sic gene start to grow on an
epithelial surface. To investigate whether
-defensin and LL-37 have
affinity for SIC, these peptides and a control peptide (GCP28) based on
a sequence in H-kininogen (18), were applied to Immobilon filters that
were probed with 125I-labeled SIC (if not indicated
otherwise SIC is from the M1 strain AP1). Fig.
1A shows that SIC interacts
with the antibacterial peptides, an observation that was confirmed also
by experiments where SIC and M1 protein were applied to plastic wells,
followed by the addition of
-defensin or LL-37 and antibodies to the
peptides. M1 protein was chosen as a control. It was isolated from the
same strain of S. pyogenes (AP1) as SIC (7), and although
the protein is predominantly associated with the cell wall, it is also
released from the bacterial surface by proteolytic cleavage (29). In the experiments summarized in Fig. 1B,
-defensin and
LL-37 showed affinity for SIC, whereas the interaction with M1 protein
was at background level.

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Fig. 1.
SIC interacts with antibacterial
peptides. A, various amounts of the antibacterial
peptides -defensin and LL-37 and the peptide GCP28 derived from
human H-kininogen were applied to a PVDF membrane. The membrane was
incubated with radiolabeled protein SIC (2 × 105
cpm/ml) for 3 h and autoradiographed for 3 days. B,
microtiter plates were coated with protein SIC or M1 protein at 2.9 nM, followed by incubation with -defensin or LL-37 (58 nM). Binding was detected with specific antibodies against
-defensin and LL-37, respectively. The bars represent the
mean ± S.E. of at least three experiments.
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|
Next we investigated the antibacterial effect of
-defensin and LL-37
on the AP1 strain. In these experiments the bacteria were washed and
resuspended in buffer prior to the addition of the peptides to exclude
that SIC was present during the incubation period. As shown in the
left panel of Fig. 2, the
peptides killed the bacteria. The concentration required for 100%
killing was ~0.4 µM for both peptides. The inhibitory
effect of SIC was then tested at a bactericidal concentration (0.448 µM) of the peptides and the results (see right
panel, Fig. 2) show that SIC blocks the antibacterial activity of
-defensin and LL-37. The inhibition curves indicate that SIC is
about 10 times more efficient in blocking LL-37 than
-defensin. M1
protein was also tested (at a maximum of 0.72 µM) but
showed no inhibitory activity.

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Fig. 2.
SIC protects S. pyogenes
against antibacterial peptides. S. pyogenes
strain AP1 (2 × 106 cfu/ml) was incubated with the
antibacterial peptides -defensin ( ) or LL-37 ( ) at indicated
concentrations for 2 h at 37 °C in buffer, and cfus were
determined (left panel). The bactericidal effect of
-defensin ( ) or LL-37 ( ), at a concentration of 448 nM, was inhibited with various concentrations of protein
SIC (right panel). Experiments were repeated at least three
times, and representative experiments are shown.
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When grown to stationary phase, the growth medium of S. pyogenes of the M1 serotype contains large quantities (10-15
µg/ml) of protein SIC (7). To inactivate the gene encoding protein SIC, AP1 bacteria were subjected to an allelic replacement mutagenesis strategy. One mutant, SIC
, was obtained, and it secreted
minute amounts of protein SIC to the growth medium as compared with
wild-type AP1 bacteria (Fig. 3A). The expression of
sic from the AP1 and SIC
strains was
investigated at different stages of growth using Northern blotting. As
shown in Fig. 3B, the expression of sic reaches
its maximum at late logarithmic growth phase, and no expression was
detected at early stationary phase. The same filter was hybridized with
a 16 S probe, showing that the same amount of RNA was applied to each
well (data not shown). In experiments where the bacteria were washed
before the addition of the antibacterial peptide LL-37, the mutant
strain SIC
was found to be as sensitive to LL-37 as the
wild-type strain AP1 (see Fig. 2, left panel). In a series
of experiments we then investigated whether SIC produced by growing M1
bacteria could protect the organisms against LL-37 (these experiments
required substantial amounts of antibacterial peptide, and only LL-37
was tested). The wild-type AP1 strain and the mutant strain
SIC
were grown to early logarithmic growth phase where
the concentration of SIC produced by AP1 bacteria was 0.85 µg/ml
growth medium as determined by ELISA (Fig. 3C). The
secretion of protein SIC by the mutant SIC
was below the
detection level (Fig. 3C). At this point different amounts
of LL-37 were added. As shown in Fig. 3D, SIC
bacteria were more sensitive to LL-37 as compared with the
SIC-producing strain AP1. The concentration of LL-37 required to kill
50% of SIC
bacteria in these experiments was 22.3 µM, while at this concentration only 20% of the
wild-type bacteria were killed. Lower concentrations of LL-37 killed
20% of SIC
bacteria, but the AP1 strain was unaffected.
The amount of protein SIC produced was determined at different time
points during growth. While the concentration of SIC produced by AP1
bacteria in this experimental system, reached its maximum (4.75 µg/ml) at early stationary phase (6 h), the amount of protein SIC in
the growth medium of the mutant strain SIC
was below the
level of detection (Fig. 3C). The results suggest that SIC
secretion provides protection against antibacterial peptides already at
an initial stage of infection.

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Fig. 3.
Secretion of protein SIC during growth of
S. pyogenes inhibits the antibacterial activity of
LL-37. A, growth media from S. pyogenes
strain AP1 and the SIC mutant were precipitated with
trichloroacetic acid. The precipitated proteins were subjected to
SDS-PAGE analysis. One gel was stained with Coomassie Blue
(Stain), and one gel was blotted to a PVDF membrane and
probed with a polyclonal anti-SIC antiserum (Blot).
B, total RNA samples from the AP1 and SIC
strains were isolated from bacteria in early logarithmic phase
(EL), late logarithmic phase (LL), or early
stationary phase (ES), subjected to Northern blotting, and
probed with a probe hybridizing with sic. C, AP1
bacteria (SIC+) and the mutant strain
SIC were grown in microtiter plates, and the
concentration of protein SIC was determined at different time points by
ELISA. D, bacteria were grown in microtiter plates and
various amounts of LL-37 were added to AP1 (SIC+) and
SIC bacteria at early logarithmic phase (indicated with
an arrow). Bacterial growth was determined by measuring the
absorbance at 490 nm at different time points. Growth medium
alone ( ); 5.55 µM LL-37 ( ); 11.1 µM
LL-37 ( ); 22.3 µM LL-37 ( ). Experiments were
repeated at least three times, and a representative experiment is
shown.
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As mentioned, SIC variants are produced by S. pyogenes
strains of M serotypes 1, 12, 55, and 57 (7, 8). Using the same
isolation protocol as for SIC from M1 bacteria (7), SIC was purified from the growth medium of M12 and M55 organisms. Analogous to the M1
strain (7), the M12 and M55 strains produced 10-15 µg of SIC/ml of
growth medium when grown to stationary phase. Protein SIC was also
purified from two additional strains of the M1 serotype, U15 and U17.
The purified variants shown in Fig.
4A were tested for their
ability to interfere with the antibacterial activity of
-defensin
and LL-37. As shown in Fig. 4B, all SIC variants, but not M1
protein, blocked the activity of the peptides. The inhibition curves
show that SICM1 is 5-100 times more potent in this respect than the
protein SIC variants from the M12 and M55 strains. To investigate a
possible molecular basis for this difference in inhibitory activity,
sequencing of the genes encoding the SIC variants was performed. As
mentioned, there is a high degree of variation among sic
genes from different strains of the M1 serotype (10), and nearly 300 alleles are known. The amino acid sequences derived from the obtained
sic sequences were compared with that of protein SIC from
the M1 strain AP1 (GenBankTM accession number X92968
(7)). A deletion of a region of 29 amino acid residues in SIC from U15
and some shorter insertions and single amino acid substitutions were
observed within the M1 variants (Fig.
5A). However, the
sic genes of the M12 and M55 strains are clearly more
different, as compared with the sic genes of the M1 strains
(not shown). The sequence of SICM12 was found to be identical to a
recently published sequence (GenBank accession number AJ300679 (30)),
and as demonstrated in Fig. 5B a high degree of homology was
found when the derived amino acid sequence of SICM55 was compared with
that of SICM12. Analysis of the amino acid composition of the SIC
variants revealed differences in charged residues, where the M1
variants have a higher negative net charge compared with SICM12 and
SICM55. This could explain the reduced capacity of SICM12 and SICM55 in
blocking the activity of
-defensin and LL-37. It is noteworthy that
the four SIC-producing M serotypes (1, 12, 55, and 57) are all known to
be associated with poststreptococcal glomerulonephritis, suggesting a
role for SIC in this condition (7, 8). Moreover, the fact that M1
strains dominate in cases of invasive disease (6) and that SICM1 is the
most potent inhibitor of
-defensin and LL-37, support the notion
that the interference of SIC with antibacterial peptides could
facilitate S. pyogenes invasion through mucosal and skin
barriers.

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Fig. 4.
Inhibitory effect of different SIC variants
on the antibacterial activity of -defensin and
LL-37. A, different variants of protein SIC (1 µg)
were subjected to SDS-PAGE (10% gel) and stained with Coomassie Blue.
Protein SICM1 was purified from S. pyogenes strains U15,
U17, or AP1. The distantly related variants SICM12 and SICM55 were
purified from S. pyogenes strains of M serotypes 12 and 55, respectively. B, S. pyogenes strain AP1
was incubated with antibacterial peptides (448 nM) for
2 h ( -defensin) or 1 h (LL-37) in the presence of various
amounts of proteins SICM1AP1 ( ), SICM1U15 ( ), SICM1U17
( ), SICM12 ( ), SICM55 ( ), or M1 protein ( ).
Experiments were repeated at least three times, and representative
experiments are shown.
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Fig. 5.
Structure of the investigated SIC
variants. A, insertions, deletions, and amino acid
substitutions in the SICM1U15 and SICM1U17 amino acid sequences are
shown as compared with the SICM1AP1 sequence (GenBank accession number
X92968 (7)). Unbroken lines represent homologous sequences,
vertical bars and single-letter amino acid abbreviations
represent substitutions indicated at their position in the SICM1AP1
sequence. B, amino acid differences between the SICM55 and
the SICM12 sequences are shown by single-letter amino acid
abbreviations and indicated at their position in the SICM12
sequence.
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Over the past 10-20 years research in many laboratories has firmly
established the fundamental role played by antibacterial peptides in
the initial clearance of pathogenic bacteria. The demonstration that
SIC not only interferes with complement function but also inactivates
antibacterial peptides further underlines the significance of the
innate immune system. The data of the present study also emphasize the
highly complex molecular interplay between S. pyogenes and
its human host. Although pathogenicity and virulence are polygenic
properties, the results indicate that SIC could represent an important
virulence determinant. A previous investigation (31) showed that an
isogenic M1 mutant strain in which the sic gene had been
inactivated, was significantly less efficient in colonizing the
throat of mice as compared with the wild-type strain. Here we find,
using a mouse model of subcutaneous infection, that the mutant strain
SIC
is significantly attenuated in virulence compared
with the wild-type AP1 strain (Table I).
Combined these data suggest that SIC promotes early stages of infection
by inactivating antibacterial peptides. The results of the present work
may also explain the unique variability of the sic gene and
the fact that the M1 serotype is the serotype most frequently connected
with invasive S. pyogenes infection.
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Table I
Subcutaneous challenge of mice with S. pyogenes strains AP1 and
SIC
NMRI mice were injected subcutaneously with 104 AP1 or
SIC bacteria, and mice were followed for 10 days.
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 |
ACKNOWLEDGEMENTS |
Ingbritt Gustafsson and Ulla Johannesson are
acknowledged for excellent technical assistance.
 |
Addendum |
In relation to the present work it is
noteworthy that protein SIC also inhibits the antibacterial activities
of lysozyme and secretory leukocyte proteinase inhibitor, proteins that
are part of the mucosal innate immune system. These data were reported by Fernie-King et al. (32) after the submission of this manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the Swedish Research
Council (Projects 7480, 13471, and 14379), the Foundations of Crafoord,
Bergvall, and Österlund, and the Royal Physiographic Society.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Cell and
Molecular Biology, Lund University, BMC, B14, Tornavägen 10, S-221 84 Lund, Sweden. Tel.: 46-46-222-8569; Fax: 46-46-157756; E-mail:
Inga-Maria.Frick@medkem.lu.se.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M301995200
 |
ABBREVIATIONS |
The abbreviations used are:
TH, Todd Hewitt
broth;
PBS, phosphate-buffered saline;
cfu, colony forming units;
PBST, PBS Tween;
ELISA, enzyme-linked immunosorbent assay;
PVDF, polyvinylidene difluoride.
 |
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