Streptococcus pyogenes is an important human pathogen
causing a number of acute suppurative infections such as erysipelas,
necrotizing fasciitis, and pharyngitis. These Gram-positive bacteria
also cause a serious toxic shock-like syndrome, whereas
glomerulonephritis and rheumatic fever are serious poststreptococcal
sequelae. To elude the host defense and establish an infection, S.
pyogenes has developed multiple molecular mechanisms. Some of
these are dependent on genes of the mga regulon. The genes of
this regulon are under the control of a positive regulator gene,
previously called mry (Caparon and Scott, 1987) or virR (Simpson et al., 1990). According to a recent agreement,
this regulator should now be called mga, multigene regulator
of group A streptococcus.
Based on structural variations in the
antiphagocytic M protein (for references, see Fischetti(1989) and
Kehoe(1994)), S. pyogenes can be divided into more than 80
different serotypes. Since the late 1980s unusually severe S.
pyogenes infections have been reported world-wide, infections
which have predominantly been associated with the M1 serotype (for
references, see Martin and Single (1993) and Musser et
al.(1993)). This serotype is also connected with
glomerulonephritis and rheumatic fever. The strain studied here, AP1,
is of the M1 serotype. In AP1, the regulatory gene mga is
followed by emm1, the gene for M1 protein. Immediately
downstream of emm1 is sph (Åkesson et
al., 1994), the gene encoding an IgGFc-binding M protein-related
molecule called protein H (Gomi et al., 1990; Åkesson et al., 1990). Located adjacent to sph is a
previously uncharacterized gene. This gene and its product are the
subject of the present report. The data described imply that this
extracellular protein, in the following referred to as protein SIC,
plays a role in S. pyogenes pathogenicity and virulence
through previously unknown molecular mechanisms.
EXPERIMENTAL PROCEDURES
Bacterial Strains, Bacteriophage, and
Plasmids
Protein SIC and the sic gene were isolated
from S. pyogenes strain AP1 (40/58) of the M1 serotype from
the Institute of Hygiene and Epidemiology, Prague, Czech Republic.
Forty-eight additional strains of different M serotypes were also
obtained from this institute. These strains have been described (Ben
Nasr et al., 1995). S. pyogenes strains of serotypes
M3, M33, M42, M52, M61, and M67 were kindly provided by Dr. U.
Sjöbring, Lund University, and a collection of 35
M1 strains isolated in Sweden 1980-89 were kindly provided by Dr.
Stig Holm, Umeå University. One S. pyogenes strain of
serotype M1 and two of type M57 were from the late Dr. L. W.
Wannamaker. The sic gene was cloned from a
EMBL3 clone
described previously (Åkesson et al., 1994). Plasmid
vectors pUC18/19 (Yanisch-Perron et al., 1985) and pK18/19
(Pridmore, 1987) were used in subcloning experiments. PCR (
)products were cloned into the inducible secretion vector
pHD389 (Dalböge et al., 1989). The Escherichia coli strain JM109 was used as host for recombinant
plasmids. Streptococci were grown in Todd-Hewitt broth supplemented
with 0.2% yeast extract and E. coli in LB broth.
Proteins and Antisera
Recombinant M1 protein,
protein PAB, and streptococcal cysteine proteinase (SCP) were purified
as described previously (Åkesson et al., 1994; de
Château and Björck, 1994;
Berge and Björck, 1995). Human serum albumin (HSA)
was from Sigma. The monoclonal antibody MCaE11 directed against a
neoantigen of polymerized C9 within the C5b-C9 membrane attack complex
(MAC) of complement (Mollnes et al., 1985b) was kindly
provided by Dr. T. E. Mollnes, The National Hospital, Oslo, Norway.
Antisera against purified streptococcal protein SIC and SCP were raised
in rabbits. Sheep anti-rat clusterin (polyclonal IgG fraction)
cross-reacting with human clusterin was purchased from Quidel (San
Diego, CA). Goat antisera against human histidine-rich glycoprotein
(HRG) was a kind gift from Dr. William T. Morgan, University of
Missouri, Kansas City, MO. Peroxidase-conjugated goat anti-rabbit IgG
(Bio-Rad) and donkey anti-sheep IgG (ICN) were used as secondary
antibodies.
Purification of Protein SIC
Streptococcal protein
SIC was purified by growing AP1 bacteria to midlog phase (A
= 0.5) and precipitating the culture
supernatant with 30% ammonium sulfate. The resulting precipitate was
resuspended to 1/100 of the starting volume with 20 mM Tris,
pH 7.5, and dialyzed against the same buffer. This material was applied
to a Mono Q column on a fast protein liquid chromatography system
(Pharmacia, Uppsala, Sweden). Using a linear NaCl gradient, a single
sharp peak was eluted at 0.3 M NaCl. Corresponding fractions
were pooled, concentrated, and subjected to gel filtration
chromatography on a Superose 12 column (Pharmacia) in PBSA (0.03 M phosphate, 0.12 M NaCl, 0.02% NaN
, pH
7.2). Fractions containing the suspected protein SIC were pooled, and
the identity of the protein was confirmed by NH
-terminal
amino acid sequencing and later by Western blotting using antisera
against protein SIC. The yield was 0.5-2 mg of purified protein
SIC per liter of culture. Recombinant protein SIC was purified from an E. coli strain JM109 harboring the sic gene in
pHD389. The strain was grown to midlog phase at 30 °C before
inducing protein production by raising the temperature to 40 °C for
4 h. The culture supernatant was collected, and purification of protein
SIC was performed as described above. In order to examine the presence
of protein SIC in other group A streptococcal strains, cultures grown
to midlogarithmic phase were precipitated with 30% ammonium sulfate.
The precipitates were washed with ice-cold ethanol, dissolved in 100
µl of SDS-PAGE sample buffer, and subjected to Western blot
analysis using rabbit antiserum against protein SIC.
Purification of Clusterin and HRG
For the
purification of clusterin and HRG, citrated fresh human plasma
supplemented with 1 mM benzamidine and 0.4 mM
phenylmethylsulfonyl fluoride was used. Clusterin was purified by a
method modified from a previous study (Wilson and Easterbrook-Smith,
1992). Thus, human plasma was differentially precipitated with
polyethylene glycol (12-23%, w/v). The precipitate was then
passed over an IgG-Sepharose column (Pharmacia Biotech Inc.). The
column was washed with PBSA, and bound proteins were eluted with 0.1 M glycine-HCl, pH 2.0. The material was dialyzed against 20
mM Tris, pH 7.5, and subjected to a Mono Q column. Using a
linear NaCl gradient, a protein peak eluted at 0.3 M NaCl was
found to contain a single band of 80 kDa when analyzed by SDS-PAGE
under nonreducing conditions. The material gave rise to a single band
of 35 kDa when run under reducing conditions in the presence of 5%
-mercaptoethanol. The bands were identified as clusterin in
Western blot experiments using anti-clusterin antibodies. HRG was
purified using the protocol of Saigo et al.(1989). Briefly,
plasma was absorbed with CM-Sephadex (Pharmacia). The matrix was washed
extensively with distilled water, and adsorbed proteins were eluted
with 0.5 M NH
HCO
. After dialysis
against PBS, the material was applied to a HiTrap Heparin column
(Pharmacia). The column was washed with PBS, and proteins were eluted
with 1.0 M NaCl. The eluate was dialyzed against 20 mM Tris, pH 7.5, and subjected to ion exchange chromatography on a
Mono Q column. A linear NaCl gradient was used, and the peak at 0.25 M NaCl was collected. The resulting preparation consisted of
two major bands of 70 and 63 kDa when examined by SDS-PAGE under
nonreducing conditions. The identity of the bands with HRG was
confirmed by Western blotting using anti-HRG antisera.
Polymerase Chain Reactions
PCR was performed using Taq DNA polymerase (Promega). For amplification of the sic gene for cloning, primers S1 and S2 (Fig. 2), corresponding
to base pairs (bp) 97-123 and bp 928-905 of the sic sequence (Fig. 1), were used. To investigate the occurrence
of sic in other group A streptococcal isolates, PCR was
performed with the S1 and S2 primer with an annealing temperature at 20
°C below the T
of the shortest primer (46
°C). Templates for the reactions were prepared by resuspending a
single bacterial colony in 100 µl of water, vortexing the sample
vigorously, and then boiling it for 5 min. Cell debris was removed by
centrifugation, and 5 µl of the resulting supernatant were used in
a 50-µl reaction. In order to determine the relationship of sic to other genes of the streptococcal mga regulon, PCRs
with two different primer pairs outlined in Fig. 2were
performed. In one reaction, the H1 primer corresponding to the
conserved membrane anchoring motif of cell wall proteins of
Gram-positive cocci; bp 1357-1381 of the sph sequence
(Gomi et al., 1990) and the S2 primer corresponding to the end
of the sic sequence were used. In another reaction, the S3
primer corresponding to bp 871-890 of the sic sequence (Fig. 1) and the C1 primer from the signal sequence of the C5a
peptidase gene, bp 888-868 in the scpA sequence (Chen
and Cleary, 1990), were used. Reactions were performed with AP1
chromosomal DNA as the template.
Figure 2:
A map of the mga regulon in
strain AP1 and a schematic outline of the structure of protein SIC. A
region of the streptococcal chromosome containing genes of the mga regulon is depicted in the upper part of the figure. The
relative location and size of the genes were determined by nucleotide
sequencing and PCR (Gomi et al., 1990; Åkesson et
al., 1994; B.-M. Kihlberg, J. Cooney, M. G. Caparon, A.
Olsén, and L. Björck,(1995)
Footnote 2). The location of oligonucleotide primers used for the PCR
experiments in this work are marked with arrows. In the lower part of the figure, protein SIC and its domains are
shown. Numbers below the schematic representation correspond
to the first amino acid of these domains.
Figure 1:
Nucleotide sequence
and deduced amino acid sequence of the sic gene from S.
pyogenes strain AP1. The first 188 nucleotides representing the
sequence between the protein H gene (sph) and the sic gene and the first 131 nucleotides of the coding sequence have
been published (Gomi et al., 1990). In this work, the sequence
from nucleotide 7 in the coding sequence was determined. The start of
the signal sequence (Ss), the NH
-terminal short
repeat region (SRR), and the tandem repeats R1-R3 are indicated with arrows. The short repeats (I-V) are marked with bars. Possible -35
and -10 promoter sequences and the ribosomal binding site (RBS) are denoted with dashed
lines.
Cloning Techniques and Sequence Determination
The
PCR product amplified for the cloning of the sic gene was
generated with primers containing recognition sites for NarI
and XbaI. This fragment of 830 bp was purified, digested with NarI and XbaI, and cloned into pHD389 digested with
the same enzymes. Standard cloning procedures were used (Sambrook et al., 1989). Restriction enzymes and T4 DNA ligase were from
Promega. DNA sequencing was performed with Sequenase (Pharmacia)
according to the manufacturer's instructions. Ordered sets of
deletions in template plasmid DNA were prepared using exonuclease Bal31 (Promega). Computer search for sequence homology was
made in the GenBank and EMBL data bases. Analysis of sequence data was
performed using the GeneWorks program (IntelliGenetics, Mountain View,
CA).
Electrophoresis and Electroblotting
SDS-PAGE was
performed as described (Neville, 1971) using gels of 8% or 12%
acrylamide content and 3.3% cross-linking. Before loading, samples were
boiled for 3.5 min in an equal volume of buffer containing 2% SDS.
Samples run under reducing conditions were boiled in the presence of 5%
2-mercaptoethanol. Western blotting was performed by transferring
proteins to polyvinylidene difluoride (PVDF) membranes (Immobilon,
Millipore) as described by Towbin et al.(1979). After
blocking, membranes were incubated for 1 h with antisera against
protein SIC (1:200, v/v), HRG (1:500, v/v), or antibodies toward
clusterin (5 µg/ml), followed by a peroxidase-labeled secondary
antibody (1:1000 v/v). For visualization of bound antibody, membranes
were incubated in 0.02% (w/v) 3-amino-9-ethylcarbazol, 0.06% (v/v)
H
O
in 50 mM sodium acetate buffer, pH
5.0.
Affinity Chromatography
In plasma absorption
experiments, citrated fresh human plasma was mixed with the protease
inhibitors benzamidine and iodoacetic acid to final concentrations of 5
mM each. 5 ml of undiluted plasma was immediately added to 1
mg of protein SIC coupled to Sepharose. After end to end rotation for 4
h at room temperature, the protein SIC-Sepharose was washed extensively
with PBS (200 times the column volume), and the proteins bound to the
column were eluted with 0.1 M glycine-HCl, pH 2.0. As a
control, 5 ml of plasma was absorbed simultaneously with
glycine-Sepharose. pH was adjusted to 7.5 by adding 1 M Tris,
and the eluates were concentrated about 100-fold. Purified radiolabeled
clusterin, HRG, or HSA (4
10
cpm in 1.0 ml of PBS)
were run on columns of Sepharose coupled with protein SIC, M1 protein,
or HSA. After washing with PBS, bound proteins were eluted with 0.1 M glycine-HCl, pH 2.0, and the radioactivity in the fractions
was measured in a
counter.
ELISA
Indirect ELISA (Engvall and Perlmann, 1971)
was performed by coating microtiter plates (Maxisorb, Nunc, Denmark)
overnight at 4 °C with serial dilutions of protein SIC (starting
concentration 2 µg/ml). The plates were washed in PBST and
incubated with clusterin (5 µg/ml) or HRG (1.25 µg/ml) diluted
in PBST containing 1% gelatin (PBSTG). Bound proteins were detected by
purified antibodies to clusterin (10 µg/ml) or antisera against HRG
(1:2000, v/v), and binding was visualized by a horseradish
peroxidase-conjugated secondary antibody against rabbit or sheep IgG
(1:5000, v/v). All incubations were done at 37 °C for 1 h and
followed by a washing step. Substrate solution, 0.1% (w/v)
2,2`-azinobis(3-ethylbenzthiazolinesulfonate), 0.012% (v/v)
H
O
in 100 mM citric acid, 100
mM NaH
PO
, pH 4.5, was added, and the
change in absorbance at 405 nm was determined after 30 min.A
competitive ELISA was performed using the same procedure specified
above except for the following: microtiter plates coated with either
clusterin (1.5 µg/ml) or HRG (0.5 µg/ml) were incubated with a
mixture of protein SIC (2 µg/ml for binding to clusterin; 1
µg/ml for binding to HRG) and serial dilutions of the competitor
protein (starting concentration 20 µg/ml). Bound protein SIC was
detected by a specific rabbit antisera (1:1000 v/v) followed by a
secondary antibody toward rabbit IgG (1:3000 v/v) and
2,2`-azinobis(3-ethylbenzthiazolinesulfonate)/
H
O
.
Binding of protein SIC to C5b-C9
complexes generated in serum was detected with a capture ELISA modified
from Mollnes et al. (1985a). Microtiter plates were coated
with 1.0 µg/ml MCaE11, a monoclonal antibody against a neoantigen
of polymerized C9. Serum diluted 1/50 in PBSTG was added to the wells
followed by serial dilutions of protein SIC or SCP (starting
concentrations 10 µg/ml), and the plates were incubated for 3 h at
room temperature. Diluted serum was also preincubated with protein SIC
or SCP at various concentrations for 3 h, after which generation of
C5b-C9 complexes was stopped by addition of EDTA at 10 mM. The
incubation mixtures were then transferred to antibody-coated microtiter
plates for analysis. Detection of protein SIC and SCP was performed as
described above using specific rabbit antisera diluted 1/1000.
Complement-mediated Hemolytic Assays
Hemolytic
assays in free solution were performed essentially as reported with
target cells added in excess (Nilsson and Nilsson, 1984). Sheep
erythrocytes, treated with rabbit antibody (NBL, Stockholm, Sweden) to
yield optimally sensitized cells (EA), were used to measure classical
pathway function. Assays were performed with Veronal-buffered saline
(VBS) containing 0.15 mM Ca
and 0.5 mM Mg
. Alternative pathway-mediated hemolysis of
guinea pig erythrocytes (GpE) was performed in VBS containing EGTA at a
final concentration of 16 mM and Mg
at 4
mM. Various quantities of protein SIC were added to human
serum diluted to a concentration causing 10% lysis of erythrocytes.
After incubation for 30 min at room temperature, EA or GpE were added
at a concentration of 5
10
cells per ml and the
mixture was incubated at 37 °C for 20 min. The reaction was stopped
by adding a 15-fold volume of cold VBS containing 10 mM EDTA.
After centrifugation, hemolysis was measured as the absorbance of the
supernatant at 541 nm. The final human serum dilutions were 1:40 for
assays of the classical pathway and 1:20 for the alternative pathway.
The serum used was free from antibodies toward protein SIC.
Complement-mediated hemolysis in gel was studied according to the
procedure described by Truedsson et al.(1981) using EA or GpE
in agarose as target cells. 5 µl of undiluted human serum and 1.5
µg (in 5 µl) of protein SIC or protein PAB were applied to
adjacent wells in the gels with a distance of 2-4 mm.
Additionally, 1.5 µg of bacterial proteins were added after 2, 4,
6, and 8 h of incubation. After incubation of the gels at 4 °C for
16 h, lysis was induced at 37 °C for 3 h.
Other Methods
NH
-terminal amino acid
sequence analysis of proteins electrotransferred to PVDF membranes were
performed on an Applied Biosystems 470A gas-liquid solid phase
sequenator. Clusterin, HRG, and HSA were labeled with
I
using the Bolton and Hunter reagent (Amersham Corp., Buckinghamshire,
United Kingdom). The specific activities were between 3 and 10 µCi
per µg of protein.
RESULTS
Sequencing of the sic Gene
The previously
reported sequence of the protein H gene (sph) revealed the
first 138 bp of an open reading frame 188 bp downstream of the stop
codon for sph (Gomi et al., 1990). To determine the
structure of this possible downstream gene, a phage clone,
1:4
(Åkesson et al., 1994), harboring both the M1 gene (emm1) and sph was employed for subcloning. A 2.2-kbp SspI/EcoRI fragment of
1:4 that contained the
open reading frame except for the first six nucleotides was cloned into
the vector pUC19. Sequencing of this subclone revealed that the entire
open reading frame was 915 bp encoding a putative amino acid sequence
of 305 residues (Fig. 1). The first 32 amino acid residues
represents a typical bacterial signal sequence including a positively
charged NH
-terminal region, a hydrophobic core, and a polar
cleavage region. The predicted mature protein has 273 amino acid
residues and a calculated molecular mass of 30.677 kDa. The most
characteristic feature of the sequence is a central repeat region
consisting of three tandem repeats of 29, 29, and 21 amino acid
residues each (R1-R3), showing 90-95% internal homology. The
sequence preceding the R repeats also contains repeats although these
are only five or nine amino acids long. In Fig. 1, five
different repeats in this short repeat region (SRR) are indicated. They
appear only twice each, and, except for repeat IV, they are not in
tandem. Instead, some of the repeats are overlapping. The sequence
located COOH-terminal of the R repeats is characterized by a high
proline content, and, in a short stretch (residue 200-220), the
prolines are evenly spaced every third or fourth residue.
Computer-assisted analysis of the sequence predicted the protein to be
highly hydrophilic with a pI of 4.2. A thorough search of the data
bases with the entire sequence, or fragments of the sequence including
all kinds of repeats, did not reveal any obvious homology either at the
nucleotide or the amino acid level.
Location of sic in the mga Regulon of AP1
A
further mapping of sic in the mga regulon of the AP1
strain was accomplished by PCR of chromosomal DNA with two sets of
primers (Fig. 2). A reaction with the H1 primer corresponding to
the sequence of M-like genes encoding the consensus membrane-anchoring
motif (Fischetti et al., 1990) and the S2 primer corresponding
to the 3` end of sic generated a fragment of 1.2 kbp. This
confirms the intergenic distance of 188 bp between sph and sic as determined by sequencing. The PCR performed with the S3
primer from the 3` end of sic and the C1 primer corresponding
to a part of the signal sequence of the C5a peptidase gene (scpA) generated a 2.2-kbp product. These experiments
demonstrate that sic is located 188 bp downstream of sph and approximately 2.1 kbp upstream of scpA (Fig. 2).
Cloning and Expression of Protein SIC in E. coli and
Purification of the Protein from Streptococcal Culture
Medium
Oligonucleotides were constructed from the sic sequence to generate a PCR fragment corresponding to the mature
protein. This fragment was amplified and cloned into the inducible
expression vector pHD389. After growing the clone and inducing
expression, a periplasmic lysate was examined by SDS-PAGE which showed
the overexpression of a 34-kDa protein as compared to the host E.
coli strain. Even higher amounts of the 34-kDa product were
detected when analyzing the culture medium after precipitation with 80%
ammonium sulfate. The protein was purified using precipitation of the
culture medium with 30% ammonium sulfate, ion exchange chromatography,
and gel filtration (Fig. 3B). The purified product was
seen as a single band on SDS-PAGE, and the yield was about 5 mg of
purified protein per liter of culture.
Figure 3:
SDS-PAGE analysis of protein SIC after
consecutive purification steps. The purification of the wild-type
protein SIC from S. pyogenes is shown in A, and the
purification of the recombinant protein from E. coli is shown
in B. The material in lanes 1 was precipitated from
culture medium with 80% ammonium sulfate followed by precipitation with
30% ammonium sulfate (lanes 2). Lanes 3 show protein
SIC after an additional purification step on Mono Q, whereas the final
product (lanes 4) was obtained after gel filtration on a
Superose 12 column. Molecular mass markers are
indicated.
Both the presence of a signal
sequence and the absence of cell wall anchoring and membrane-spanning
sequences in the gene suggested that the protein is secreted by
streptococci of the AP1 strain. Consequently, a purification scheme
similar to that used for the recombinant protein was applied to the
culture medium of streptococcal AP1 bacteria, and a protein with
identical migration on SDS-PAGE was obtained (Fig. 3A).
The estimated amount of protein SIC in a 1-liter culture was
10-15 mg from which 1-2 mg of purified protein was
obtained. NH
-terminal amino acid analysis of the protein
showed that the first five amino acids (ETYTS) were identical with the
start of the mature protein as indicated by the nucleotide sequence.
Thus, the gene product of sic was shown to be expressed by the
AP1 strain as an extracellular protein.
Absorption of Plasma Proteins with Immobilized Protein
SIC
Being a secreted product, protein SIC is most likely active
outside the bacterial cell. On the streptococcal chromosome, the sic gene is found in a regulon encoding proteins which
interact with host molecules, particularly plasma proteins. In order to
examine if protein SIC also has affinity for plasma proteins, the
molecule was coupled to Sepharose and used to absorb human plasma.
After extensive washing, the proteins bound to the protein
SIC-Sepharose were eluted with glycine buffer, pH 2.0. The eluted
material was lyophilized, resuspended in PBS at 50 times the
concentration, and examined on SDS-PAGE. As seen in Fig. 4, two
additional bands appeared as compared to the background pattern
obtained after absorption with glycine-Sepharose. The bands were
electroblotted onto a PVDF membrane, excised from the membrane, and
subjected to NH
-terminal amino acid sequencing. The band of
80 kDa (labeled I in Fig. 4) showed the presence of two
residues in equimolar yields after each sequencing cycle. The two
sequences (DQTVSDNELQ and SLMPFSPYEP) were identical with the
NH
-terminal sequences of the
and
chains of the
plasma protein clusterin (Jenne and Tschopp, 1989; Kirszbaum et
al., 1989). The band of 70 kDa (labeled II in Fig. 4) had the sequence VSPTDCSAVE, identifying the protein as
HRG (Koide et al., 1986). Plasma absorption experiments were
performed with recombinant or streptococcal protein SIC coupled to
Sepharose, and the same results were obtained.
Figure 4:
SDS-PAGE analysis of proteins eluted from
protein SIC-Sepharose after plasma absorption. Proteins eluted from
glycine-Sepharose (lane A) or protein SIC-Sepharose (lane
B) following incubation with human plasma. The samples were
separated by SDS-PAGE (8%) under nonreducing conditions. Molecular mass
markers are shown to the left. The bands of 80 kDa and 70 kDa
in lane B are indicated with I and II,
respectively.
Further Analysis of the Interaction between Protein SIC
and the Plasma Proteins Clusterin and HRG
The binding of protein
SIC to clusterin and HRG was now further analyzed. Firstly, clusterin
and HRG were purified from human plasma. The observations made with
human plasma were then re-examined by affinity chromatography of
radiolabeled clusterin and HRG on protein SIC-Sepharose. About 30% of
the
I-clusterin and about 60% of the
I-HRG
were retained after chromatography on a protein SIC-Sepharose column.
In contrast, less than 5% of iodine-labeled albumin was eluted from the
column (Fig. 5). Additionally, less than 5% of
I-clusterin or
I-HRG was bound to columns
of M1 protein or HSA-Sepharose (data not shown). The interactions were
also tested by indirect ELISA in which the binding of clusterin or HRG
to protein SIC was shown to be concentration-dependent (Fig. 6).
Additionally, competitive ELISAs with clusterin or HRG as the coated
proteins and protein SIC as the probe were performed. In these
experiments, the binding of protein SIC to clusterin was blocked by HRG
although less efficiently than by clusterin itself (Fig. 7A). Similarly, the binding of protein SIC to HRG
was inhibited by clusterin (Fig. 7B). The results
indicate overlapping or closely located binding sites for the two
plasma proteins in protein SIC.
Figure 5:
Affinity chromatography of radiolabeled
proteins on protein SIC-Sepharose. 4
10
cpm in 1.0
ml of PBS of clusterin (A), HRG (B), or HSA (C) was applied to a protein SIC-Sepharose column. After
washing with the application buffer, proteins bound to the column were
eluted with 0.1 M glycine, pH 2.0. The radioactivity of the
fractions was determined.
Figure 6:
Binding of clusterin and HRG to protein
SIC in indirect ELISA. Microtiter plates were coated with dilution
series of protein SIC (
) or protein PAB (
). 5 µg/ml
clusterin (A) or 1.25 µg/ml HRG (B) were applied
followed by specific antibodies to these proteins and a
peroxidase-labeled secondary antibody. The absorption at 405 nm is
presented in arbitrary units.
Figure 7:
Competition for the binding of protein SIC
to clusterin and HRG. Competitive ELISAs were performed on microtiter
plates coated with 1.5 µg/ml clusterin (A) or 0.5
µg/ml HRG (B). Dilutions of clusterin (
), HRG
(
), or HSA (
) were mixed with an equivolume (100 µl)
of protein SIC (2 µg/ml in A and 1 µg/ml B),
and the mixtures were applied to the wells of the titer plates. Protein
SIC bound to the immobilized proteins was detected with specific rabbit
polyclonal antiserum followed by a peroxidase-labeled secondary
antibody. The results are expressed as percent of protein SIC bound in
the presence of a competitor relative to the specific binding in the
absence of a competitor (100%).
Inhibition of Complement-mediated Hemolytic Activity by
Protein SIC
Together with vitronectin, clusterin and HRG bind to
the C5b-C9 complex in serum and thereby regulate the cytotoxic action
of MAC (Tschopp et al., 1993; Chang et al., 1992).
Clusterin partly inhibits hemolysis, while HRG modulates complement
function in a biphasic fashion. To assess a possible role for protein
SIC in classical pathway-mediated hemolysis, the protein was incubated
with diluted human serum for 30 min at 20 °C prior to addition of
EA. A dose-dependent inhibition of hemolysis was obtained (Fig. 8A). To exclude nonspecific degradation of
complement components during preincubation of serum, an unrelated
bacterial protein, the albumin-binding protein PAB of Peptostreptococcus magnus (de Château and
Björck, 1994) was used as a negative control. The
effect of protein SIC on alternative pathway-mediated hemolysis was
examined using GpE as target cells in serum chelated with EGTA and
supplemented with Mg
. As shown in Fig. 8B, the effect of protein SIC on hemolysis of GpE
was comparable to the effect on hemolysis of EA, consistent with
interference at the C5b-C9 level. Similar results were obtained when
effects of protein SIC were studied in hemolytic gels with EA and GpE
as target cells (Fig. 8C). When protein SIC was applied
adjacent to wells containing serum, the zone of lysis caused by serum
was partly eclipsed. In contrast, protein PAB had no inhibitory effect
on hemolysis.
Figure 8:
Inhibition of complement-mediated
hemolysis. Diluted human serum was incubated with various
concentrations of protein SIC (
) or protein PAB (
) followed
by addition of sensitized sheep erythrocytes (A) or guinea pig
erythrocytes (B). Hemolysis was measured by determining the
absorbance of supernatants at 541 nm. Inhibition of lysis was
calculated by dividing the absorbance with the value obtained when
incubating serum with buffer (PBS). Data represent the mean from three
separate experiments and ±S.E. is indicated. In C, 5
µl of undiluted human serum was applied to wells in agarose gels
containing sensitized sheep erythrocytes (I) or guinea pig
erythrocytes (II). Protein SIC or protein PAB were applied (6
µg) in the adjacent wells as indicated. Gels were incubated at 4
°C overnight and hemolysis was induced by incubation at 37 °C
for 3 h.
Binding of Protein SIC to C5b-C9 Complexes
The
interaction between protein SIC and the terminal complement proteins in
serum was examined with a capture ELISA. In the first layer, a
monoclonal antibody was used that detects an epitope of polymerized C9
that is formed during assembly of the C5b-C9 complex. Incubation of
serum gives a high level of such complexes binding to the antibody due
to spontaneous in vitro activation of complement (Mollnes et al., 1985a). In Fig. 9, an experiment is shown where
serum and protein SIC simultaneously were added to and incubated with
the solid-phase antibody. The amount of bound protein SIC was then
measured. In contrast to the control protein, SCP, protein SIC is
incorporated into the C5b-C9 complex generated in serum. No protein SIC
binding was detected in the absence of serum or in serum containing
EDTA. Further experiments were carried out to ensure that deposition of
complement proteins due to activation by the immunoglobulin-coated
microtiter plates (Zwirner et al., 1989) did not influence the
results. Thus, serum was preincubated with protein SIC. After 3 h, EDTA
was added and the incubation mixtures were transferred to the
antibody-coated plates for analysis. The results obtained were
virtually identical with those shown in Fig. 9.
Figure 9:
Binding of protein SIC to C5b-C9
complexes. A monoclonal antibody directed against a neoantigen of
polymerized C9 within C5b-C9 was used to coat microtiter plates (1
µg/ml). Dilution series of protein SIC (
) or SCP (
)
were mixed with human serum in a final dilution of 1:50 and applied to
the wells. Incubation was also done with a dilution series of protein
SIC in buffer (
). Protein SIC and SCP were detected by specific
antibodies to these proteins and a peroxidase-labeled secondary
antibody.
Distribution of Protein SIC in Strains of S.
pyogenes
A collection of 55 group A streptococcal strains of
different M types were tested for the presence of the sic gene
and the expression of protein SIC. At the DNA level, sic was
identified by PCR using primers from the start of the coding sequence
and the repeat regions and by performing the reaction under low
stringency conditions. The expression of protein SIC was tested by
growing the strains to midlogarithmic phase, precipitating the culture
media with 30% ammonium sulfate, and examining the precipitate in a
Western blot using polyclonal antisera against protein SIC. The strains
of M types 1 and 57 were positive both in the PCR and in Western blot
experiments. The rest of the strains gave no PCR products and were
negative in the Western blot analyses. After the initial screening, 35
additional M type 1 isolates and two M type 57 strains were tested. All
of these strains contained sic and expressed protein SIC. The
results suggest that the sic gene is highly restricted among
various M serotypes, whereas within these serotypes all isolates have
and express the gene.
DISCUSSION
Analogous to other proteins encoded by genes under the
control of mga, protein SIC contains repeated sequences.
However, the sequence of protein SIC, including the repeats, shows no
homology to previously sequenced genes. It is also noteworthy that in
contrast to all other described products of the mga regulon,
protein SIC does not have the typical structural features of cell wall
proteins in Gram-positive bacteria; i.e. a COOH-terminal
region anchored to the cell wall through an LPXTG motif
(Fischetti et al., 1990; Schneewind et al., 1992,
1995), followed further toward the COOH terminus by a hydrophobic
membrane-spanning domain and a tail of mostly positively charged amino
acid residues. The missing cell wall anchor, the occurrence of a
typical signal sequence, and the fact that considerable amounts of
protein SIC are found in the growth medium suggest that the molecule is
secreted and has extracellular function(s).
Previous work has
demonstrated several interactions between components of the complement
system and proteins encoded by genes of the mga regulon.
Members of the M protein family (M protein, protein Arp, protein Sir,
and protein H) have been reported to bind complement factor H, CD46,
and/or the C4b-binding protein (Horstmann et al., 1988; Okada et al., 1995; Thern et al., 1995), (
)three
proteins with regulatory functions in the complement system.
Furthermore, the C5a peptidase (Wexler et al., 1983), which
can be released from the streptococcal cell wall by a cysteine
proteinase produced by the bacteria (Berge and
Björck, 1995), cleaves the C5-derived fragment C5a
and destroys its chemoattractant activity for polymorphonuclear
leukocytes (Wexler et al., 1985). In the present study, the
specific interactions between protein SIC and the plasma proteins
clusterin and HRG directed our attention to the final cytolytic step in
the complement cascade. Clusterin is known to inhibit the hemolytic
activity of complement by binding to MAC (Tschopp et al.,
1993; Tschopp and French, 1994), whereas the influence of HRG on MAC is
biphasic, inhibitory, or stimulatory, depending on the experimental
conditions (Chang et al., 1992). As demonstrated in the
present study, protein SIC was inhibitory to hemolysis in classical
pathway as well as alternative pathway systems. Furthermore, protein
SIC was shown to be incorporated into C5b-C9 complexes formed in serum.
Although other mechanisms may also be considered, the findings suggest
that the anticomplementary action of protein SIC is focused on the
terminal cytotoxic functions of complement.
The metabolically inert
erythrocyte is a very sensitive target for MAC whereas most pathogenic
bacteria including streptococci are resistant to complement-mediated
cytolysis. However, apart from its cytolytic activity, MAC also has
proinflammatory effects by stimulating the production and release of
inflammatory mediators such as reactive oxygen metabolites, metabolites
of arachidonic acid, and cytokines (for references see Morgan(1989)).
Bacterial products affecting the various functions of MAC, directly or
indirectly, could therefore influence the host-parasite relationship.
The molecular complexity necessary to establish and maintain this
relationship makes it difficult to predict the consequences of any
isolated interaction. However, in the case of pathogenic and virulent
bacteria like S. pyogenes, the balance is disturbed, and there
is circumstantial evidence (see below) that protein SIC may contribute
to the imbalance of the host-parasite relationship in S. pyogenes infections.
To our knowledge, protein SIC is the first
bacterial protein reported to interact with clusterin or HRG. Mycobacterium tuberculosis, however, selectively absorbs
clusterin from human plasma, (
)suggesting that this
important human pathogen could express surface molecules with affinity
for clusterin. As mentioned, glomerulonephritis represents a medically
significant sequelae following infections with S. pyogenes. In
these cases, certain M serotypes are more common, including the two
protein SIC-producing serotypes M1 and M57 (Holm, 1988). In
post-streptococcal glomerulonephritis, immunoglobulin deposits are
found in the glomeruli. Interestingly, MAC is regarded as a mediator of
glomerular injury in immune complex-related disease (Couser et
al., 1985), and clusterin was found to be co-localized with MAC in
biopsies from glomerulonephritic kidneys (French et al.,
1992). Also, plasma depleted of clusterin (to <30%) enhanced
proteinuria and deposition of MAC components in perfused kidneys
(Saunders et al., 1994). The association of protein SIC to
nephritogenic M serotypes, and its binding of clusterin in human plasma
makes it interesting to test whether protein SIC can induce kidney
damage in an animal model.
Since the late 1980s, a world-wide
increase of hyperacute, toxic, and often lethal S. pyogenes infections has attracted public attention also (Nowak, 1994).
These systemic infections have been associated particularly with
streptococci of the M1 serotype, and the observation that all M1
strains tested, including isolates from Swedish patients with toxic and
severe infections (Holm et al., 1992), carry and express the sic gene, supports the notion that protein SIC plays a role in
pathogenicity and virulence. The present work and future studies on
protein SIC may therefore clarify molecular mechanisms which could be
used as targets to prevent and treat S. pyogenes infections.