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(Received for publication, March
29, 1994; and in revised form, September 16, 1994) From the
In this report we have analyzed the binding of collagen to Streptococcus pyogenes strain 6414. This binding was rapid,
specific, and involved a limited number of receptor molecules (11,600
copies per cell). When the proteins in a streptococcal lysate were
blotted onto a nitrocellulose filter and probed with Interactions of cells with the surrounding extracellular matrix
play important roles in numerous physiological and pathological events.
In higher animals, cell-matrix interactions involve binding of cells to
collagens, proteoglycans, and various glycoproteins such as fibronectin
and laminin. Collagens are widely distributed proteins in vertebrate
tissues and at least 14 genetically different types of collagen have
been discovered. In the organisms the collagens play a structural role
and influence biological processes such as cell
attachment(1, 2) , proliferation(3) , as well
as cell differentiation during organogenesis (4) and
hematopoiesis(5) . Membrane proteins with collagen binding
properties have been suggested to act as collagen receptors and have
been purified from osteoblastoma cells(6) ,
fibroblasts(7) , endothelial cells(8) ,
platelets(9) , and chondrocytes(10) . Several
studies have shown that a variety of pathogenic bacteria interact with
extracellular matrix components including fibronectin (11) ,
fibrinogen(12) , laminin(13) , and collagen. For
instance strains of coagulase-negative staphylococci ( Within the genus Streptococcus,
there are species, mainly among the groups A, B, C, and G, which bind
collagens(19) . This binding has been postulated as a factor
contributing to the development of a number of infections. For example
binding of Streptococcus mutans strains to collagen has been
proposed to play a role in the pathogenesis of root
caries(20) , and the ability of Streptococcus pyogenes to bind collagen type IV may be an important virulence factor in
determining post streptococcal glomerulonephritis(21) . A
collagen receptor from streptococci has previously not yet been
isolated or characterized. In this study we report on the isolation and
characterization of a 57-kDa collagen-binding protein from S.
pyogenes 6414 which can act as an adhesin and mediate the
adherence of streptococcal cells to collagen rich tissues.
Bovine serum albumin, ovalbumin, fetuin, bovine
IgG, protein A, and protein G were from Sigma. Iodogen was from Pierce
Chemical Co. Todd Hewitt Broth was supplied by Difco. Carrier-free
After
harvesting, bacteria were suspended in 0.13 M sodium chloride,
0.02% sodium azide, and 10 mM sodium phosphate, pH 7.4 (PBS), (
For Western ligand blotting
assay the membranes were incubated with 3% bovine serum albumin in PBS
for 1 h at 22 °C and then probed overnight with 5 For immunoblotting assays, the nitrocellulose
sheets were blocked with 3% albumin in TBS (20 mM Tris, 0.5 M NaCl, pH 7.5) then probed with IgG overnight at 4 °C,
followed by washing with TTBS (0.5 M NaCl, 20 mM Tris/HCl, 0.05% Tween 20, pH 7.5). Subsequently the sheets were
incubated with anti-mouse IgG antibodies conjugated with horseradish
peroxidase (Bio-Rad) diluted 1:1000 in TTBS containing 1.0% albumin.
For adhesion assays, microtiter
wells were coated with 50 µl of collagen type II (100 µg/ml)
and blocked with bovine serum albumin as reported above. The wells were
then overlaid with 10-20 µl of a suspension of
The binding
of Heat-killing of streptococcal cells did not alter the binding
kinetics and similar amounts of collagen bound to live and killed
bacteria. Therefore, heat-treated bacteria were used throughout this
study, and the cells were routinely incubated with the ligand for 60
min. The specificity of collagen binding by streptococci was
examined by incubating bacterial cells with When increasing concentrations of native and denatured type
II collagen and gelatin were tried as inhibitors of
Figure 1:
Effect of native and denatured collagen
on
Incubation
of streptococci with increasing concentrations of
Figure 2:
Saturability of binding of
In a similar study using cells of S.
zooepidemicus, strain S
Figure 4:
Analysis of proteins obtained at different
steps of collagen receptor purification. A, fractions obtained
from affinity and gel filtration chromatography were subjected to
electrophoresis on a 10% SDS-PAGE gel in non-reducing conditions and
stained with Coomassie Brilliant Blue. Lane 1, unfractionated
lysate of S. pyogenes 6414; lane 2, unbound material
(Ia); lane 3, material washed out by 0.5 M NaCl
(IIa); lane 4, material eluting with 2 M guanidinium
chloride (IIIa); lane 5, pool Ib, and lane 6, pool
IIb, from Sephacryl S-200 HR. Arrows and numbers on
the left indicate the migration distances and molecular masses
of standard proteins. B and C, immunoblot detection
of collagen-binding proteins with anti-57-kDa protein antibodies (B) and with a nonimmune IgG (C). Bound IgG was
detected with peroxidase-conjugated goat anti-mouse-immunoglobulin. D, materials from the collagen receptor purification steps
after separation in the polyacrylamide gel were electroblotted onto
nitrocellulose membranes and probed with
To isolate these proteins
the whole lysate obtained by sonication of bacterial cells was loaded
onto a collagen-Sepharose affinity matrix equilibrated with PBS. The
column was washed with 10 mM phosphate buffer containing 0.5 M NaCl and proteins adsorbed to the affinity matrix were
eluted with 2 M guanidinium chloride in PBS, dialyzed against
water, and lyophilized (Fig. 3A).
Figure 3:
Purification of collagen- binding protein
from S. pyogenes 6414. A, affinity chromatography on
collagen-Sepharose. A lysate of S. pyogenes 6414 was passed
over a type II collagen-Sepharose 4B column (2.8
Analysis by
polyacrylamide gel electrophoresis showed that the material bound to
collagen-Sepharose and subsequently eluted with guanidinium chloride
contained a predominant component of molecular mass 57-kDa, a minor
protein of 130-kDa and a mixture of low molecular mass peptides (Fig. 4A, lane 4). Further purification of the
major protein was achieved by gel filtration chromatography on a column
of Sephacryl S-200 HR, equilibrated with 2 M guanidinium
chloride supplemented with 0.1% n-octyl- When a streptococcal lysate was passed over a gelatin-Sepharose
column and the column subsequently eluted with guanidinium chloride
none of the above polypeptides were seen in the eluate. Moreover, when
the flow-through from the gelatin column was then applied to a
collagen-Sepharose column, a 57-kDa protein bound to the column and was
eluted by guanidinium chloride demonstrating a preference of the 57-kDa
protein for native type II collagen-Sepharose compared to the gelatin
matrix.
The presence of
collagen-binding components of similar molecular weight was
demonstrated in experiments in which samples from different steps of
purification were separated by electrophoresis, electroblotted onto a
nitrocellulose membrane and then probed with To test if the 57-kDa
purified protein represents a soluble form of the collagen receptor,
the protein was analyzed for its ability to competitively inhibit
ligand binding to intact bacteria. Binding assays in which increasing
amounts (2.5-35 µg) of isolated 57-kDa protein were added to
incubation mixtures containing S. pyogenes 6414 cells and
Figure 5:
Cellular location of 57-kDa protein. A, a lysate obtained from
Figure 6:
Competition of
Figure 7:
Scatchard plot analysis of collagen
binding to solid-phase adsorbed collagen receptor. Microtiter wells,
coated with 2 µg of 57-kDa protein, were incubated with increasing
concentrations of
Figure 8:
Immunoblot of collagen-binding proteins
from S. pyogenes and S. zooepidemicus.
Collagen-binding proteins isolated by affinity chromatography of
lysates of S. pyogenes 6414 (lane 1) and S.
zooepidemicus S
Figure 9:
Kinetics of attachment of S. pyogenes strain 6414 to collagen-coated microtiter wells. Collagen-coated
surfaces (5 µg/well) were overlaid with 3.3
Figure 10:
Attachment of S. pyogenes 6414
to wells coated with different collagen types. Bacteria (6.6
The proteins tested as competitors of
radiolabeled collagen binding to streptococci were also used as
potential inhibitors of streptococcal adherence to collagen coated
substrate. Among the proteins analyzed only collagen type II and the
solubilized 57-kDa protein showed inhibitory activity (Fig. 11).
Therefore, these data suggest that collagen may serve as a substrate of
streptococcal adherence and the 57-kDa protein can act as a collagen
adhesin.
Figure 11:
Influence of various proteins on the
adherence of S. pyogenes strain 6414 to collagen coated wells.
Bacteria (6.6
We have demonstrated that the binding of collagen type II to
cells of S. pyogenes 6414 exhibits the properties of a typical
receptor-ligand interaction. Binding of collagen was specific and
saturable, and cells bound exogenous collagen with high affinity.
Incubation of bacteria with proteolytic enzymes resulted in a rapid
loss of collagen binding suggesting the protein nature and surface
location of the receptor. On the basis of these preliminary results
we then isolated a 57-kDa collagen-binding protein from a streptococcal
lysate by affinity chromatography on collagen-Sepharose followed by gel
filtration chromatography. The collagen-binding protein behaved as one
would expect of a collagen receptor. Binding of collagen type II to S. pyogenes was inhibited in a concentration-dependent manner
by the purified 57-kDa protein. Furthermore, the isolated receptor
protein bound collagen with the same high affinity as intact cells, and
this binding was inhibited by unlabeled collagen but not by unrelated
proteins. Conflicting results were found concerning reversibility of
collagen binding to intact cells and isolated receptor molecule
adsorbed onto microtiter wells. This could be explained if we assume
that the conformation of the solubilized receptor differs from that of
receptor on the surface of bacteria. The 57-kDa protein could be
labeled by external At the present
time we do not know what relationship, if any, the 57-kDa protein may
have with the other collagen-binding components (molecular mass of
130-150 kDa) identified in the lysate of S. pyogenes.
Further studies are needed to establish the relationship between the
two proteins. Recent studies have shown the presence of
collagen-binding receptors on other species of bacteria. The Dr
fimbriae found on uropathogenic Escherichia coli strains with
the serotype 075 have been found to bind type IV collagen(30) ,
while the enteropathogenic Yersinia enterocolitica and Yersinia pseudotubercolosis adhere to various collagen
molecules through the YadA protein(31) . A collagen-binding
component with a relative molecular mass of 135,000 Da has been
isolated from S. aureus(16) . The streptococcal
collagen-binding protein we have isolated has structural and functional
properties distinct from the staphylococcal receptor. The difference in
size of the two receptors suggests structural uniqueness. Furthermore,
an antibody raised against the staphylococcal collagen receptor and
shown to effectively inhibit the binding of It still remains to be seen whether this in vitro interaction promoted by the 57-kDa protein facilitates the
establishment of streptococcal infections and thus contributes to the
pathogenicity of this microbe.
Volume 270,
Number 1,
Issue of January 6, 1995 pp. 347-353
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-labeled collagen, a prominent collagen-binding protein
of 57 kDa was identified as well as minor 130-150-kDa components.
The major 57-kDa protein was isolated by affinity chromatography on
collagen-Sepharose followed by gel filtration chromatography. The
57-kDa protein purified from S. pyogenes was used to raise a
monospecific antibody which also reacted with a collagen-binding
protein of similar molecular size isolated from Streptococcus
zooepidemicus. The two collagen-binding proteins from streptococci
have a similar amino acid composition and isoelectric points. Isolated
collagen-binding protein was specifically recognized by
I-collagen in a solid-phase binding assay and displayed
an affinity for the ligand quite similar to that exhibited by intact
bacteria (K
= 3.1 versus 3.5
10M, respectively).
Surface-labeled bacteria attached to microtiter wells coated with
different collagen types and the 57-kDa protein blocked the adhesion to
collagen substrate. We propose that the 57-kDa protein is an adhesin
involved in the attachment of streptococci to host tissues.
)and
strains of Staphylococcus aureus, particularly those isolated
from patients with septic arthritis or osteomyelitis, have been
reported to express collagen receptors(14) . The binding of
collagen to staphylococci has been characterized in some
detail(15) , and the isolation(16) , cloning, and
sequencing of a collagen receptor from S. aureus has been
performed(17) . Furthermore, evidence that the staphylococcal
collagen receptor functions as a colonization factor of cartilage and
as a potential virulence determinant in septic arthritis has been
reported(18) .
Chemicals
Type II collagen was purified from
bovine nasal septum as described by Strawich and Nimmi(22) .
Collagen was denatured by heating an aliquot of the collagen stock
solution at 60 °C for 30 min immediately before using. Isolation of
native collagen types I-IV was as described
previously(15) . Fibronectin was purified from human
plasma(23) .I (specific activity, 15 mCi/µg) was purchased from
Radiochemical Centre, Amersham, UK.
Bacterial Cultures
The strain used for this study, S. pyogenes 6414, was obtained from Dr. M. Hook (Institute of
Extracellular Matrix Biology, Texas A& University, Houston, TX). Streptococcus zooepidemicus strain S
III and S. pyogenes strain Sp 1-4065 were provided by Dr. M. Lindberg
(Swedish University of Agricultural Science, Uppsala, Sweden). The
strains were grown in Todd-Hewitt Broth at 37 °C for 16 h.
)washed, and adjusted to a cell density of 10 cells/ml using a standard curve relating the A
to the cell number determined by counting cells in a
Petroff-Hausser chamber. The cells were then heat-killed at 88 °C
for 10 min and then stored at -20 °C until used.
Radiolabeling of Collagen and Bacteria
Collagen
was labeled by the IODO-GEN method following the procedure recommended
by the manufacturer (Pierce). The specific activity of the
radioactively labeled ligand was estimated to be 3 10
cpm/µg. Radiolabeling of bacteria was performed as
reported(24) . The specific activity of the bacterial
suspension was 10
cpm/3.7 10 cells of S. pyogenes 6414.Binding of
The binding of I-Collagen to
Bacteria
I-labeled collagen to
streptococci was quantitated as described previously(11) .
Briefly, 5
10
bacteria were incubated with 5
10
cpm of I-labeled collagen in 0.5
ml of PBS containing 0.1% bovine serum albumin and 0.1% Tween 80 to
block aspecific binding to cells and tubes. The mixture was rotated in
an end-over-end mixer for 1 h at 22 °C. The reaction was stopped by
the addition of 3 ml of ice-cold PBS containing 0.1% Tween 80, and the
tubes were centrifuged at 1400
g for 15 min. After
aspiration of the supernatant, the remaining pellet was analyzed for
radioactivity in a -counter. Radioactivity recovered in the tubes
incubated in the absence of bacteria (background) was subtracted from
that of the samples containing bacteria. The background radioactivity
(600-800 cpm) was similar to that obtained in controls where
I-labeled collagen was incubated with bacteria in the
presence of 100 µg of unlabeled collagen. Samples and controls were
run in duplicate.
Solubilization of Collagen-binding Proteins
Cells
of S. pyogenes 6414 were suspended in 50 mM Tris-HCl,
pH 7.6, containing 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mMN-ethylmaleimide, to a density of 5 10 bacteria/ml. The suspension was sonicated (4
1.2 min),
and bacterial debris were removed by centrifugation (30 min
10,000 g). The supernatant was further clarified by
centrifugation for 1 h at 90,600 g and then stored at
-20 °C until used.Incubation of Bacteria with Trypsin
Streptococci
(10 cells/ml) were suspended in phosphate buffer and
digested with trypsin (1 µg/ml) at 37 °C for 30 min. The
reaction was stopped by heating for 3 min at 100 °C. Cells were
pelleted by centrifugation, washed, suspended in NaCl/P
,
and finally assayed for collagen binding. Trypsin-treated cells showed
a 75% reduction in collagen binding ability relative to control cells.
The supernatant from the trypsin-treated cells (denoted trypsinate) was
employed for immunoblotting assays.Electrophoresis and Blotting
Electrophoresis in
10% SDS-polyacrylamide gel electrophoresis (PAGE) was carried out
according to Blobel and Dobberstein(25) . Radioactive
components were visualized by autoradiography of dried gels with X-Omat
A-R film (Eastman Kodak Co.). Blotting was performed essentially using
the procedure of Towbin et al.(26) . Proteins
separated by electrophoresis were electrotransferred for 2 h at 200 mA
onto nitrocellulose membranes (Bio-Rad). 10 cpm of I-labeled collagen type II containing 0.1%
albumin and 0.1% Tween 80. The membranes were washed extensively with
0.1% Tween 80 in PBS, dried, and exposed at -70 °C using a
X-Omat A-R film.
Amino Acid Analysis
The material isolated from
collagen-Sepharose was subjected to SDS-PAGE and electrophoretically
transferred to Immobilon-P membrane (Millipore, Bedford, MA) according
to the procedure described by Matsudaira (27) and stained with
Coomassie Brilliant Blue. The band corresponding to the 57-kDa protein
was excised and subjected to gas-phase hydrolysis in 6 N HCl
containing 1% (v/v) phenol at 105 °C for 24 h. Amino acid analysis
was carried out after precolumn derivatization procedure as reported by
Cohen and Michaud(28) . using a Jasco (Japan Spectrometry)
amino acid analyzer equipped with 980-PU pump and a 820-FP detector.Amino-terminal Sequence
The 57-kDa protein
electrophoretically adsorbed on Immobilon-P was excised and covalently
attached to the membrane by using the SequeNet kit from Millipore and
sequenced by a Millipore model 6625 protein sequencer.Solid-phase Binding Assay
Adhesion of streptococci
to surface coated collagen and binding of collagen to
surface-immobilized 57-kDa protein were performed in microtiter plates
(P.E.G.T. assay strips, Costar, Europe Ldt., Badhoevedorp, The
Netherlands). In binding experiments wells were coated with 20 µl
of protein solution (100 µg/ml), incubated overnight at 4 °C,
and then subjected to blocking with 200 µl of 1% bovine serum
albumin for 1 h at 37 °C. After washing with PBS containing 0.1%
Tween 20 the wells were incubated with the indicated amounts of
radioactively labeled collagen for 2 h at 37 °C. After extensive
washing the amount of bound I-labeled collagen was
quantitated in a
-counter.
I-labeled bacteria (3.3
10
cells/ml),
incubated at 37 °C for 2 h, washed extensively with PBS containing
0.1% Tween 20, and counted. All of the samples were corrected for
background values corresponding to the radioactivity recovered in wells
coated with albumin alone.Preparation of Antibodies to 57-kDa Protein
BALB/c
mice (6-week-old females) were immunized by intraperitoneal injection
with 15 µg of isolated 57-kDa protein emulsified in Freund's
complete adjuvant. Three booster injections of the same material in
incomplete adjuvant were administrated at 2-week intervals. Ten days
following completion of the fourth cycle of injections, the mice were
sacrificed, and the blood was collected. The blood was allowed to clot
and spun at 2000 g for 10 min. The IgG fraction of the
serum was purified on a column of protein A-Sepharose (Pharmacia,
Uppsala-Sweden), and adsorbed IgG antibodies were eluted with 3 M MgCl, dialyzed against PBS, and stored frozen in small
aliquots.
Characterization of Collagen Binding to S. pyogenes
Strain 6414
To study the streptococcal collagen receptor we
selected a strain, S. pyogenes 6414, which in preliminary
studies was shown to bind a high amount of collagen. This strain grown
in brain-heart infusion or Todd-Hewitt broth more effectively bound I-collagen than when grown in Luria broth.
I-labeled collagen was very rapid and essentially
completed before 10 min of incubation. Prolonged incubation did not
result in additional binding of
I-labeled collagen.
I-labeled
type II collagen in the presence of excess amounts of a variety of
unlabeled proteins. Addition of unlabeled type II collagen to the
incubation mixture effectively blocked the binding of labeled ligand.
Other proteins tested, including
1-acid glycoprotein, fetuin,
ovalbumin, fibronectin, protein G, protein A, and rabbit IgG antibody
to the collagen receptor on S. aureus, did not affect ligand
binding.I-collagen binding to bacteria we found that the native
collagen was a more potent inhibitor compared to the denatured forms (Fig. 1) indicating that native collagen displayed a higher
affinity than denatured collagen for the streptococcal collagen binding
component. The binding of
I-labeled collagen to S.
pyogenes was essentially irreversible, i.e. iodinated
collagen which bound to bacteria during a preliminary incubation period
of 1 h was only marginally displaced from the cells on addition of 100
µg of unlabeled collagen to the incubation mixture.
I-collagen binding to S. pyogenes 6414.
Bacteria were incubated for 1 h with
I-labeled collagen
in the presence of increasing concentrations of native (
),
denatured collagen type II (), and gelatin (
), and the
amount of bound radiolabeled collagen was determined. Inhibition is
expressed as percentage of I-labeled collagen bound to
bacteria in the absence of any potential
inhibitor.
I-labeled collagen showed that the cells could be
saturated with labeled ligand (Fig. 2). The amount of ligand
bound to the bacteria increased to a maximum of 570 ng of collagen per
1
10 cells of S. pyogenes 6414. If we
assume collagen has a molecular weight of 2.85 10 and that collagen is bound only to specific receptors, we can
calculate an average number of 11,600 binding sites per cell. Since
Scatchard plot analysis requires the binding reaction to be reversible,
the necessary requirements to calculate the K
value are not fulfilled. However, an apparent dissociation
constant of 3.5 10M can be
estimated from the concentration required for half-maximal binding of
the ligand.
I-labeled collagen to S. pyogenes 6414.
Heat-killed bacteria (1
10) were incubated with
increasing amounts of I-labeled collagen (specific
activity, 14,000 cpm/µg) for 1 h. Background values were determined
for each concentration of added collagen and subtracted from the
incubation mixtures containing bacteria.
III, binding of radiolabeled
collagen was highly specific, saturable, and with a calculated apparent K value of 5 10M, which is of the same order of that found in S.
pyogenes 6414.
Solubilization and Purification of Collagen-binding
Proteins from S. pyogenes 6414
Mild digestion of streptococcal
cells with trypsin markedly reduced binding of I-labeled
collagen, suggesting that the bacterial collagen receptor contained a
protein component. To assess whether the interaction of collagen with
bacteria is mediated by specific protein components, a crude lysate
obtained by sonication of bacteria was fractionated by SDS-PAGE and
subjected to Western blot analysis with
I-collagen.
Western ligand blotting showed that the major collagen-binding proteins
in the lysate have molecular masses of 57 and 130-150 kDa (see Fig. 4D, lane 1).
I-labeled
collagen. Lanes in B, C, and D are numbered
as reported in A. Lane 7 in B is a lysate from the
collagen receptor-negative strain Sp
1-4065.
8 cm)
equilibrated in 20 mM phosphate buffer, pH 7.4. After washing
the column with 0.5 M NaCl in phosphate buffer, bound proteins
were eluted with 2 M guanidinium chloride. Fractions were
pooled as indicated by the bars and dialyzed against water. B, gel filtration chromatography on Sephacryl S-200 HR.
Freeze-dried material (pool IIIa) from the affinity chromatography step
was dissolved in a small volume of phosphate buffer containing 2 M guanidinium chloride and 0.1% n-octyl-
-D-glucopyranoside and eluted at a rate
of 22 ml/h through a column of Sephacryl S-200 HR (0.8 117 cm)
equilibrated in the same buffer. Fractions were pooled as indicated by
the bars.
-D-glucopyranoside (Fig. 3B). This chromatography step resulted in the
separation of the major 57-kDa component from the low molecular mass
proteins (Fig. 4A, lanes 5 and 6). Preliminary Characterization of the 57-kDa
Protein
An antiserum generated by immunizing mice with purified
57-kDa protein was found to react with a single band both in the whole
lysate and in the fractions purified by affinity chromatography and gel
filtration chromatography steps, with mobility identical to the antigen (Fig. 4B). An additional faint band corresponding to
the 130-kDa polypeptide was detected in the material retained by the
collagen-Sepharose matrix, suggesting that this component is
immunologically related to the 57-kDa protein. The 57-kDa protein was
not present in lysate generated from the collagen receptor-negative
strain Sp 1-4065 of S. pyogenes when probed in Western blot
analysis with immune IgG (Fig. 4B, lane 7).
Furthermore, nonimmune IgG did not give a detectable signal with the
material blotted as reported in Panel B (Fig. 4C). When the immune IgG were included in a
cell/collagen binding assay they did not inhibit the binding of I-ligand to intact bacteria.
I-labeled
collagen (Fig. 4D). In the lysate, as well as in the
materials adsorbed on collagen-Sepharose and further purified by gel
filtration chromatography most of the radiolabeled collagen that bound
nitrocellulose was associated with a 57-kDa component. Other components
in the molecular range of 130-150 kDa were labeled with the
ligand. The M
130,000 protein may correspond to
the band detected after Coomassie Blue staining of the gel as shown in Fig. 4A, lane 4.I-labeled collagen, were performed. Binding was inhibited
by the purified 57-kDa protein in a dose-dependent manner suggesting
that this component competed with the cell bound collagen receptor for
available sites in the ligand molecule. These data indicate that the
57-kDa protein is a soluble form of a collagen receptor. Amino acid
composition analysis (Table 1) of the purified protein revealed a
high molar percentage of leucine (14.5%) and Glu/Gln (combined: 11.8%).
The NH
-terminal amino acid sequence of the protein was
determined (Table 2) and found to represent a unique sequence
when compared to amino acid data sequences stored in the Swiss-Prot
data bank. Electrophoretic analysis (Fig. 4A) and
staining with antibodies (Fig. 4B) indicated that the
isolated collagen-binding protein may be composed of two closely spaced
bands with molecular masses of 57- and 53-kDa. To investigate the
relationship of these two components, the bands were excised from the
nitrocellulose membrane and separately sequenced.The identity of the
NH-terminal sequences suggests that the smaller component
was derived from the 57-kDa protein due to endogenous proteolysis
during extraction and/or purification.
Cellular Location of the 57-kDa Protein
To
determine whether the isolated 57-kDa protein is located on the surface
of S. pyogenes an experiment was performed in which bacteria
were I-labeled externally, sonicated, and the
corresponding lysate loaded on a column of collagen-Sepharose.The
column was washed with phosphate buffer, followed by 0.5 M NaCl, and the material bound to the affinity matrix was eluted
with 2% SDS. An autoradiogram of electrophoresed material bound and
eluted from the column showed the presence of a 57-kDa protein as a
major component along with several labeled smaller proteins (Fig. 5A). More conclusive evidence that the 57-kDa
protein is present at the surface of bacteria was obtained by probing
material released by trypsin from cells of S. pyogenes 6414
with an anti-receptor antibody. A mixture of peptides ranging from 43
to 35 kDa was detected, whereas the trypsinate from S. pyogenes Sp 1-4065 did not reveal any detectable amount of anti-receptor
antibody reactive protein. Furthermore, a preimmune antibody did not
react with trypsin released proteins from S. pyogenes 6414 (Fig. 5B).
I surface-labeled
cells of S. pyogenes 6414 was loaded on a column of
collagen-Sepharose. The column was washed with NaCl/P
followed by 0.5 M NaCl. Material bound to the affinity
matrix was eluted with 2% SDS, analyzed by SDS-PAGE and visualized by
autoradiography. B, trypsinates of strain 6414 (lane
1) and strain Sp 1-4065 (lane 2) after electrophoretic
separation in non-reducing conditions were transferred to
nitrocellulose membranes and probed with an anti-57-kDa protein
antiserum. Immunostaining with the second antibody was performed as
reported under ``Materials and Methods.'' In lane 3 the reaction of trypsinate from strain 6414 with nonimmune mouse
IgG is reported as a control.
Complex Formation of Collagen with Solid-phase Adsorbed
57-kDa Protein
Further evidence of the collagen binding activity
of the 57-kDa protein was obtained in assays where the purified protein
coated on microtiter wells was shown to bind I-labeled
collagen. Different unlabeled proteins were then tried as potential
inhibitors. As expected, binding of
I-labeled collagen to
the immobilized 57-kDa protein was strongly inhibited by unlabeled
collagen type II, whereas other proteins tested, e.g. fibronectin, fetuin, fibrinogen, bovine IgG, ovalbumin, and
albumin did not affect binding (Fig. 6) demonstrating the
specificity of the collagen-receptor interaction. A major portion (up
to 70%) of
I-labeled collagen bound to immobilized
receptor could be displaced by subsequent addition of a large excess of
unlabeled ligand, demonstrating a reversibility of ligand-receptor
interaction in this system. Under the equilibrium conditions of the
microtiter plate assay Scatchard analysis of collagen binding to
solid-phase absorbed 57-kDa protein fitted a straight line suggesting
the presence of one class of binding sites for collagen
ligand(29) . The calculated value of the dissociation constant
of the complex was 3.1
10M (Fig. 7).
I-collagen
binding to solid-phase adsorbed collagen receptor by non-collagen
proteins. Microtiter wells coated with 2 µg of 57-kDa protein were
incubated with 10
cpm of I-labeled collagen
in the presence of 2 µg of the proteins indicated. After 2 h, wells
were washed and the ligand binding was quantitated as described under
``Materials and Methods.'' Data are expressed as the
percentage of collagen binding relative to controls incubated in the
absence of potential inhibitors. Vertical bars show the S.D.
of triplicate samples.
I-labeled collagen as indicated. The
specific binding of ligand was plotted according to the method of
Scatchard (correlation coefficient = 0.97). The binding data for
kinetic analysis were calculated by a nonweighted linear regression
computer program. The inset shows the saturation binding
isotherm of collagen to the receptor protein. B/F, bound over
free collagen. Each data point is the average of three
samples.
Comparison of Collagen-binding Proteins from S. pyogenes
and S. zooepidemicus
We also isolated a collagen-binding protein
from S. zooepidemicus S III, a member of group C
streptococci, associated with infections in animals. The protein
migrated on SDS-PAGE with an apparent M of 57,000,
similar in size to the collagen-binding protein purified from S.
pyogenes (data not shown). The two proteins were found to be
homologous by at least two criteria. First, amino acid analysis
indicates that both the proteins have a similar amino acid composition (Table 1) and are acidic as indicated by isoelectric points (pI
5.0). Second, the proteins were shown to be immunologically related
since antisera raised against the collagen-binding protein from S.
pyogenes cross-reacted with the collagen-binding protein from S. zooepidemicus (Fig. 8), and antisera raised against
the collagen-binding protein from S. zooepidemicus recognized
the collagen receptor from S. pyogenes (data not shown).
III (lane 2) were subjected
to SDS-PAGE under non-reducing conditions, transferred to
nitrocellulose, and incubated with anti-57-kDa protein antibody. Bound
antibody was detected as reported.
Collagens as Substrates of Bacterial Adhesion
The
ability of S. pyogenes to bind collagen type II with high
affinity prompted us to analyze the role of this interaction in
bacteria attachment to collagen-containing substrates. Radiolabeled
bacterial cells were incubated in microtiter wells coated with type II
collagen or bovine serum albumin. Adhesion of streptococci to type II
collagen containing substrata was time-dependent and reached a maximum
after 2 h of incubation, whereas negligible amount of bacteria had
attached to bovine serum albumin after 3 h of incubation (Fig. 9). Moreover, I-labeled streptococci adhered
efficiently to surfaces coated with all collagens tested (types I, II,
III, and IV) but not to ovalbumin (Fig. 10). This broad
specificity indicates that the streptococcal receptor recognizes a
common structure in the different collagen molecules. On the other
hand, streptococci showed a marginal attachment to either denatured
collagen or isolated
-chains, suggesting the importance of the
triple-helical structure in the collagen substrates for adherence of
streptococcal cells.
10 cells of a suspension of I-labeled bacteria and
incubated for the indicated periods of time. After extensive washing
with phosphate-buffered saline containing 0.1% Tween 80, adherence of
bacteria was determined by counting the wells in a
-counter.
Symbol
shows bacterial adherence to 5 µg/well of bovine
albumin.
10 cells)were incubated for 2 h at 37 °C in wells
coated with 5 µg of native collagen, denatured collagen type II,
gelatin, and isolated chains of collagen type I. After extensive
washing the number of attached cells was quantitated. The values are
averages of incubations performed in
triplicate.
10 cells) were surface labeled with I and incubated for 2 h at 37 °C with collagen-coated
microtiter wells in the presence of 2 µg of the proteins indicated
at the left. Data are averages of incubations in triplicate.
The amount of attached cells is given as a percent of the number of
cells that attached to wells in the absence of
proteins.
I-iodination of bacteria, and a
streptococcal trypsinate was positively detected by an anti-57-kDa
protein antiserum suggesting a surface location of the isolated
collagen receptor. Taken together these results strongly suggest that
the isolated 57-kDa protein is a cell surface receptor and responsible
for the binding of collagen to streptococcal cells.
I-labeled
collagen to S. aureus cells did not interfere with the binding
of collagen to streptococcal cells. The staphylococcal collagen
receptor has been suggested as a cartilage colonization factor, because
it is needed not only for the adhesion of the bacteria to
collagen-containing substrates, but also to cartilage, a tissue rich in
collagen(18) . The preliminary results reported in this study
indicate that the 57-kDa collagen-binding protein may behave as an
adhesin. In fact, incubation of bacteria with the collagen-coated
microtiter wells in the presence of the soluble receptor protein
resulted in a specific, significant inhibition of bacterial attachment.
))
We are indebted to Dr. Magnus Hook who provided
encouragement and critical reading of the manuscript and Dr. Fabrizio
Ceciliani for amino acid analysis. We also thank Anna Vai for typing
the manuscript, Dr. Giampaolo Minetti for helping with elaboration of
graph and numerical data, and Dr. Katharine Dyne for revising the text.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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