(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 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
10
M, 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.
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 ()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) .
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 I (specific activity, 15 mCi/µg) was purchased from
Radiochemical Centre, Amersham, UK.
After
harvesting, bacteria were suspended in 0.13 M sodium chloride,
0.02% sodium azide, and 10 mM sodium phosphate, pH 7.4 (PBS), ()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.
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 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.
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 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.
The binding
of 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.
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 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.
When increasing concentrations of native and denatured type
II collagen and gelatin were tried as inhibitors of 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.
Figure 1:
Effect of native and denatured collagen
on 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.
Incubation
of streptococci with increasing concentrations of 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
10
M can be
estimated from the concentration required for half-maximal binding of
the ligand.
Figure 2:
Saturability of binding of 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.
In a similar study using cells of S.
zooepidemicus, strain S III, binding of radiolabeled
collagen was highly specific, saturable, and with a calculated apparent K
value of 5
10
M, which is of the same order of that found in S.
pyogenes 6414.
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 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.
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 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.
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--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).
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 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.
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 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.
Figure 5:
Cellular location of 57-kDa protein. A, a lysate obtained from 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.
Figure 6:
Competition of 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.
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 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.
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 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.
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 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.
Figure 10:
Attachment of S. pyogenes 6414
to wells coated with different collagen types. Bacteria (6.6
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.
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 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.
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 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.
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 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.
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.