From the
We have identified a discrete collagen-binding site within the
Staphylococcus aureus collagen adhesin that is located in a
region between amino acids Asp
Staphylococcus aureus cells can colonize many different
host tissues and cause various types of infections such as
endocarditis, pneumonia, wound infections, osteomyelitis, and septic
arthritis. Adherence of staphylococci to host tissues involves a family
of adhesins that recognize extracellular matrix components and which we
have named MSCRAMMS (Microbial Surface Components Recognizing Adhesive
Matrix Molecules)
(1) . The expression of specific MSCRAMMs
appears to be needed for the colonization of different types of
tissues. For example, staphylococcal strains recovered from the joints
of patients diagnosed with septic arthritis or osteomyelitis almost
invariably express a collagen-binding MSCRAMM, whereas significantly
fewer isolates obtained from wound infections express this
adhesin
(2) . Similarly, S. aureus strains isolated from
the bones of patients with osteomyelitis often have an MSCRAMM
recognizing the bone-specific protein, bone sialoprotein
(BSP)
(3) .
We have previously reported the cloning,
sequencing, and expression of a gene cna, encoding a S.
aureus collagen-binding MSCRAMM
(4) . The cna gene
encodes an 133-kDa adhesin that contains structural features
characteristic of surface proteins isolated from Gram-positive
bacteria. We have demonstrated that the collagen-binding MSCRAMM is
required and sufficient for the adherence of S. aureus to
collagen-coated artificial substrates as well as to cartilage, a tissue
rich in type II collagen
(2) . All strains expressing the
collagen-binding MSCRAMM were able to adhere to cartilage, whereas
those strains lacking the MSCRAMMs did not adhere. Preincubation of
S. aureus with polyclonal antibodies raised against the
purified adhesin or saturation of the cartilage substrata with soluble
recombinant collagen-binding MSCRAMM resulted in a complete inhibition
of bacterial attachment
(2) .
S. aureus colonization
of the articular cartilage within the joint space appears to be an
important factor contributing to the development of septic arthritis.
The importance of the collagen-binding MSCRAMM in the pathogenesis of
septic arthritis was examined by comparing the virulence of two sets of
S. aureus isogenic mutants in an animal
model
(5) . Greater than 70% of mice injected with CNA
Recently, we have localized the
ligand-binding site within the N-terminal half of the collagen-binding
MSCRAMM
(6) . By analyzing the collagen binding activity of
recombinant proteins corresponding to different segments of the
MSCRAMM, we identified a 168-amino-acid long protein fragment
(corresponding to amino acid residues 151-318) that had
appreciable collagen binding activity. Short truncations of this
protein in the N- or C terminus resulted in a loss of ligand binding
activity but also resulted in conformational changes in the protein as
indicated by circular dichroism spectroscopy. These results raised the
possibility that the ligand-binding site of the MSCRAMM is contained
within a short segment of amino acids and that flanking sequences are
required for the proper folding of these residues in the ligand-binding
site.
In the current study we have sought to further define the
ligand-binding site in the S. aureus collagen-binding MSCRAMM
and have identified a 25-amino-acid peptide that directly inhibits the
binding of S. aureus to
After the completion of synthesis, peptide resins containing the
coupled peptides were washed thoroughly with dimethylformamide,
ethanol, and ether, then dried in a vacuum desiccator. Trifluoroacetic
acid containing anisole, thioanisole, and water (2.5% each) was used to
cleave the peptides from the resin as well as to assist in the removal
of side chain protecting groups. The resins were filtered and the
peptides precipitated with cold anhydrous ether. The precipitate was
washed an additional three times with anhydrous ether and dried.
Peptides were analyzed by reverse-phase HPLC on a Waters 625 Liquid
Chromatography system using a C
Introduction of potential inhibitors involved different
preincubation steps including: (A) incubation of bacterial
cells for 30 min at 25 °C with IgG purified from immune and
preimmune sera from rabbits immunized with CBD(151-297) to assess
the inhibitory activity of the antibody. (B) A two-step
preincubation procedure to determine the ability of synthetic peptides
to neutralize the inhibitory activity of the antibody. First, 100
µg of each peptide was incubated with
On-line formulae not verified for accuracy
where j represents the jthclass of
binding sites and K
Rabbits were immunized
with CBD(151-297) as described under ``Experimental
Procedures.'' Sera were also collected prior to immunization and
tested for reactivity to CBD(151-297). The reactivity of the
antisera with different segments of CBD(151-297) was tested in an
ELISA using a series of eight 25-amino-acid long synthetic peptides
() with partially overlapping sequences as targets. As
shown in Fig. 1, purified IgG reacted strongly with peptides 2,
3, 5, 6, and 7 and weakly with peptides 1, 4, and 8. When preimmune IgG
was tested with the CBD peptides, little reactivity could be detected.
The different peptides have not been compared with respect to their
quantitative binding to the microtiter plate, and therefore the data
presented in Fig. 1should not be used for a quantitative
comparison of their immunological properties. However, the relative
immunological reactivity of the different peptides correlated closely
with their antigenic index using the algorithm of Jameson and
Wolf
(16) .
The BIAcore sensorgrams
for the binding of the different mutants (20 µM) to
immobilized collagen type II are shown in Fig. 7. Under the
conditions described in this study CBD(30-529) exhibits complex
multiphasic interactions when analyzed using the BIAcore. True
equilibrium was not obtained during the injection time period due to
the slow dissociation rate (k
From the Biosensor
results it is apparent that residues Asn
The ability of
microorganisms to express an MSCRAMM that can specifically recognize
and bind to a collagenous substrata may represent a significant
virulence factor for the initiation of many different types of
infections. Eighty to 90% of nongonococcal bacterial arthritis is
monoarticular, and in most of these cases S. aureus is the predominant pathogen
(18) . Localization of S.
aureus to the joint and the subsequent colonization of the
collagenous tissues appear to be a critical initial step in the
development of septic arthritis. Scanning electron micrographs of
tissues removed from both animals and humans with septic arthritis
demonstrated the presence of S. aureus closely associated with
cartilage matrix surfaces, particularly collagen fibers (19). Previous
studies have shown that expression of the collagen-binding MSCRAMM is
both necessary and sufficient for S. aureus to adhere to
cartilage
(2) . Moreover, S. aureus strains lacking the
collagen-binding MSCRAMM were less virulent than their isogenic
parental strains in a mouse model of septic arthritis (5). Bacteria
other than S. aureus have also been shown to express
collagen-binding MSCRAMMs. Strains of enteropathogenic Yersinia were
shown to bind several collagen types with a high
affinity
(20, 21) . Collagen binding is mediated by YadA,
a plasmid-encoded cell surface-associated multifunctional protein that
was identified as a virulence factor
(21) . Yersinia spp. have also been implicated as initiators of reactive
arthritis
(22) . Interestingly, immunofluorescence techniques
have been used to demonstrate the presence of a number of Yersinia
enterocolitica 0:3 antigens in synovial fluid cells
(23) and tissues
(24) from patients with
Yersinia-triggered reactive arthritis. Although the presence
of the YadA protein in these samples has not been specifically
demonstrated, it is possible that its collagen binding ability may
allow localization to the joint thereby contributing to the pathology
of reactive arthritis. Many uropathogenic E. coli express
O75X, a frimbriae that is highly specific for the 7S fragment of
collagen type IV
(25) . Purified 075X fimbriae injected into rats
preferentially localized from the circulation to the glomerular
basement membrane. 075X fimbriae binding to collagen type IV within the
glomerular basement membrane is postulated to contribute to the
pathogenesis of chronic glomerulonephritis
(26) . Other bacteria
that are reported to express collagen-binding MSCRAMMs include
Klebsiella pneumonia(27) , Streptococcus mutans (28), group A, B, C, and G Streptococci
(29) ,
Lactobacilli (30), and Trypansoma cruzi(31) .
In addition to adhesins from prokaryotic organisms, a variety of
proteins expressed by eukaryotes have collagen binding activity. These
proteins include cell surface receptors such as the
We have previously
localized the collagen-binding domain from the 133-kDa S. aureus collagen-binding MSCRAMM to a 168-amino-acid long segment
(CBD151-318)
(6) . Data presented in this article
demonstrate that the collagen-binding domain is contained within amino
acid residues Asn
The underlined nucleotides indicate the location of the
mutation. Oligonucleotides were synthesized by the Gene Technologies
Laboratory, Institute of Developmental and Molecular Biology, Texas
A& University, College Station, Texas.
We thank A. Höök for technical assistance.
and Tyr
.
Polyclonal antibodies raised against a recombinant form of the collagen
adhesin inhibited the binding of collagen type II to S.
aureus. When overlapping synthetic peptides mimicking segments of
the adhesin fragment were tested for their ability to neutralize the
inhibitory activity of the antibody only one peptide, CBD4 was found to
be active. CBD4 bound directly to collagen and at high concentrations
inhibited the binding of collagen to S. aureus. A synthetic
peptide derivative of CBD4 lacking 2 carboxyl-terminal residues
(Asn
, Tyr
) had no inhibitory activity. The
importance of these residues for collagen binding was confirmed by
biospecific interaction analysis. Mutant adhesin proteins
N
A and Y
A exhibited dramatic
changes in collagen binding activity. The dominant dissociation rate
for the binding of mutant adhesin protein N
A to
immobilized collagen II decreased almost 10-fold, while the
Y
A and the double mutant exhibited even more
significant decreases in affinity and apparent binding ratio when
compared to the wild type protein.
strains (i.e. a clinical isolate expressing the
collagen-binding MSCRAMM or a negative strain into which the cna gene had been introduced) developed clinical signs of arthritis,
whereas less than 27% of the animals showed symptoms of disease when
injected with CNA
strains (i.e. a strain
lacking the cna gene or a strain in which the cna gene had been inactivated through homologous recombination). Taken
together these results demonstrate that the collagen-binding MSCRAMM
plays an important role in the pathogenesis of septic arthritis induced
by S. aureus.
I-labeled collagen type
II. Furthermore, site-directed mutagenesis of the collagen-binding
MSCRAMM has revealed 2 specific residues critical for ligand binding
activity.
Bacteria and Growth Conditions
Escherichia
coli strain JM101 was used as the bacterial host for plasmid
cloning and protein expression. Luria Broth (Life Technologies, Inc.)
was used as the culture medium for E. coli JM101. S.
aureus strain Phillips, originally isolated from a patient
diagnosed with osteomyelitis, was used in the collagen binding studies.
S. aureus was cultured in brain heart infusion broth (Difco,
Detroit, MI) at 37 °C with constant rotation, harvested by
centrifugation, and resuspended in phosphate-buffered saline
(PBS,(
)
10 mM phosphate, 150 mM NaCl, 0.02% azide, pH 7.4). Bacteria were resuspended to a final
density of 1
10
cells/ml as determined by
comparing the OD
of the sample with a standard curve
relating OD
to cell number.
Collagen
Bovine collagen type II was a gift from
Dr. Richard Mayne, University of Alabama at Birmingham.
NaI, specific activity 15 mCi/µg, was obtained from
Amersham Corp. Collagen was labeled with
I by the
chloramine-T method (7) as described previously
(8) . Estimated
specific activity of type II collagen ranged from 2
10
to 5
10
counts/min/µg protein.
Peptides
Synthetic peptides () were
synthesized by a solid-phase method on a p-benzyloxybenzyl
alcohol resin using Fmoc chemistry and a model 350 Multiple Peptide
Synthesizer from Advanced ChemTech Inc. (Lousiville, KY). Fmoc-amino
acids were purchased from Advanced ChemTech Inc. All coupling reactions
were mediated through diisopropylcarbodiimide in presence of
1-hydroxy-benzotriazole as the coupling reagent. Deprotection of Fmoc
group was accomplished with a solution of 40% piperidine in
dimethylformamide. Side chains were protected with the following
groups: t-butyl (Asp/Glu and Ser/Thr/Tyr),
t-butyloxycarbony (Lys),
2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg) and trityl (Asn/Gln/His).
analytical column. The
purity of the peptides as assessed by HPLC was greater than 90%.
Peptides (5 mg/ml) were solubilized in dimethyl sulfoxide and diluted
into the appropriate assay buffer prior to their use in binding and
inhibition assays.
Antibodies
Polyclonal antibodies against the
purified recombinant collagen-binding MSCRAMM segment
CBD(151-297) were produced in rabbits (HTI Bio-Products, Ramona,
CA). The IgG fraction of serum (CBD(151-297) IgG) was
purified by affinity chromatography on a column of immobilized Protein
A/G (Pierce) according to the manufacturer's recommendations.
Construction of Expression Plasmids
A series of
expression plasmids were constructed using the vector pQE-30 (Qiagen
Inc., Chatsworth, CA). Recombinant proteins expressed from this vector
contain a N-terminal tail of 6 histidine residues. The derivation of
expression constructs pQE-504 (CBD(151-318)) and pQE-441
(CBD(151-297)) have been previously described
(6) . To
construct pQE-1500 (CBD(30-529)), a cna fragment was
amplified from S. aureus FDA 574 genomic DNA
(4, 6) by polymerase chain reaction (PCR) together with flanking
oligonucleotides, primer A and primer D (). PCR and the
subsequent cloning of the amplified gene product into the expression
plasmid were performed as described earlier
(4, 6) .
Site-directed Mutagenesis
Plasmid DNA purified
from E. coli containing pQE-1500 served as the template in the
mutagenesis studies. Residues Asn and Tyr
in CBD(30-529) were changed to alanine residues
individually or as a pair by overlap extension PCR
(9) using the
oligonucleotides listed in . In two separate reactions,
flanking PCR primer A (BamHI restriction site incorporated
into the oligonucleotide) and primer D (SalI restriction site
incorporated into the oligonucleotide) were used in conjunction with
appropriate internal primers B and C, respectively, to generate PCR
products AB and CD. The PCR products were purified by agarose gel
electrophoresis and the two overlapping PCR fragments AB and CD were
combined with flanking primers A and D and used in a second PCR
reaction. The amplified PCR product was agarose gel purified (EluQuik;
Schleicher & Schuell), digested with BamHI and
SalI, ligated into expression vector pQE-30, and transformed
into E. coli JM101 cells. Transformants were screened for the
proper plasmid construction by restriction digest analysis. The DNA
sequence in the region of the mutation was determined by the
dideoxy-chain termination method
(10) using
[
-
S]dATP (Amersham) and Sequenase 4.0
(United States Biochemical Corp., Cleveland, OH). Mutants possessing
the correct nucleotide changes were selected for further investigation.
Expression and Purification of Recombinant
Proteins
Recombinant collagen adhesin proteins,
CBD(151-297), CBD(151-318), CBD(30-529), and the
site-directed mutants N232Y, Y233A, and N232A:Y233A constructed from
CBD(30-529) were expressed in E. coli and purified to
homogeneity using immobilized metal chelate affinity
chromatography
(6) . Only freshly purified proteins were used for
the analysis when it was observed that the proteins exhibited altered
binding characteristics upon prolonged storage.
The binding
of I-Collagen Binding Assay
I-labeled collagen type II to S. aureus strain Phillips in the presence of different potential inhibitors
was quantified essentially as described previously
(8) . Briefly,
I-collagen was added to a tube containing 10
cells of S. aureus in a final volume of 0.5 ml of PBS
containing 0.1% BSA and 0.1% Tween 80, pH 7.4, and incubated at 25
°C, end-over-end for 1 h. After centrifugation at 1350
g for 15 min, the supernatants were removed by centrifugation
and radioactivity associated with the bacterial pellets was quantitated
in a gamma counter. Duplicate samples were used for each data point.
CBD(151-297) IgG
(12 µg) for 30 min, and then 10
S. aureus cells were added. The incubation was continued for an additional
30 min at which time the
I-labeled collagen was added.
(C) To test the ability of synthetic peptides to bind to
collagen and inhibit bacterial binding, the peptides were preincubated
with
I-labeled collagen for 30 min at 25 °C before
addition of 10
S. aureus cells. Because these
experiments were conducted over several months the amount of
I-labeled collagen bound by S. aureus and the
extent of inhibition by
CBD(151-297) IgG varied somewhat
from experiment to experiment. However, within a specific experiment
extremely small variations were observed.
ELISA
The reactivity of CBD(151-297)
IgG, purified from preimmune and immune rabbit sera, was tested against
the synthetic CBD peptides () by ELISA. Immulon-4 96-well
plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with 50
µl of each peptide (100 µg/ml in PBS) overnight at 4 °C.
The plates were washed three times with PBS, containing 0.05% Tween 20
(PBST). A 0.1% BSA solution was added to the wells to block any
remaining protein binding sites. After 1 h at room temperature, the
wells were washed again three times with PBST, and 50 µl of
CBD(151-297) IgG (80 µg/ml) was added to the wells and
allowed to bind for 90 min at room temperature. The wells were again
washed with PBST and alkaline phosphatase-conjugated goat anti-rabbit
IgG (50 µl) (Bio-Rad) diluted 1:3000 in PBST was incubated with the
peptide-coated wells for 90 min at room temperature. The samples were
washed, and 100 µl of the alkaline phosphate substrate
p-nitrophenylphosphate (Sigma) was added to the wells for 50
min at 37 °C. The A
was monitored in a
microplate reader (Molecular Devices Corp., Menlo Park, CA).
A
of the BSA-coated wells was used to assess
nonspecific binding and was subtracted from A
of
the peptide samples. Each peptide was tested in triplicate.
Circular Dichroism
Average secondary and tertiary
structures of the recombinant collagen-binding MSCRAMM proteins were
monitored by far-UV and near-UV circular dichroism (CD) on a Jasco J720
spectropolarimeter. The spectropolarimeter was calibrated with a 0.1%
(w/v) 10-camphorsulfonic acid-d solution. Four spectra,
representing CD data from 190 to 250 nm (far-UV CD) were recorded at 25
°C in a 0.2-mm path length quartz cell and averaged. Likewise, four
spectra representing CD data from 250 to 320 nm (near-UV CD) were
collected at 25 °C in a 1.0-cm path length quartz cell and
averaged. Protein concentrations of 12 µM in 10 mM Tris, pH 7.5, were used for near- and far-UV CD data acquisitions.
Molar ellipticity was expressed as degcm
/mol.
Biospecific Analyses
Surface plasmon resonance
measurements utilizing the BIAcore system (Biosensor AB, La Jolla, CA)
were used to determine the equilibrium constants and kinetic rates for
the interaction of CBD(30-529) and the mutant derivatives with
immobilized bovine type II collagen. Sensor chip CM5 and the Amine
Coupling Kit for the immobilization of bovine type II collagen were
also obtained from Biosensor AB. Briefly, the detector monitors mass
changes on a biospecific surface containing immobilized ligand
molecules (L) in real time using the optical phenomenon
surface plasmon resonance
(11) . The binding of analyte
(A) (present in solution) to immobilized ligand is monitored
versus time for a series of analyte concentrations. HBS (10
mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA,
0.05% P-20 surfactant) was the buffer used for the binding studies.
Concentration ranges for monitoring the binding of each analyte
depended on the measured affinities and are reported in I.
The association phase begins when the analyte is injected over the
immobilized ligand; flow rate 10 or 20 µl/min. The dissociation
phase begins when the injection of analyte ends and is characterized by
a decrease in the response values with time. The dissociation of
analyte from ligand was measured using the higher concentrations in the
indicated ranges in order to minimize rebinding. Dissociation rates
were measured using two different concentrations of analyte and five
different flow rates from 15 to 450 µl/min; this was done in order
to ensure that the complex, multiphasic rates reported were independent
of analyte concentration and flow rate. Regeneration of the
collagen-coated matrix was carried out by passing 100 mM HCl
over the sensor chip surface for 5.0 min, followed by HBS buffer flow.
Immobilization Procedures
Pepsin-treated bovine
type II collagen was covalently coupled to research grade sensor chip
CM5 via primary amine groups using the following protocol.
Carboxymethylated dextran was activated by derivatization with 50
mMN-hydroxysuccinimide mediated by treatment with
200 mMN-ethyl-N`-[(dimethylamino)propyl]-carbodiimide
for 4.0-5.0 min. Following activation, a 25-30 µg/ml
solution of collagen type II in 10 mM maleic acid, pH 6, was
passed over the surface for 4.0-5.0 min at 5 µl/min and
resulted in between 900-1500 response units (RU) of immobilized
collagen. Non-covalently associated collagen was washed from the matrix
by HBS buffer flow, and unreacted sites on the activated dextran
surface were blocked by treatment with 1 M ethanolamine
hydrochloride for 5 min.
Data Analysis
A plot of equilibrium response
versus analyte concentration ([A]) can be
analyzed via traditional Scatchard analysis
(v/[A] versus v)
(6, 12) where v represents the molar binding ratio (moles
A/moles L). The slope of this line gives the
association constant (K) with the x intercept
being the maximum apparent binding ratio (BR
). In our
case, these plots were often biphasic
(6) . Another method to
determine equilibrium constants from these biphasic plots is to
non-linearly curve fit the data to the expression
j
represents the
dissociation constant of the jthclass. Both
Scatchard analysis and the non-linear curve fit gave comparable
solutions when used to fit the equilibrium data. Dissociation rate
constants were determined from non-linear curve fitting
analysis
(14, 15) of the acquired data using software
specifically designed for BIAcore data and available in the
BIAevaluation 2.1 package (Pharmacia Biosensor).
RESULTS
Antibodies to MSCRAMM Fragments Inhibit Collagen
Binding to S. aureus
In previous studies, we attempted to
identify the ligand-binding site of the S. aureus collagen-binding MSCRAMM by examining the binding activity of
recombinant MSCRAMM fragments of progressively decreasing size.
Truncations beyond a 168-amino-acid long segment, CBD(151-318),
resulted in loss of collagen binding activity but also affected the
folding of the resulting proteins as indicated by CD spectroscopy. Thus
it is possible that the ligand-binding site is contained within a short
segment of CBD(151-318), but due to the improper folding of the
protein the collagen-binding site is not in an active form. To explore
this possibility, we decided to try an antibody
inhibition-neutralization approach. A similar strategy was used
successfully to monitor the purification of the native collagen-binding
MSCRAMM from S. aureus cells
(8) . To generate an
inhibiting antibody, we used as an antigen CBD(151-297), a
recombinant version of the largest segment that did not bind collagen
and exhibited an altered conformation. In this way, we would minimize
generating inhibiting antibodies which recognize conformational
dependent epitopes. An inhibiting monoclonal antibody generated against
a biologically active collagen-binding MSCRAMM recognized a
conformational dependent epitope and was of limited use in identifying
the binding site.(
)
Figure 1:
Reactivity of
CBD(151-297) IgG with synthetic CBD peptides. The ability of
CBD(151-297) IgG to recognize individual CBD peptides was
quantitated by ELISA. Microtiter wells were coated with CBD peptides
(100 µg/ml) in PBS. After incubation with the peptides, the wells
were washed, and the remaining binding sites were blocked with BSA.
Purified IgG (80 µg/ml) from immune
CBD(151-297) sera
(shaded boxes) or preimmune sera (open boxes) was
then added to the peptide-coated wells. The amount of IgG bound was
detected by the addition of alkaline phosphatase conjugated goat
anti-rabbit IgG as described under ``Experimental
Procedures.'' The amino acid sequences of the peptides are shown
in Table I.
Purified CBD(151-297) IgG inhibited the
binding of S. aureus to
I-labeled collagen in a
dose-dependent manner (Fig. 2). The amount of
I-collagen bound by 10
bacterial cells was
reduced over 50% by 5 µg and essentially completely inhibited by 10
µg of the purified immune IgG. Conversely, antibodies purified from
preimmune sera did not possess significant inhibitory activity
(Fig. 2). These results suggest that the
CBD(151-297)
antibodies recognize epitopes at or close to the active site of the
MSCRAMM, thereby directly inhibiting or sterically interfering with
collagen binding. A Synthetic Peptide Neutralizes the Inhibitory Activity of
CBD(151-297) IgG-The different synthetic CBD peptides
described above and shown to react with the
CBD(151-297) IgG
were assayed for their potential to neutralize the inhibitory activity
of the antibody. The
CBD(151-297) IgG (12 µg) was
preincubated with a single dose (100 µg) of each peptide in step
one. The S. aureus cells were then added and the preincubation
continued. Finally, the
I-labeled collagen type II was
added. As shown in Fig. 3, peptide CBD4 neutralized 68% of
CBD(151-297) IgG inhibitory activity, while the other
peptides tested had little or no effect. These results suggested that
only antibodies recognizing epitopes present in CBD4 were able to
inhibit collagen binding to bacteria. Although other peptide sequences
were more immunogenic than CBD4, the antibodies recognizing the
corresponding epitopes were not inhibitory. These data suggest that the
ligand-binding site of the MSCRAMM is located close to or within the
sequence covered by peptide CBD4.
Figure 2:
Purified CBD(151-297) IgG
inhibits the binding of S. aureus to collagen type II. S.
aureus was preincubated with increasing amounts of
CBD(151-297) IgG (
), or IgG purified from preimmune
sera (
) prior to the addition of
I-labeled
collagen type II. Collagen binding to bacteria was quantitated as
described under ``Experimental Procedures.'' Results are
expressed as mean ± standard deviation. Each sample point was
done in duplicate.
Figure 3:
Peptide neutralization of
CBD(151-297) IgG inhibitory activity. Overlapping
25-amino-acid long CBD peptides (100 µg) derived from
CBD(151-297) were assayed for their potential to neutralize the
ability of
CBD(151-297) IgG (24 µg/ml) to inhibit the
binding of S. aureus to
I-labeled collagen type
II. For details see ``Experimental Procedures.'' The amino
acid sequences of the peptides are shown in Table
I.
To further investigate the
interaction between peptide CBD4 and CBD(151-297) IgG and
its affect on collagen binding, a fixed concentration of the antibody
was incubated with an increasing amount of peptide CBD4 (Fig. 4).
To make the assay more sensitive, we chose an
CBD(151-297)
IgG concentration (12 µg/ml) which resulted in a 50% reduction in
collagen binding to 10
S. aureus cells. Thus, a
relatively small reduction in inhibition could be easily detected. At
low concentrations, the peptide appears to neutralize the inhibitory
activity, and in this experiment 100 µg of peptide CBD4 restored
the level of collagen binding to S. aureus observed in absence
of
CBD(151-297) IgG. Somewhat surprisingly, addition of more
peptide CBD4 resulted in a dose-dependent decrease in collagen binding
to S. aureus (Fig. 4).
Figure 4:
Effect of
peptide CBD4 on the ability of CBD(151-297) IgG to inhibit
the binding of S. aureus to collagen type II. An increasing
amount of peptide CBD4 was added to
CBD(151-297) IgG (12
µg/ml), and the amount of
I-labeled collagen bound by
S. aureus was quantitated as described under
``Experimental Procedures.'' The solid vertical bar indicates the amount of
I-labeled collagen bound by
S. aureus in the absence of any inhibitor. Results are
expressed as mean ± standard deviation. Each sample point was
done in duplicate.
Peptide CBD4 Directly Inhibits Collagen Binding to S.
aureus
To assess the role of amino acids 209-233 in
collagen binding, peptide CBD4 was tested for the ability to directly
inhibit the binding of I-collagen to S. aureus.
Peptide CBD7 which reacted strongly with
CBD(151-297) IgG in
the ELISA assay (Fig. 3) was also tested. As shown in
Fig. 5
, when increasing amounts of peptide CBD4 were incubated
with
I-collagen prior to the addition of S.
aureus, binding to collagen was inhibited in a dose-dependent
manner. Five µM CBD4 inhibited binding by over 50%.
Peptide CBD7 had no inhibitory effect when it was preincubated with
I-collagen. These data suggest that peptide CBD4 can bind
soluble
I-collagen and that CBD4 contains the residues
that represent a collagen-binding site within the MSCRAMM protein.
Figure 5:
Dose-dependent inhibition of
I-labeled collagen binding to S. aureus by
peptide CBD4. Increasing amounts of peptide CBD4 (
) or peptide
CBD7 (
) were added to
I-labeled collagen, and the
binding to S. aureus was evaluated as described under
``Experimental Procedures.'' Results are expressed as mean
± standard deviation. Each sample point was done in
duplicate.
Identification of Critical Residues within CBD4 Necessary
for Collagen Binding Activity
To identify residues within CBD4
necessary for collagen binding we synthesized several smaller
overlapping peptides (). A series of peptides that
contained truncations of 5, 10, or 15 amino-terminal residues and one
peptide that contained a 2 amino acid deletion at the carboxyl terminus
were made. The peptides (10 µM) were assayed for their
ability to inhibit the binding of I-labeled collagen to
S. aureus (Fig. 6). Peptide CBD4 inhibited collagen
binding by 76%, whereas all peptides containing amino-terminal
truncations had little activity at the concentration tested. These data
indicate that when as few as 5 residues are removed from the active
site peptide, the ability to bind collagen is lost. Moreover, deletion
of only the 2 carboxyl-terminal amino acids causes a complete loss of
biological activity. This suggests that amino acids at the C terminus
of CBD4, Asn
or Tyr
or both are integral
member(s) of the active site of the collagen MSCRAMM.
Figure 6:
Effect of CBD4 synthetic peptide
derivatives on the binding of collagen binding to S. aureus.
Binding of S. aureus to I-labeled collagen was
evaluated in the presence of peptides (10 µM). The total
amount of collagen bound was quantitated in the absence of any peptide.
Results are expressed as mean ± standard deviation. Each sample
point was done in duplicate. Amino acid sequences of the peptides are
shown in Table I.
Collagen Binding Activity of Defined MSCRAMM
Mutants
The importance of amino acid residues Asn and Tyr
in the MSCRAMM for collagen binding was
examined by creating specific mutants of CBD(30-529) and
characterizing the interactions of these mutants with immobilized type
II collagen using the BIAcore. In the mutations made, the identified
amino acid residues were replaced individually,
(N
A, Y
A) or as a pair,
(N
A:Y
A) with alanine, a residue
that is not expected to interfere with the existing secondary
structure. The corresponding base changes were made using overlap
extension PCR as described under ``Experimental Procedures''
and the sequence changes confirmed experimentally. The recombinant
proteins containing the mutations were purified to homogeneity by metal
ion chelating chromatography. Structural analyses of the isolated
proteins by near- and far-UV CD spectroscopy suggested that no
significant changes in secondary or tertiary structure had occurred as
a result of the mutations (data not shown).
). The association
phase exhibits multiphasic binding with an interaction characterized by
a fast k
apparent at the start of the injection
followed by a second phase characterized by a much slower
k
. The dissociation phase gives information
about the k
, and this rate is independent of the
number of binding sites, analyte concentration, and flow rate. Analysis
of these data indicates at least a three-component dissociation with
the two fastest rates greater than 10
s
and the slowest k
at 5
10
s-1 (I). The association phase
contains the information to determine the association rate
(k
) of the interaction, but it is also influenced
by the dissociation rate, the number of binding sites for each
interaction, and the concentration of the analyte. Because of the
complexity of this interaction and the absence of measurable
equilibrium data, it was not possible to determine the binding constant
(K
), apparent binding ratio (BR
), and
k
.
Figure 7:
BIAcore analysis of the interaction
between recombinant collagen adhesin constructs and collagen.
Sensorgrams indicating relative response versus time for the
binding of CBD(30-529) and mutant proteins
NA, Y
A, and
N
A:Y
A to immobilized collagen
type II. The concentration of each protein assayed was 20
µM.
In a comparison of the three mutant proteins
with wild type CBD(30-529), it is readily apparent that each of the
introduced mutations affected the collagen binding properties of the
generated proteins (Fig. 7, I). While the shape of
the binding sensorgram remains essentially the same for mutant
NA, analysis of the dissociation phase indicates
that the slowest dissociation rate has increased almost 10-fold.
Although a dissociation constant (K
) cannot be
determined, it appears that the affinity of this mutant for collagen
type II has also been influenced by this mutation. Both the
Y
A and the double mutant bind immobilized type II
collagen weakly, and analysis of the available equilibrium information
indicates dissociation constants greater than 10
M and fast dissociation rates (>10
s
). The Y
A mutant exhibits
biphasic binding to collagen type II, but the highest rate could not be
accurately determined because of protein concentration restrictions.
Analysis of the data obtained with the double mutant produces a
monophasic binding constant when evaluated by either Scatchard analysis
or Equation 1. Additionally, the BR
has decreased to
approximately two to three high affinity sites.
and Tyr
are important for both the affinity and the specificity of
CBD(30-529)'s binding to type II collagen. There does not,
however, seem to be an additive effect of the double mutation when
compared to the single mutations. It is possible that the individual
MSCRAMM binding sites on type II collagen are heterogeneous thus giving
rise to the multiphasic character of the measured interaction.
DISCUSSION
The adhesion of microorganisms to host tissues is the
critical first step in the development of most infections. It has
become increasingly clear that eukaryotic adhesive extracellular matrix
components, that are normally involved in cell-matrix interactions
within the host, also serve as ligands for pathogenic microorganisms.
Collagens are major constituents of most extracellular matrices and
different specialized tissues are usually characterized by a specific
repertoire of different types of collagens
(17) . For example,
dentin from root surfaces contains predominately collagen type I,
cartilage mainly consists of type II collagen, and collagen type IV is
exclusively found in basement membranes.
(32) and
(33, 34) integrins,
anchorin II
(35) , and extracellular matrix proteins such as the
propolypeptide of von Willebrand factor
(36) ,
fibronectin
(37) , decorin
(38) , and
thrombospondin
(39) . Although the number of proteins that have
been described to bind collagen is quite large, the localization of
specific collagen-binding sites within those polypeptides has been
analyzed in only a few cases. The YadA protein from Y.
enterocolitica contains a stretch of 22 amino acids located in the
hydrophobic amino terminus that is necessary for binding collagen types
I, III, IV, and V. A dodecapeptide
(36) , derived from the
propolypeptide of bovine von Willebrand factor
(40) , has been
shown to contain collagen binding activity.
and Tyr
. In addition,
site-directed mutagenesis of this region revealed that residues
Asn
and Tyr
are critical for high affinity
collagen binding. Moreover, mutated proteins containing alanine
substitutions at Asn
and Tyr
positions
exhibited altered collagen-binding kinetics compared to the wild-type
protein. Residues Asp
-Gly
are apparently
also important for collagen binding since a truncated version of
peptide CBD4 lacking these residues lost its collagen binding activity.
Furthermore, an MSCRAMM mutant in which we had deleted residues
Gly
, Gly
did not bind collagen. However,
this mutated protein had undergone considerable conformational changes
compared to the wild-type protein as indicated by CD analyses (data not
shown). Therefore, the importance of these residues in direct collagen
binding is unclear. To our knowledge this is the first report where
specific amino acids within a collagen-binding protein have been
identified that are directly involved for collagen binding activity.
Studies are underway to determine the location of the critical residues
in the folded collagen-binding MSCRAMM.
Table:
Synthetic peptides derived from the sequence of
the S. aureus collagen-binding MSCRAMM
Table:
Oligonucleotides used for site-directed
mutagenesis
Table: 0p4in
Concentration of MSCRAMM protein used
in the binding studies (µM).(119)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.