(Received for publication, July 13, 1995)
From the
A monoclonal antibody 3A10, generated from a mouse immunized
with the Streptococcus dysgalactiae fibronectin (Fn) binding
protein FnbA, was isolated, and its effect on ligand binding by the
antigen was examined. The epitope for 3A10 was localized to a
previously unidentified Fn binding motif (designated Au) just
N-terminal of the repeat domain which represents the primary ligand
binding site on FnbA. Fn binding to Au was enhanced by 3A10 rather than
inhibited. This effect was demonstrated in two different assays. First,
in the presence of 3A10 the Au-containing proteins and synthetic
peptide more effectively competed with bacterial cells for binding to
Fn. Second, 3A10 dramatically increased the binding of biotin-labeled
forms of the Au-containing proteins to Fn immobilized on a blotting
membrane. Pure 3A10 IgG did not recognize the antigen by itself, and Fn
was required for the immunological interaction between the antibody and
the epitope. This induction effect of Fn was shown in both Western blot
and enzyme-linked immunosorbent assay in which immobilized
Au-containing molecules were probed with 3A10 in the presence of
varying concentrations of Fn. Specificity analyses of 3A10 revealed
that the monoclonal also recognized a ligand binding motif in a Streptococcus pyogenes Fn binding MSCRAMM but not the
corresponding motifs in two related adhesins from Staphylococcus
aureus and S. dysgalactiae. Furthermore, 3A10 stimulated
Fn binding by S. pyogenes cells. These results together with
subsequent biophysical studies presented in the accompanying paper
(House-Pomepeo, K., Xu, Y., Joh, D., Speziale, P., and
Höök, M.(1996) J. Biol.
Chem. 271, 1379-1384) indicate that the ligand binding sites
of Fn binding MSCRAMMs have little or no secondary structure. However,
on binding to Fn, they appear to undergo a structural rearrangement
resulting in a defined structure rich in sheet and expressing a
ligand-induced binding site for antibodies such as 3A10.
Bacterial adherence to the host tissue is recognized as the
first step in the pathogenesis of most infections. Adherence is
mediated by bacterial surface components called adhesins which
recognize and bind to specific structures in the host tissue. One
family of adhesins recognize extracellular matrix components, and
members of this adhesin family have been collectively called MSCRAMMs ()(microbial surface components recognizing adhesive matrix
molecules; for reviews, see (1) and (2) ). Several
fibronectin (Fn) binding MSCRAMMs have been isolated and characterized
from different Gram-positive bacteria. Genes encoding Fn binding
MSCRAMMs from Staphylococcus aureus(3) , Streptococcus pyogenes(4, 5) , and Streptococcus dysgalactiae(6) have been cloned and
sequenced. The deduced amino acid sequences revealed 60-100-kDa
proteins with very similar structural organization. The N-terminal
signal sequence is followed by a long stretch of unique sequence which
in some cases is interrupted by two copies of a
30-amino acid long
segment. The ligand binding site is located just N-terminal of a
proline-rich domain, which is believed to anchor the proteins in the
cell wall. This domain is followed by the sequence LPXTGX which is a cell wall targeting signal(7) , a stretch of
hydrophobic residues representing a transmembrane unit and a short
C-terminal cytoplasmic domain containing a cluster of positively
charged residues. The primary Fn-binding sites on these MSCRAMMs
consist of 30-42 amino acid long motifs repeated 3-4 times,
and most of the repeated units contain a consensus sequence (6, 8) . In a recent communication(9) , we
reported that recombinant proteins corresponding to the repeat regions
from the different Fn binding MSCRAMMs are all capable of inhibiting
binding of Fn to different Gram-positive bacteria, including S.
aureus, S. dysgalactiae, and S. pyogenes.
Furthermore, studies using individual synthetic peptides revealed that
a number of the repeat units retain Fn-binding activity, and interfere
with binding of Fn by all of the Gram-positive species tested. These
data suggest that the binding sites in Fn for the different MSCRAMMs
are either overlapping or closely spaced on the matrix protein.
The S. aureus Fn binding MSCRAMM contains an additional ligand
binding site in a 30-amino acid long segment which encompasses the
consensus sequence and is located N-terminal of the repeat region. This
segment can also interact with Fn and its N-terminal domain (designated
N29)(10) . The present study identifies a new ligand binding
sequence called Au located just N-terminal of the A repeats of the S. dysgalactiae MSCRAMM, FnbA.
Because of the pivotal role
that tissue adherence plays in the pathogenic process, bacterial
adherence has been identified as a target in new strategies to prevent
and treat infections. It is possible that adhesins can be used as
vaccines to generate antibodies which in addition to participating in
the conventional host defense, also can block tissue adherence.
Previous studies using Fn binding MSCRAMMs as an antigen have given
inconclusive results(11, 12) , and it is unclear if
generated antibodies can block ligand binding and adherence. In this
study, we report on a monoclonal antibody (designated 3A10) which
recognizes the newly identified ligand binding site Au in the S.
dysgalactiae MSCRAMM FnbA only when Au is bound to Fn.
Furthermore, the monoclonal antibody 3A10 appears to enhance ligand
binding to recombinant proteins or synthetic peptides containing the Au
sequence. This activity is similar to those of anti-LIBS
(ligand-induced binding sites) antibodies described for the platelet
integrin
(13, 14) . This
family of antibodies appear to recognize a conformation of a receptor
induced by ligand binding.
The antigen was mixed with complete Freund's adjuvant (Sigma) for the primary immunization, incomplete adjuvant for the next three injections, and in saline only for the final immunization. Three days after the last injection, the splenic lymphocytes were isolated and fused with Spe/0 Ag.14 mouse myeloma cells at the ratio 5:1 using 50% polyethyleneglycol 4000. Supernatants of the resulting hybridomas grown in selective media, (hypoxantine/aminopterin/thymidine) were first tested in an ELISA for the presence of antibodies reactive with FnbA-coated microtiter wells (Costar, Cambridge, MA). Positive hybridomas were then subcloned by limited phase dilution into 96-well plates. A clone designated 3A10 was subcloned and grown to high density in RPMI 1640 medium containing 10% (v/v) fetal bovine serum and antibiotics.
To produce ascites fluid,
cells were harvested from late-log phase cultures, washed with sterile
phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM NaHPO
, and 1.4
mM KH
PO
, pH 7.3) and 5
10
cells were injected intraperitoneally into
Pristane-primed mice. The collected ascites fluid was clarified by
centrifugation and when so indicated antibodies were purified on
protein G-Sepharose as recommended by the manufacturer (Pharmacia,
Uppsala, Sweden). Using a Mouse Isotyping Kit (Bio-Rad), the purified
IgG was determined to be of the IgG
isotype.
Labeling of
Fn N29 with [I]iodine was performed according
to the chloramine-T method of Hunter et al.(18) .
Peptides were synthesized on an Advanced ChemTech multisynthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as described(8) . The amino acid sequence of the synthetic peptide Au is STPEGPTEGENNLGGQSEEITITEDSQSGM and corresponds to aa residues 806-835 of FnbA. The amino acid numbering of this and other constructs are based on the sequence of FnbA reported by Lindgren et al.(6) .
Oligonucleotides were obtained from the Advanced DNA Technologies Laboratory, Texas A& University (College Station, TX).
Figure 1: Mapping of the 3A10 epitope. A, domain organization of FnbA and its truncated constructs. A1, A2, and A3 are the Fn-binding motifs of the primary ligand-binding site. Au is the newly-identified ligand-binding motif described in the text. The plasmids pSDF102, pSDF102c14, and pSDF102c18 are pUC18-based constructs containing DNA encoding the indicated region of the protein(6) . PAQ5, PAQ8, and PAQ14 are polyhistidine fusion proteins expressed from pQE30-based plasmids containing the corresponding DNA fragments. Peptide Au is a synthetic 30-mer peptide (aa 806-835 of FnbA according to Lindgren et al.(6) ) mimicking the Au motif. B, Coomassie Blue-stained gel (i) and Western blot (ii) of the lysates of E. coli harboring the indicated plasmids. The membrane containing the transferred proteins was probed with 3A10 ascites fluid. C, Coomassie Blue-stained gel (i) and Western blots (ii) of the PAQ proteins. The Western blots were probed with 3A10 ascites fluid in the absence (-Fn) and presence (+Fn) of added 10 µg/ml Fn.
Construction and purification of rFNBD-A, rFNBD-B, rFNBD-D, rFNBD-P, and CBD(151-318) was described earlier(9) .
To further identify the epitope recognized by 3A10, additional
pQE30-based plasmids encoding the polypeptides corresponding to the
segments described in Fig. 1A were constructed. In the
pQE-based constructs the insert DNA is expressed as a fusion protein
with a short N-terminal segment containing a stretch of histidine
residues which allows purification by Ni-chelating
chromatography(21, 22) . PAQ5 contains a 81-amino acid
long fragment of the MSCRAMM located N-terminal of the previously
identified ligand binding A repeats. PAQ8 corresponds to the A repeats
and an additional 90-amino acid long N-terminal sequence. PAQ14
contains the A repeats but lacks the N-terminal sequence. When analyzed
by SDS-PAGE, the purified PAQ proteins migrated slower than expected
according to their respected mass (Fig. 1C, i); however, this behavior is consistent with our previous
observations of similar constructs(9) . The PAQ proteins are
very acidic with isoelectric points of 4.6 (PAQ5), 3.8 (PAQ8), and 3.8
(PAQ14), which results in a relatively poor binding of SDS and abnormal
migration in SDS-PAGE(23) . The molecular masses of PAQ5, PAQ8,
and PAQ14 calculated from the amino acid sequences are 10,112, 23,682,
and 18,401 daltons, respectively. Electrospray mass spectroscopy
analysis of the purified PAQ proteins produced spectra containing
single major peaks. The electrospray mass spectroscopy determined
molecular masses of the recombinant proteins correspond exactly to
those deduced from their respective amino acid sequences.
When recombinant PAQ proteins were fractionated by SDS-PAGE, transferred to a supporting membrane, and probed with 3A10 ascites fluid, only PAQ8 reacted with the antibody (Fig. 1C, ii). However, when 65 µg of porcine Fn was added to the 5-ml antibody solution in which the membrane was incubated, also PAQ5 but not PAQ14 reacted with the antibody. Furthermore, the antibody signal from the PAQ8 protein was stronger when Fn was added. Thus, these experiments demonstrated that the 3A10 epitope is located within the 81-amino acid long segment present in PAQ5 and PAQ8 but not in PAQ14. The effect of Fn on the antibody binding is discussed in detail below.
Figure 2: Sequence alignment of the P1, Au, and A2. The Au sequence corresponds to aa 806-835 of FnbA from S. dysgalactiae. P1 and A2 are Fn-binding motifs present in Sfb from S. pyogenes and FnbA(9) . The bold-faced letters in P1 and A2 represent residues identical with residues found in Au. Asterisk (*) represent homologous residues.
Figure 3:
Inhibition of bacterial binding of I-N29 by the PAQ proteins and peptide Au. Binding of
I-labeled N29 to S. pyogenes strain 64/14, S. dysgalactiae strain S2, and S. aureus strain
8325-4 in the presence of varying concentrations of the potential
inhibitors, PAQ5 (circles), PAQ8 (squares), PAQ14 (open triangles), and peptide Au (filled triangles).
Bacterial binding in the absence of inhibitor was taken as 100%. Error bars indicate the variations between duplicate data
points.
The Fn binding activity of the PAQ proteins was
also demonstrated by a Western blot type assay in which I-labeled N29 was used as a probe and allowed to bind to
the PAQ proteins that had been fractionated by SDS-PAGE and transferred
to a supporting membrane. N29 bound strongly to PAQ8 and PAQ14 (Fig. 4B), but not enough
I-N29 bound to
the immobilized PAQ5 to generate a detectable signal. However, when 75
µg of IgG purified from 3A10 ascites fluid (``3A10 ascites
IgG'') was added to the 5 ml of
I-N29 solution in
which the membrane was incubated, binding of the labeled ligand to PAQ5
was markedly increased and readily detectable (Fig. 4B). Taken together, these data suggest that the
epitope for 3A10 is located in the 81-residue long upstream segment
which contains the newly-identified Fn binding motif Au.
Figure 4:
Affinity Western blots of PAQ proteins.
The PAQ proteins (PAQ5, PAQ8, and PAQ14) were fractionated by SDS-PAGE
on a 12% polyacrylamide gels and stained with Coomassie Blue (A) or transferred to a supporting membrane. The membrane was
probed with I-labeled N29 (B) in the presence
(+3A10) or absence (-3A10) of 10 µg/ml
3A10 ascites IgG.
Figure 5:
Monoclonal antibody 3A10 enhances the Fn
binding activity of protein/peptides containing the Au sequence. S.
aureus cells (strain 8325-4) suspended in 0.5 ml of PBSTB were
incubated with I-labeled N29 and varying concentrations
of PAQ5 (A), PAQ8 (B), PAQ14 (C), and
peptide Au (D) were in the presence (square) and
absence (circle) of 100 µg/ml 3A10 ascites IgG. Bacterial
binding in the absence of the inhibitors and 3A10 was taken as 100%. Error bars indicate the difference between duplicate data
points.
The potentiating effect of the 3A10
antibody on the Fn binding activity of Au-containing proteins/peptides
was measured as a function of antibody concentration. S. aureus cells were incubated with I-labeled ligand and
either PAQ protein or Au peptide where the concentration of protein or
peptide was set at a level which cause 10-40% inhibition of
ligand binding in the absence of 3A10 IgG. As the concentration of 3A10
ascites IgG was increased from 0 to 120 µg/ml, the inhibitory
activity of PAQ5 (at 0.75 µg/0.5 ml) was enhanced in a
concentration-dependent manner from 10% up to 85% (Fig. 6A). The inhibition by 2 µg of PAQ8 was
increased in a similar way from 40 to 80% by 3A10 ascites IgG (Fig. 6B), whereas the inhibitory activity of PAQ14,
which lacks the 3A10 epitope, was only marginally affected by the
antibody (Fig. 6C). The Fn binding activity of the
synthetic peptide Au was also dramatically enhanced by 3A10 in a
concentration dependent manner. In the absence of 3A10 antibody, the Au
peptide at 100 µg/ml caused <10% inhibition of bacterial binding
of
I-N29. This inhibition was enhanced up to 80% in the
presence of the highest concentration of 3A10 ascites IgG tested (Fig. 6D). The 3A10 antibody alone had only a marginal
effect on the binding of N29 to S. aureus (Fig. 6A). The response of peptide Au to 3A10
further demonstrates that the binding sites for both Fn and 3A10 reside
in the 30-residue synthetic peptide.
Figure 6:
Dose-dependent effect of 3A10. Cells (5
10
) of S. aureus Cowan 1 suspended in 0.5
ml of PBSTB were incubated with indicated concentrations of PAQ5, PAQ8,
PAQ14, and peptide Au and
I-labeled N29 (the PAQ
proteins) or Fn (peptide Au) in the presence of varying concentrations
of 3A10 ascites IgG. Inhibition (%) = {1 - (binding
in the presence indicated inhibitor and 3A10)/(binding in the absence
of inhibitor and 3A10)}
100. Error bars indicate
the difference between assays performed in
duplicate.
The enhancement of Fn binding activity of PAQ5 by 3A10 was also demonstrated in a Western blot type assay. Fn and N29, separated on SDS-PAGE, were transferred to a membrane and allowed to interact with biotin-labeled PAQ proteins in the absence or presence of 3A10. Binding of PAQ5 to both intact Fn and N29 immobilized on the membrane was wholly dependent on the presence of 3A10 (Fig. 7B). Both PAQ8 and PAQ14 bind Fn and N29 also in the absence of 3A10 which is expected since both constructs contain the A1, A2, and A3 repeat domains of the primary ligand binding site.
Figure 7:
Effects of 3A10 on binding of the PAQ
proteins to immobilized ligand. Fn and N29 separated by nonreducing
SDS-PAGE on 10% polyacrylamide gels were stained with Coomassie Blue (A) or electroblotted onto supporting membranes (B).
The blots were probed with biotin-labeled PAQ proteins in the absence
(-3A10) or presence (+3A10) of 10
µg/ml 3A10 ascites IgG. A high molecular weight (60,000) band
is observed in the lanes of N29. This fragment which is not observed in
the stained gel may have resulted from incomplete digestion of Fn
during the N29 preparation.
Figure 8: Fn-dependent interaction between 3A10 and FnbA. Lysates of E. coli harboring pSDF100 or pSDF200 separated by SDS-PAGE on 10% polyacrylamide gels were stained with Coomassie Blue (A) or electroblotted onto supporting membranes (B). The membranes were probed with 10 µg/ml 3A10 ascites IgG in the absence (-Fn) or presence (+Fn) of added Fn (5 µg/ml). Bound antibody was detected as described under ``Experimental Procedures.''
The requirement of Fn for the interaction of 3A10 with the Au sequence was further demonstrated in an ELISA assay (Fig. 9). The 3A10 IgG failed to bind to PAQ proteins coated on microtiter wells in the absence of Fn. Binding of 3A10 IgG to PAQ5 and PAQ8 but not PAQ14 was obtained by the addition of soluble Fn in conjunction with the antibody (Fig. 9A). Full-length FnbA did not react with 3A10 IgG in the absence of Fn, but bound the antibody in the presence of Fn or its N-terminal fragment N29 (Fig. 9, B and C). These effects of Fn were dose-dependent. Thus the epitope recognized by 3A10 is formed by an interaction of the Au motif in FnbA with Fn regardless of if the Au motif is present in a synthetic peptide, a short recombinant protein, or the full-length MSCRAMM.
Figure 9: ELISA analyses of immobilized FnbA and its derivatives. PAQ5, PAQ8, PAQ14 (A), or FnbA (B and C) was immobilized onto microtiter wells and probed with 100 µl of 180 pg/ml (A) or 10 µg/ml (B and C) monoclonal 3A10 IgG in the presence of varying concentrations of Fn (A and B) or N29 (C). Bound 3A10 was detected as described under ``Experimental Procedures.'' Error bars indicate standard error in triplicate or quadruplicate assays.
Figure 10: Specificity of 3A10. The rFNBD proteins and CBD(151-318) (9) fractionated by SDS-PAGE on a 10% polyacrylamide gel were stained with Coomassie Blue (A) or blotted onto a supporting membrane (B). The blot was probed with 3A10 ascites fluid.
The effect of 3A10 on Fn binding by bacteria was examined by
incubating cells of S. aureus, S. dysgalactiae, or S. pyogenes with I-labeled N29 in the presence
or absence of purified 3A10 IgG (Fig. 11). The monoclonal 3A10
dramatically enhanced the amount of
I-labeled N29 bound
to the S. pyogenes cells, while only marginally stimulating
N29 binding to S. aureus and S. dysgalactiae. It is
important to note that FnbA is not expressed in the S. dysgalactiae strain grown under conventional culture conditions(6) ,
which explains the low enhancement in Fn binding to the S.
dysgalactiae cells. Therefore, the result shown in Fig. 11is consistent with the difference in reactivity of 3A10
with the rFNBD proteins observed in the Western blot (Fig. 10).
The results demonstrate that 3A10 can recognize an intact MSCRAMM
expressed on the surface of a bacterial cell and enhance Fn binding to
bacteria presumably by stabilizing the MSCRAMM-ligand complex.
Figure 11:
Effect of 3A10 on Fn binding to bacterial
cells. Binding of I-labeled N29 to bacterial cells were
measured in the absence (solid bar) and presence of 4.8
µg/ml 3A10 IgG (hatched bar). Binding in the absence of
IgG was taken as 100%. Error bars indicate the difference
between the assays performed in duplicate.
The presence of a previously unidentified ligand binding site in the Fn binding MSCRAMM FnbA from S. dysgalactiae is demonstrated in this study. This ligand binding site, Au, is contained within a 30-amino acid residue segment which ends 8 residues N-terminal of the previously identified primary binding domain consisting of the three Fn binding motifs A1, A2, and A3. The Au sequence, present in a synthetic peptide or in a recombinant fusion protein also binds Fn as shown by its ability to inhibit Fn binding to S. aureus cells.
The epitope for the monoclonal antibody 3A10 has been located to the
Au sequence. In the absence of Fn, however, 3A10 does not recognize the
Au sequence in a recombinant full-length MSCRAMM, in truncated
proteins, or in a synthetic peptide. The epitope recognized by 3A10
appears to be conformation dependent and formed by the binding of Au to
Fn. Thus 3A10 can be said to recognize a LIBS in the Fn binding MSCRAMM
FnbA. Recent biophysical analysis (17) has shown that the
primary ligand binding domain present in the recombinant protein
rFNBD-A (9) has little, if any, secondary structure and no
tertiary structure, and occurs largely as a random coil. However, on
binding to Fn, rFNBD-A appears to undergo a substantial structural
change, adopting a defined conformation rich in -sheet structure.
Although the Au sequence has not been directly examined in similar
studies, it seems reasonable to assume that, on ligand binding, this
segment also undergoes a conformational change from ``random
coil'' to a defined structure contains the epitope for 3A10.
The monoclonal antibody 3A10 also enhances the Fn binding activity of a synthetic peptide or recombinant proteins containing the Au sequence. Presumably, this is caused by 3A10 stabilizing the conformation induced in Au on binding to Fn. Although the A1, A2, and A3 sequences also presumably undergo ``induced-fit'' conformational changes on binding to Fn, 3A10 does not recognize these sequences nor does it stabilize their ligand complex. Furthermore, 3A10 ascites fluid reacts strongly with rFNBD-P and weakly with rFNBD-B and rFNBD-D. Taken together, these results suggest that the 3A10 epitope is composed of specific amino acid residues present in Au and the P1/P2 motif (Fig. 2) which form a specific conformation on binding to Fn.
Frelinger et al. (13, 14) described a
number of monoclonal antibodies for LIBS expressed on the platelet
integrin only when the integrin
forms a complex with the ligand fibrinogen or ligand mimetics. These
antibodies were isolated from mice immunized with a mixture of the
integrin and a RGD-containing peptide, a ligand mimetic. The anti-LIBS
antibodies recognizing
or the Fn
binding MSCRAMM appear to bind to cell surface ``receptors''
which undergo fairly extensive conformational changes on ligand
binding. In the case of the platelets, this conformational change
appears to be of physiological importance and is part of the activation
process involved in platelet aggregation.
It is tempting to speculate that the conformational changes in the Au sequence induced by ligand binding and manifested by establishing the 3A10 epitope is a reflection of an important process evolved in bacteria to avoid immunological interference of microbial adherence to Fn. Our attempts to generate blocking polyclonal antibodies using various recombinant forms of Fn binding MSCRAMMs, or synthetic peptides as antigens have been largely unsuccessful. We do not know if the antigens used generate blocking antibodies together with enhancing anti-LIBS antibodies which compromised the effect of the blocking antibodies, or if the animals are unable to generate any blocking antibodies at all. If the MSCRAMM did not undergo a conformational change on ligand binding, the generated antibodies to the MSCRAMM could interfere with or be indifferent to ligand binding. A conformational rearrangement in the MSCRAMM on Fn binding makes it possible to generate anti-LIBS antibodies which stabilize the ligand MSCRAMM complex resulting in enhanced Fn binding and substrate adhesion. This would clearly represent an advantage to the microbe since the generated antibodies could enhance adherence to host tissue rather than to inhibit this critical step in tissue colonization and the pathogenic process. Studies are in progress to further analyze the immunological response to Fn binding MSCRAMMs.