From the Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, New York 10021
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ABSTRACT |
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The plasmin(ogen) binding property of group A
streptococci is incriminated in tissue invasion processes. We have
characterized a novel 45-kDa protein displaying strong plasmin(ogen)
binding activity from the streptococcal surface. Based on its
biochemical properties, we confirmed the identity of this protein as
-enolase, a key glycolytic enzyme. Dose-dependent
-enolase activity, immune electron microscopy of whole streptococci
using specific antibodies, and the opsonic nature of polyclonal and
monoclonal antibodies concluded the presence of this protein on the
streptococcal surface. We, henceforth, termed the 45-kDa protein, SEN
(streptococcal surface enolase). SEN is found
ubiquitously on the surface of most streptococcal groups and serotypes
and showed significantly greater plasmin(ogen) binding affinity
compared with previously reported streptococcal plasminogen binding
proteins. Both the C-terminal lysine residue of SEN and a region
N-terminal to it play a critical role in plasminogen binding. Results
from competitive plasminogen binding inhibition assays and
cross-linking studies with intact streptococci indicate that SEN
contributes significantly to the overall streptococcal ability to bind
plasmin(ogen). Our findings, showing both the protected protease
activity of SEN-bound plasmin and SEN-specific immune responses,
provide evidence for an important role of SEN in the disease process
and post-streptococcal autoimmune diseases.
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INTRODUCTION |
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Streptococcus pyogenes is responsible for a wide variety of human diseases that range from suppurative infections of the throat (pharyngitis), skin (impetigo), and underlying tissues (necrotizing fasciitis), to an often fatal toxic shock syndrome, and the post-streptococcal sequelae, rheumatic fever, and acute glomerulonephritis. Bacterial surface proteins play a major role in these disease processes by exhibiting a wide range of functions. As data have become available, it is clear that most surface proteins found on Gram-positive bacteria, particularly those on group A streptococci, have a great deal of structural similarities (1, 2). Proteins for which the function(s) has been defined have been found to be multifunctional, whereas in others a function has only been attributed to one of two or more domains (2, 3). Thus, the multifunctional characteristics of these surface proteins increase the complexity of the Gram-positive surface beyond what has been previously imagined.
We recently described one such multifunctional protein, streptococcal surface dehydrogenase (SDH),1 as a major surface protein on group A streptococci and other streptococcal groups which is structurally and functionally related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4). SDH also has an ADP-ribosylating activity (5) and exhibits multiple binding activities to several mammalian proteins such as fibronectin and cytoskeletal proteins (4). A structurally and enzymatically similar streptococcal protein, Plr, was also identified on group A streptococci, based on its ability to bind plasmin (6). SDH, however, is a weak plasmin-binding protein (4). During our studies characterizing the SDH molecule, we reported that a 45-kDa protein was also found in high amounts on the surface of group A streptococci (4). While determining the relative plasmin binding activity of SDH with respect to other streptococcal surface proteins, we found that the 45-kDa protein had in fact strong plasmin binding activity.2
The plasmin(ogen) system displays a unique role in the host defense by dissolving fibrin clots and serving as an essential component to maintain homeostasis and vascular potency (7-9). Studies on the ability of Gram-positive bacteria to subvert the fibrinolytic activity of human plasmin(ogen) to their own advantage for tissue invasion have been largely focused on pathogenic streptococci and were described first as early as 1933 by Tillet and Garner (10). This property was subsequently attributed to the plasmin(ogen) activator, streptokinase (11), an extracellular 48-kDa protein secreted in culture supernatants (12). The role of pathogenic bacteria in tissue invasion utilizing this system has recently been reviewed (13).
In the present communication, we describe purification and
characterization of the 45-kDa protein and show that it is the major
plasmin(ogen) binding molecule on the surface of streptococci. We also
show that this protein has significant sequence similarity with one of
the important glycolytic enzymes, -enolase, found generally in the
cytoplasm. While bound on the surface of group A streptococci, this
45-kDa protein is found to retain its
-enolase activity, hence we
named it SEN (streptococcal surface enolase). It is distinct from the 48-kDa streptokinase (12, 14), the 35.8-kDa SDH
(4), the 41-kDa Plr (6), or the 45-kDa plasminogen-binding protein, PAM
(15), all of which have been reported to bind plasmin to varying
degrees.
-Enolase has not been previously identified on the surface
of bacteria; however, it has been shown to be expressed on the surface
of neuronal (16), cancer (17), and some hematopoietic cells (18, 19) as
a novel plasmin(ogen) receptor. Here, in addition to the structural and
functional characterization of SEN, we also describe the biological
activity of SEN, the functional consequence of plasmin(ogen) binding to
SEN, and the enzymatic activity of SEN-bound plasmin.
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EXPERIMENTAL PROCEDURES |
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Bacteria--
Group A -hemolytic streptococcal strains of
various M types and standard strains used for streptococcal grouping
were from The Rockefeller University Culture Collection (New York, NY)
and are listed as follows: M2(D626), M4(F694), M6(D471), M9(F690), M11(F743), M14(T14/46), M15(D176A), M22(D943), M25(B554),
M35(C171), M40(C270), M44(C757), M49(B910), M51(A291), M58(D632),
M60(D630), M61(D336), M62(D458), M63(D459), M66(D794), group B
(0902), group C (C74), group D (D76), group E (K131), group F (F68C),
group G (D166B), group H (F90A), group L (D167B), and group N (C559). These strains were grown overnight in Todd-Hewitt broth (Difco) and
washed once with 50 mM ammonium bicarbonate followed by two washes in 50 mM phosphate buffer, pH 6.1, to eliminate the
presence of any soluble streptokinase, which may interfere with
analysis. Type M6 (D471) streptococci (4) were used for the isolation of SEN, whereas the other strains were used to study the prevalence of
the plasmin-binding 45-kDa related proteins in different streptococcal groups and group A serotypes.
Human Plasminogen and Plasmin-- Purified human plasminogen and plasmin (lysine-plasmin) were purchased commercially (Sigma). Plasmin was also generated from plasminogen by incubation with urokinase (20 units/ml, Sigma) in HBS gel buffer (50 mM HEPES/NaOH, pH 7.4, containing 1 mM MgCl2, 0.15 mM CaCl2, and 0.1% of gelatin) containing 40 mM lysine. Conversion of plasminogen to plasmin was maximal after 1 h at 37 °C. This method consistently converted more than 95% of the single chain zymogen molecule plasminogen to the heavy and the light chain of the plasmin molecule as reported earlier (20).
Radioiodination of Plasminogen and Plasmin--
Purified
plasminogen and plasmin were radioiodinated with Na125I (17 Ci mg1, NEN Life Science Products) by the chloramine-T
method, using IODO-BEADs (Pierce) as described (4). The labeled
proteins were separated from free iodine by passage over a G-25 column (PD-10, Amersham Pharmacia Biotech) and collection in HBS gel. The
labeled proteins were stored at
70 °C. Plasmin was also generated from the 125I-radiolabeled plasminogen by incubation with
urokinase (20 units ml
1, Sigma) in HBS gel that contained
40 mM lysine (20). More than 95% of the radioactivity was
found to be retained with the plasmin. Thus, the specific radioactivity
of the labeled plasmin and plasminogen was found to be essentially the
same. Furthermore, the specific radioactivity of commercially available
purified plasmin (Sigma) and urokinase-generated plasmin was also the
same. Typically, specific radioactivity of the 125I-labeled
plasmin/plasminogen was achieved in a range of 1.2-2.0 × 106 cpm µg
1 protein.
Blot Overlay System for Plasmin(ogen) Binding--
Proteins in
the bacterial cell wall extracts were resolved by 12% SDS-PAGE gels
and blotted electrophoretically onto a PVDF membrane as described (21,
22). Blots were incubated at room temperature for 3 h in a
blocking HBST gel buffer (50 mM HEPES/NaOH, pH 7.4, containing 0.15 M NaCl, 1% acidified BSA, 0.5% gelatin, 0.5% Tween 20, 0.04% NaN3) and probed for 4 h at
room temperature in the HBST gel buffer containing 2.0 mM
PMSF and 125I-labeled human plasminogen or plasmin 3 × 105 cpm ml1. The probed blots were washed
several times with half-strength HBST gel buffer containing 0.35 M NaCl, dried, and autoradiographed by exposure to Kodak
X-OMAT AR film with an intensifying screen for 15 h at
70 °C.
Extraction of Streptococcal Cell Wall-associated Proteins with
Lysin or Mutanolysin--
M6 strain D471 was grown to stationary phase
at 37 °C for 18 h in 4-6-liter batches of Todd-Hewitt broth.
Bacteria were pelleted by centrifugation, washed, and resuspended in 50 mM phosphate buffer (1/50th of the original culture volume)
containing 30% raffinose and 5 mM dithiothreitol and 5 mM EDTA. Streptococcal cell wall extracts using lysin (an
amidase) enzyme (128 units ml1) was carried out as
described (21) and was dialyzed against 50 mM Tris/HCl, pH
8.0, and concentrated 10-fold using Centriprep-10 concentrators (Amicon
Inc., Beverly, MA). The muralytic enzyme, mutanolysin (20 µg
ml
1, Sigma), was used to prepare cell wall extracts of
each grouping strain suspended in 50 mM Tris/HCl buffer, pH
6.8, containing 30% raffinose as described (4).
Purification of the 45-kDa Protein--
The dialyzed and
concentrated cell wall extracts were sequentially precipitated with
ammonium sulfate at 40, 60, and 80% saturation. The precipitated
proteins were then dialyzed against 50 mM Tris/HCl, pH 8.0, and concentrated to an appropriate volume. The proteins in the dialyzed
preparations were resolved by SDS-PAGE, electroblotted onto a PVDF
membrane, and probed with labeled plasmin(ogen). A strong plasmin(ogen)
binding activity was found to be mainly associated with a 45-kDa
protein of the sequentially fractionated cell wall extract with
40-60% saturation of ammonium sulfate (Fig.
1, A and B). For
further purification, 40-60% ammonium sulfate precipitates were used
as starting material. The dialyzed precipitate was concentrated (Centriprep-10, Amicon) and stored at 70 °C until further use. The
concentrated sample was applied to a Mono Q FPLC column (HR10/10, Amersham Pharmacia Biotech) pre-equilibrated with 50 mM
Tris/HCl buffer, pH 8.0. After washing with 5-column volumes of this
buffer, bound proteins were eluted with a 70-ml linear NaCl gradient
from 0 to 700 mM and then with a 20-ml linear NaCl gradient
from 700 mM to 1 M. Protein elution profile in
each fraction was determined by SDS-PAGE and by Coomassie stain. A
duplicate gel was Western blotted and probed with
125I-plasmin(ogen) as described above. The 45-kDa protein
eluted at 630 mM NaCl. The pooled fractions containing the
45-kDa protein and exhibiting plasmin(ogen) binding activity were
dialyzed against the starting buffer and re-chromatographed on the Mono
Q column using the same conditions. The positive fractions were again
pooled and concentrated to a volume of <1.0 ml, using Centriprep-30
and Centricon-30 concentrators (Amicon). The concentrated sample was applied to a Superose-12 FPLC column (Amersham Pharmacia Biotech) pre-equilibrated with 50 mM Tris/HCl, pH 8.0. Fractions
containing both the 45-kDa protein and plasmin(ogen) binding activity
were pooled. These fractions were then concentrated, mixed with an equal volume of 4 M
(NH4)2SO4, and applied to a Poros
BU/M hydrophobic column (Perspective Biosystems, Cambridge, MA)
pre-equilibrated with 50 mM Tris/HCl buffer, pH 8.0, containing 2 M
(NH4)2SO4. The proteins were eluted
with a 20-ml decreasing linear gradient of
(NH4)2SO4 from 2.0 to 0.0 M. The 45-kDa protein was eluted in one fraction at 1.32 M (NH4)2SO4. The eluted
protein was then dialyzed and stored at a concentration of 250 µg/ml
at
70 °C until further use. Protein concentration was determined
by the BCA (bicinchoninic acid) method (Pierce).
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N-Terminal Sequencing and Peptide Mapping-- N-terminal amino acid sequence of the purified 45-kDa protein was determined as described (4, 23). Briefly, the purified 45-kDa protein was resolved by SDS-PAGE and electroblotted onto a PVDF membrane. The protein was visualized by staining with 0.1% Ponceau S (Sigma) in 1% acetic acid. Plasminogen and plasmin binding activity was confirmed by autoradiography. The section of the membrane containing the protein band of interest was excised, destained with double distilled water, and subjected to automated Edman degradation. Each sample contained approximately 5 µg of the protein as determined by the BCA protein estimation method (Pierce). A duplicate sample of PVDF membrane was digested with lysine-specific endopeptidase (Lys-C, sequencing grade, Boehringer Mannheim), and the resulting peptide fragments were separated by capillary electrophoresis interphased with the matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Perspective Biosystems). N-terminal sequences of the two internal peptide fragments were then determined as described above. All microsequence analyses were performed at the Protein/Biotechnology Facility of the Rockefeller University.
-Enolase Activity and Enzyme Kinetics--
The strong
N-terminal sequence homology of the 45-kDa protein with
-enolase
prompted us to investigate whether this protein is enzymatically
active.
-Enolase activity was measured essentially as described
earlier by both the coupled assay (24) and the direct assay at
A240 (25).
-Enolase Activity of Intact Streptococci--
To determine
whether the 45-kDa protein is functionally active as an
-enolase on
the streptococcal surface, an overnight culture of group A streptococci
(D471) was washed (3 ×) with 100 mM HEPES/NaOH buffer, pH
7.0, and different concentrations of streptococci were incubated with
and without 3 mM 2-PGE in 100 mM HEPES buffer,
pH 7.0, containing 10 mM MgCl2 and 7.7 mM KCl as described above. The reaction was allowed to
occur in a final volume of 1.0 ml for a period of 3 min at room
temperature, after which the bacteria were removed by centrifugation
(4000 × g for 10 min). The supernatants were analyzed
by measuring absorbance at 240 nm as described above. For the remaining
portion of the "Experimental Procedures," the 45-kDa protein will
be referred to as SEN (surface enolase).
Production and Purification of Rabbit Polyclonal Antisera Against SEN-- Polyclonal antibodies to SEN were prepared in New Zealand White rabbits immunized subcutaneously with 150 µg of purified SEN emulsified in complete Freund's adjuvant (1:1) at multiple sites. Rabbits were boosted twice, each time with 150 µg of the purified protein in incomplete Freund's adjuvant (1:1) at 3-week intervals. The rabbits were bled 10 days after the third immunization. All sera were filter-sterilized and stored at 4 °C.
To prepare SEN-specific IgG, the polyclonal serum was subjected to sequential purification on protein-A Sepharose CL-4B (Amersham Pharmacia Biotech) and SEN affinity columns. The affinity column was made by covalently linking approximately 2 mg of purified SEN to 0.5 g of affinity matrix (Ultralink 3M-Carboxy beads, Pierce) with 200 µl of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide-HCl (Pierce, 120 mg mlProduction and Purification of Mouse Monoclonal Antibodies Against SEN-- BALB/c × SJL-F1 mice were subcutaneously immunized with 30 µg of purified SEN in complete Freund's adjuvant (1:1 v/v). After 3 weeks, mice were bled and tested for antibodies to SEN by enzyme-linked immunosorbent assay and Western blot analysis using a crude cell wall extract of group A M6 strain D471. Affinity purified rabbit polyclonal antibodies against SEN were used as a positive control. Mice with high antibody titers were given a second dose of antigen intraperitoneally in distilled water. Mouse spleens were excised 3-3.5 days after the last booster. The spleen cell fusion to P3-NS1/1AG4-1(NS-1) myeloma cells was performed as described (27, 28). Hybridomas cloned by limiting dilution were grown in 2-liter rolling tissue culture flasks. From these cultures, secreted monoclonal antibodies were precipitated at 50% ammonium sulfate saturation. The precipitates were then dialyzed and purified using a protein A-Sepharose affinity column.
Location of SEN in Streptococci--
To determine the location
of SEN in streptococcal cells, an overnight culture of strain D471 was
subjected to lysine digestion in 30% raffinose buffer to extract
streptococcal cell wall-associated proteins as described above. From
the resulting protoplasts, the membrane and cytoplasmic fractions were
separated as described previously (5, 22). Proteins from each cellular
fraction were resolved by SDS-PAGE and electroblotted onto a PVDF
membrane. The presence of SEN in different cell fractions was monitored by affinity purified anti-SEN polyclonal (75.0 ng ml1)
and monoclonal antibodies (12 ng ml
1) as described (4,
5).
Immune Electron Microscopy-- Group A streptococci (D471) from the overnight TH broth cultures were harvested, washed, and adjusted to a concentration of 109 cfu/ml. An aliquot of 200 µl of the bacterial suspension was incubated with 4 µg of affinity purified anti-SEN(1A10) or anti-SDH (4F12) monoclonal antibodies for 4 h followed by a 2-h incubation with colloidal gold (5- and 10-nm sized beads for anti-SEN and anti-SDH labeled bacteria, respectively) anti-mouse IgG (Amersham Pharmacia Biotech, 1:25) at room temperature. The labeled bacteria were then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 4 h at 4 °C. The fixed labeled bacteria were then processed for transmission electron microscopy as described (29).
Prevalence of SEN in Various Group A Streptococcal M Types and
Streptococcal Groups--
Proteins from the cell wall extract of
several M serotypes after lysin digestion and those of various
streptococcal grouping strains (group A-H, L, and N) after mutanolysin
digestion were resolved by SDS-PAGE and transferred to a PVDF membrane.
The blots were blocked, probed with affinity purified anti-SEN rabbit
polyclonal antibodies (75.0 ng ml1) for 3-4 h, and the
reactive protein bands were visualized as described (4, 5).
Location and Nature of Plasmin(ogen) Binding Domain of SEN-- Purified SEN (10 µg) was treated with carboxypeptidase Y (Boehringer Mannheim) at a substrate to enzyme concentration ratio of 13.5:1 in 50 mM HEPES buffer, pH 7.0, at 37 °C for 6 h. Equal amounts of the enzyme-treated and untreated SEN were resolved by SDS-PAGE, and their plasminogen binding activity was determined by the blot overlay method as described above. In another set of similar experiments, plasmin(ogen) binding activity was measured in the presence of 0.1 M EACA or 0.1 M lysine.
Ligand Binding Assays for Plasmin(ogen) Binding to Immobilized
SEN--
The ligand binding analysis was carried out using 96-well
microtiter plates (C8 Maxi Break-apart, Nalge Nunc International, Naperville, IL). The plates were coated with 100 µl of purified SEN
in 0.05 M carbonate buffer, pH 9.6 (5 µg
ml1), for 3 h at 37 °C and were then kept at
4 °C overnight. The plates were washed and blocked with HBST gel
blocking buffer for 4 h at room temperature. A serial 2-fold
dilution of aprotinin and PMSF-treated 125I-plasminogen
(3.8 pmol) or 125I-plasmin (2.2 pmol) in a final volume of
100 µl of HBST gel buffer containing 2 mM PMSF was added
to the SEN-coated wells and incubated for 4 h at room temperature
on a shaker. The plates were then washed three times with HBST gel
buffer, and individual wells were counted in a (
-counter for
quantitative analysis of the bound plasmin(ogen). Each dilution was
tested in triplicate wells. Nonspecific binding was measured after the
addition of a 200 molar excess of unlabeled plasminogen/plasmin or 0.1 M EACA. Nonspecific binding was also evaluated in
BSA-coated plates. Nonspecific binding contributed between 4 and 10%
of the total counts without these agents. Specific binding was
calculated by subtracting nonspecific binding (10%) from the total
binding. The amount of free plasmin(ogen) was calculated by subtracting
specifically bound plasmin(ogen) from the total amount of labeled
plasmin(ogen) added. To determine the equilibrium dissociation constant
(KD), a nonlinear least square analysis of the total
count offered versus the count bound was carried out using
the curve fitting computer program from Sigma Plot. The values of bound
plasmin(ogen) versus bound/free ratio were plotted, and the
slope representing
1/KD was determined by linear
regression analysis using the formula of Scatchard (30). The specific
binding of plasmin(ogen) to SEN was also determined in a competition
assay in which a constant amount of 125I-plasminogen (0.77 pmol) and 125I-plasmin (1.37 pmol) was mixed with
decreasing concentrations of free plasmin(ogen) (5 ng to 50 µg,
i.e. up to 380 pM excess). The amount of
plasmin(ogen) bound to immobilized SEN in wells, in the absence of any
competitor, was treated as the maximum binding value. Percentage of the
maximum binding at different concentrations of the competitors was
plotted.
Cross-linking Studies-- Sulfosuccinimidyl-2-[p-azido-salicylamido]ethyl-1-3'-dithiopropionate (SASO) (Pierce, 300 µg) was iodinated (0.5 mCi 125I-Na) with IODO-GEN (100 µg), and conjugation of [azido-salicylamido]ethyl-1-3'-dithiopropionate to plasmin(ogen) (400 µg) was carried out in the dark essentially as described before (31) with minor modifications. 125I-labeled plasminogen-ASD was purified on a PD-10 column as described above. Cross-linking of 125-labeled plasminogen-ASD with intact group A streptococci was performed as follows. Overnight TH broth culture of streptococci (D471) was centrifuged, washed once with phosphate-buffered saline, and resuspended to a concentration of 2 × 1010 cfu/ml. An aliquot of (100 µl) of this streptococcal suspension was incubated with 125I-labeled plasminogen-ASD (3 × 105 cpm) in the dark under constant rotation at 37 °C for 1 h in a final volume of 150 µl. The reaction mixtures were then irradiated for an additional 30 min with UV350 nm light. The labeled bacteria were then resuspended in 50 mM phosphate buffer containing 30% raffinose, digested with mutanolysin, and further fractionated into cell walls, cytoplasm, and membranes as described (4, 5). Samples were then analyzed by SDS-PAGE under reducing conditions, followed by autoradiography.
Plasminogen Binding Activity of Intact
Streptococci--
Streptococci (5 × 109 cfu/ml, 50 µl/well) were fixed in a 96-well poly-L-lysine-coated
microtiter plate with 0.2% glutaraldehyde for 1 h at room
temperature. Unoccupied sites were blocked by 0.1 M lysine.
The plates containing streptococci were then treated overnight at
4 °C with 2% BSA in HBST gel buffer. To determine the role of the
C-terminal lysine residue in plasminogen binding, some of wells with
fixed streptococci were treated with carboxypeptidase B (5 µg/well,
Boehringer Mannheim) for a period of 4 h at 37 °C on a
microtiter plate shaker. Dose-dependent plasminogen binding activities of intact and carboxypeptidase B-treated streptococci were
measured using serial 2-fold dilutions of 125I-plasminogen
in a final volume of 100 µl of HBST gel buffer, as described above
for plasminogen binding activity of purified SEN. The plates were then
washed three times with HBST gel buffer, and individual wells were
counted in a -counter for a quantitative analysis of the bound
plasmin(ogen). Each dilution was tested in triplicate.
In Vitro Proteolytic Activity of Plasmin Bound to
SEN--
2-Antiplasmin is a fast-acting plasmin
inhibitor of plasma (32). Thus, the proteolytic activity of plasmin was
evaluated in terms of its inhibition in the presence of
2-antiplasmin when bound to various substrates. To
determine the proteolytic activity of plasmin bound to SEN, purified
SEN was immobilized onto Ultralink 3M-Carboxy beads (Pierce), using a
method similar to that as described above for the preparation of
monospecific anti-SEN antibodies. Ultralink beads containing 1 µg of
SEN were mixed with 125I-labeled plasmin (0.375 µg) or
plasminogen (0.39 µg) for 4 h in a final volume of 100 µl.
Unbound plasmin(ogen) was then removed by three washes with HBST gel
buffer. The amount of bound plasmin(ogen) was determined on the basis
of radioactive counts. The proteolytic activity of bound plasmin was
determined by measuring the cleavage of the chromogenic substrate
Val-Leu-Lys-para-nitroanilide (Sigma, 23.8 µg, 3.4 mg
ml
1) in a final reaction volume of 200 µl of HBST gel.
The change in absorbance at 405 nm was determined
spectrophotometrically (MR 4000, Dynatech Laboratories, Inc.,
Chantilly, VA). The inhibitory effect of
2-antiplasmin
on equivalent amounts of SEN-bound and free plasmin was determined by
measuring the cleavage activity on the chromogenic substrate.
Similarly, SEN-bound and free plasminogen were activated with either
tPA (600 units) or streptokinase (100 units), and the subsequent
proteolytic activity of the generated plasmin was measured in the
presence and absence of
2-antiplasmin as described
above. Blanks were run with buffer containing only substrate.
Opsonophagocytosis of Group A Streptococci in the Presence of
Anti-SEN Antibodies--
In vitro phagocytosis of group A
type M6 streptococci (strain D471) and heterologous type 14 strain
(T/14/46) by human phagocytes in the presence of polyclonal anti-SEN
antibodies was measured as described (33). Briefly, 0.4 ml of freshly
drawn heparinized blood was mixed with 0.1 ml of appropriately diluted
logarithmic phase culture streptococci (100-300 cfu ml1)
in the presence of different concentrations of anti-SEN antibodies. The
mixtures were incubated at 37 °C for 3 h either with constant slow rotation or under a stationary condition. At the end of the incubation, an aliquot was plated on proteose peptone blood agar and
incubated at 37 °C overnight. Surviving bacteria were determined from the number of
-hemolytic colonies. The experiments carried out
under stationary conditions served as an internal control.
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RESULTS |
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Identification of a Novel 45-kDa Plasmin(ogen) Binding Protein in the Group A Streptococcal Cell Wall Extracts-- By Western blot, we examined the plasmin(ogen) binding activity of proteins in a crude streptococcal cell wall extract using 125I-labeled plasmin and plasminogen (Fig. 1A). The results showed that, in addition to the weak plasminogen binding to the 39-kDa SDH molecule (4), a significantly stronger plasminogen binding occurred with a 45-kDa protein present in the streptococcal cell wall extract. We also found similar binding activities of SDH and the 45-kDa protein with 125I-labeled plasmin (Fig. 1B). These findings identify a new protein with strong plasmin(ogen) binding activity in the cell wall extract and confirm our previous report of the weak plasmin(ogen) binding activity of SDH (4).
Purification of the 45-kDa Protein-- The 45-kDa protein was partially purified from the cell wall extract by 40-60% ammonium sulfate precipitation. The protein was further purified by ion-exchange chromatography on a Mono Q column followed by a Superose-12 molecular sieve. With the latter, the peak elution volume having plasmin(ogen) binding activity corresponded to that of a standard 150-kDa IgG molecule, suggesting that the native form of the 45-kDa protein is likely a multimer. Final purification was achieved on a Poros BU/M hydrophobic column (Fig. 2A). The average yield of purified 45-kDa protein from a total of 10 liters (1.5 × 108 cfu/ml) of bacterial culture was 1.128 mg.
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N-terminal Amino Acid Sequence and Identification of the 45-kDa
Protein as an -Enolase Enzyme--
N-terminal amino acid sequence
of the 45-kDa protein revealed a single amino acid in the first 50 residues (Fig. 2B). N-terminal sequences of two internal
peptides (Pep-1, molecular mass 1712.1 Da; and
Pep-2, molecular mass 1683.5 Da) obtained after cleavage with a lysine-specific endopeptidase were also determined for 15 and 11 residues, respectively. The presence of a single amino acid at each
sequence cycle for the intact 45-kDa protein and each internal peptide
verified the homogeneity of these molecules.
-Enolase Activity and Enzyme Kinetics--
By establishing that
the sequence of the 45-kDa protein was that of
-enolase, we
investigated whether it also possessed the activity of this glycolytic
enzyme. In a coupled-enzyme assay, the 45-kDa protein converted
terminal NADH to NAD in a dose-dependent manner (Fig.
3A). This indicated the
conversion of pyruvate to lactate by lactate dehydrogenase and NADH,
confirming the conversion of phosphoglycerate to phosphoenolpyruvate by
-enolase and further to pyruvate in a sequential manner in the
presence of externally furnished pyruvate kinase and ADP. A similar
dose-dependent conversion of 2-phosphoglycerate to
phosphoenolpyruvate was exhibited by the 45-kDa protein in a direct
enzyme assay (not shown) also confirming that it is an enzymatically
active
-enolase. The latter assay was used to determine the kinetic
properties of the 45-kDa protein.
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-Enolase Activity of Intact Streptococci--
To determine
whether the
-enolase activity of the 45-kDa protein is in fact
displayed on the streptococcal surface, enzymatic activity was carried
out using intact group A streptococci by the single enzyme assay method
in the presence of 3 mM 2-phosphoglycerate. The results
shown in Fig. 3D revealed a dose-dependent
-enolase activity catalyzed by the intact streptococci. In the
absence of 2-phosphoglycerate, intact streptococci did not catalyze any detectable enzyme reaction, further suggesting that the 45-kDa protein
is expressed on the surface. In addition, to rule out the possibility
that this enzymatic activity was not due to cell lysis-related release
of enzyme, we assayed for the presence of the cytoplasmic enzymes
lactate dehydrogenase and pyruvate kinase and found no activity (not
shown). Based on these results, the 45-kDa protein is hereafter
referred to as SEN.
Subcellular Location and Prevalence of SEN in Other M Types and Streptococcal Groups-- By using affinity purified rabbit anti-SEN antibodies and monoclonal antibodies, we examined the distribution of SEN in various subcellular fractions of group A streptococci by Western blot analysis. The results showed that SEN is found in both the cell wall and cytoplasmic fractions, with negligible amounts in the membrane fraction (Fig. 4 A). Furthermore, SEN was found to be present in comparable quantities in all M types examined and in all streptococcal groups except group N (Fig. 4B). The uniform antibody reaction that showed no obvious size heterogeneity among the SENs in different M types indicates that SEN is a conserved protein in all streptococci tested.
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Plasmin(ogen) Binding Activity of SEN and Its Comparison with That of SDH-- To compare the plasmin(ogen) binding activities of purified SEN and SDH, equal quantities of the purified proteins were separately resolved by SDS-PAGE and electro-blotted onto PVDF membranes. The blots were then probed with either 125I-plasminogen, 125I-plasmin, or 125I-plasmin derived from 125I-plasminogen by urokinase. The results showed that plasmin(ogen) bound weakly to SDH compared with SEN (Fig. 5A). The results are in agreement with our previous results (4) and with those shown in Fig. 1. Furthermore, SEN consistently showed significantly higher binding affinity for plasminogen than plasmin (Fig. 5A).
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Role of SEN in Plasminogen Binding Activity of Streptococci-- In addition to enzymatic and biochemical properties suggesting that SDH (4) and SEN are located on the streptococcal surface (Figs. 2-4) and to provide additional proof of their presence on the streptococcal surface, SEN- and SDH-specific mouse monoclonal antibodies (1A10 and 4F12, respectively) were used in indirect immune electron microscopy. As shown in Fig. 6A, both SEN and SDH molecules, reacting with their specific monoclonal antibodies (4 µg/106 streptococci), were found on the surface of streptococci. Their binding patterns suggest that the distribution of these proteins is either in the form of a cluster or the epitopes recognized by specific monoclonal antibodies are not uniformly exposed on the cell surface. The latter argument is supported by the fact that even at higher concentrations of both monoclonal antibodies (up to 20 µg of IgG/106 streptococci), of the distribution of gold particles was the same as that seen with lower concentrations of monoclonals (data not shown).
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Protease Activity of Plasmin and Activated Plasminogen Bound to
Purified SEN--
The proteolytic activity of free and SEN-bound
plasmin was determined by measuring their ability to cleave the
chromogenic substrate Val-Leu-Lys-para-nitroanilide in the
presence and absence of 2-antiplasmin, a fast acting
plasmin inhibitor. The results show that plasmin, either bound to SEN
or free in solution, exhibits the same proteolytic activity (Fig.
7). However, SEN-bound plasmin was not
inactivated as rapidly in the presence of
2-antiplasmin as free plasmin. After 4 h, SEN-bound
plasmin still retained activity whereas free plasmin was nearly
completely inactivated (Fig. 7).
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Opsonophagocytosis of Group A Streptococci in the Presence of anti-SEN Antibodies-- To understand the biological role of SEN as an important streptococcal surface protein, we determined whether antibodies to SEN were able to opsonize these organisms in vitro. The opsonic activity of anti-SEN antibodies was measured in terms of the ability of streptococci to survive in blood from a nonimmune individual who lacks type-specific anti-M antibodies against the test strain. The results, as shown in Fig. 8, A and B, revealed that affinity purified anti-SEN IgG antibodies effectively opsonized and enhanced the phagocytosis of group A streptococci of two different serotypes (types 6 and 14). These results not only confirm the surface location of SEN on streptococci but also suggest that anti-SEN IgG antibodies may foster non-type-specific protection against streptococcal infection.
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DISCUSSION |
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The plasmin(ogen) binding property of pathogenic bacteria in
general, and of streptococci in particular, is suggested to be one of
the characteristics that may contribute to tissue invasion and the
overall pathogenicity of group A streptococci (13, 34). Plasminogen
activation is responsible for the degradation of intravascular clots
and extracellular proteolysis in a wide variety of physiological and
pathological processes (7-9). In this report, we identify and
characterize a novel plasmin(ogen)-binding protein, SEN, on the surface
of group A streptococci. Structurally and functionally this protein is
an -enolase, one of the key glycolytic enzymes, and is the second
glycolytic enzyme that we have identified on the surface of group A
streptococci. We reported earlier that streptococcal surface
dehydrogenase (SDH), a major protein on the surface of these organisms,
is structurally and functionally related to the glycolytic enzyme
glyceraldehyde-3-phosphate dehydrogenase and is able to bind plasmin
weakly (4). On the basis of latter activity, another group has found a
structurally and functionally similar protein, Plr (plasmin receptor),
from a clinical group A streptococcal strain (6). The identification of
this 45-kDa plasminogen-binding protein as
-enolase was based on its
strong sequence identity, both at its N-terminal end and within
two different internal peptides to other reported
-enolases.
We have identified and isolated SEN from the M6 streptococcal strain
from which SDH was originally identified and purified (4).
Functional identity of SEN in its purified form, as well as on the
streptococcal surface, was confirmed by its ability to catalyze the
conversion of 2-PGE to PEP in both direct and coupled enzyme assays.
The values for the enzyme kinetic constants, Km and
Vmax, for purified SEN (Fig. 3) are comparable
with those of other reported -enolase enzymes (35, 36). Furthermore, both monospecific polyclonal and monoclonal antibodies against SEN were
found to be important tools for its cellular location in group A
streptococci and its presence on the surface of other streptococci
(Fig. 4).
From this and several other reports (6, 14, 15), it is apparent that
group A streptococci express more than one type of plasmin(ogen)
binding receptor which acquires plasmin(ogen) through various
mechanisms (34). Clinical isolates and animal-passaged strains have
more plasmin(ogen) binding capacity then the same strains passaged in
the laboratory (37). Thus, group A streptococci expressing several low
affinity plasmin(ogen) binding molecules may bind the equivalent amount
of plasmin(ogen) as strains that express fewer high affinity
molecule(s). In addition to strain specificity, M type specificity has
also been reported for the plasmin(ogen)-binding protein, PAM, in group
A streptococci (15). Although not characterized, at least two types of
plasmin(ogen) receptors (low and high affinity) have been reported in
group G streptococci (38), a characteristic that may well be found on
other streptococcal groups. We report here that in group A streptococci, surface glyceraldehyde-3-phosphate dehydrogenase (SDH/Plr) is a weak plasminogen-binding protein and that SEN is a
strong plasminogen-binding protein (Fig. 5). By using anti-SEN antibodies, we have identified -enolase-like molecules on the surface of both encapsulated and unencapsulated strains of
Streptococcus pneumoniae, but not on
staphylococci,2 suggesting that surface
-enolase
(plasmin(ogen) binding) may be an important virulence determinant for
pathogens of the respiratory mucosa.
-Enolase is one of the key glycolytic enzymes found generally in the
cytoplasm; nevertheless, its presence on the surface of cells is not
without precedent. Several eukaryotic studies have provided evidence
that
-enolase-related molecules are expressed on the surface of
several cell lines such as U937 human monocytoid (19), human breast
tumor (17), peripheral blood monocytes, and neutrophils (18) and that
these molecules contribute about 10% of the plasminogen-binding
capacity of the cells. Recently,
-enolase has also been shown to be
present as an abundant immunodominant antigen in the cell wall of
Candida albicans (39, 40). In prokaryotes, however, the
presence of cell-surface
-enolase has not been previously reported.
Our present findings on the plasmin(ogen) binding activity of SEN (Fig.
5) differ from those of the reported eukaryotic plasminogen binding
-enolases (18) in two major respects. (i) SEN exhibits significantly
higher affinity for plasmin(ogen) (KD = 1-4
nM, Fig. 5D) as compared with that of eukaryotic enolase (KD = 0.1-2 µM). (ii) In
contrast to eukaryotic enolase, SEN exhibited more than one interaction
site for plasminogen and plasmin. Although plasminogen and plasmin
showed comparable binding affinity to SEN in a solid phase assay, the
latter consistently bound less efficiently on Western blots. We
speculate that in addition to the C-terminal lysine binding site of the
SEN molecule for plasmin(ogen), the region upstream of this site may
also be responsible for the binding. This is supported by the fact that plasmin(ogen)-binding proteins that do not possess lysine residues at
their C-terminal ends also show appreciable affinity for plasmin(ogen), possibly through other exposed lysine residue(s) (38, 41).
The high affinity of intact group A streptococci for plasmin(ogen) (KD = 0.2 nM (37)) has recently been attributed to the Plr protein, a member of GAPDH family (6); however, this activity may actually be due to the combined activity exhibited by several such binding proteins on the streptococcal surface. Recently, glyceraldehyde-3-phosphate dehydrogenase isolated and purified from Streptococcus equisimilis has been shown to have an equilibrium constant in the range of 220-260 nM for plasminogen and about 25 nM for plasmin (41). We reported earlier that SDH, also a member of GAPDH family, is a weak plasmin(ogen)-binding protein (4). In view of these reports and of other published reports showing low affinity plasminogen-binding proteins of group A streptococci (14, 15, 38), SEN may be the major plasmin(ogen)-binding protein on the surface of group A streptococci. Based on its ubiquitous presence on the surface of a variety of group A streptococcal serotypes and streptococcal groups (Fig. 4), we suggest that SEN, or an SEN-like molecule, may serve as a major plasmin(ogen)-binding molecule/receptor on the surface of nearly all pathogenic streptococci and would therefore play an important role in disease outcome.
Earlier reports that the Plr molecule (6, 42, 43) or other
plasminogen-binding proteins (15, 41) are the streptococcal plasminogen-binding receptors, are based on the plasminogen binding activity of the purified natural or recombinant proteins using different methods. It is not clear, however, if Plr (6) or SDH (4) is
in fact the streptococcal plasminogen-binding receptor and, if so,
whether this binding activity represents the observed plasminogen
binding activity exhibited by intact streptococci. Hence, in the
present communication we investigated whether plasminogen binding
activities of SEN and SDH are relevant to the observed streptococcal
plasminogen binding activity. We first confirmed their surface location
using immune electron microscopy with SEN- and SDH-specific monoclonal
antibodies (Fig. 6A). Furthermore, we found a significant
reduction in plasminogen binding activity of carboxypeptidase B-treated
intact streptococci indicating that the structural domain of SEN and/or
SDH which contains the C-terminal lysine residue is exposed to the
surface. In conjunction with these results, we found that the
plasminogen binding activity of the surface-bound SEN, but not that of
cytoplasmic SEN, was reduced after treatment with carboxypeptidase B,
suggesting that SEN was accessible to this enzyme. The observation that
the enzyme-treated streptococci also showed significant plasminogen
binding activity raises the possibility that the region which is
N-terminal to the C-terminal lysine residues of SEN (and also that of
SDH) may also play a role in plasminogen binding activity. From the
cross-linking studies that were designed to determine whether SEN, SDH,
or both the proteins play a role in streptococcal plasminogen binding, it was possible to determine that SEN is probably more exposed to the
surface as compared with SDH, since the labeled ASD from the
125I-labeled plasminogen-ASD complex was found to
cross-link to SEN rather than to SDH. A dose-dependent
inhibition of the streptococcal plasminogen binding activity, even in
the presence of low concentrations of free SEN, further confirmed that
the high affinity of SEN for plasminogen inhibits the ability of the
bacteria to bind to plasminogen. These results together indicate that
SEN serves as a primary receptor for plasminogen binding. The question
of how glyceraldehyde-3-phosphate dehydrogenase (SDH/Plr) (4, 6) and
-enolase (SEN) are transported through the cell membrane and sorted
onto the cell surface without the presence of a signal sequence remains
intriguing. Whether SEN, like eukaryotic surface
-enolases, is
transported by internal signal sequences like that found with
plasminogen activator inhibitor 2 (44) or by the post-translational
acylation method (45) needs further investigation. It is possible that
SEN and SDH are transported to the cell surface as a complex in
conjunction with a secreted protein utilizing a specialized secretory
system. What is clear, however, is that SEN and SDH, are members of the
growing group of proteins which lack signal sequences but are
transported to the surface and anchored to cells by an as yet undefined
mechanism (44).
Like GAPDH/SDH (4, 46), eukaryotic -enolase has also been shown to
be a multifunctional protein presenting a variety of activities besides
its native glycolytic activity. Functions such as being a structural
component of turtle lens, J-crystallin (47), a neurotropic factor (48),
the ability to form a stable complex with Clostridium
difficile toxin B (49), and the ability to bind to polynucleotides
(50) have been recently reported for
-enolase. If SEN, like
eukaryotic
-enolases (47-50) and SDH (4, 5), is also a
multifunctional molecule, its physiological implications are at present
equivocal.
Our finding that plasmin bound to SEN retains its proteolytic activity
even in the presence of 2-antiplasmin indicates that SEN
may be an important streptococcal virulence determinant. Particularly in infected tissues, such a characteristic would enable the
streptococcus to become more invasive by evading localization by the
host's clotting pathway (34). The fact that
-enolase has recently been found to be secreted in the growth medium (39, 51) and that
increased levels of fungal-specific
-enolase have been found in
patients with invasive candidiasis (52) suggest that a similar phenomenon may exist in cases of invasive streptococcal infection. Understanding the properties of this molecule during growth and infection may prove useful in sorting out the complex pathogenic properties displayed by group A streptococci.
-Enolase present in the cell wall of C. albicans (40) has
been designated as an abundant immunodominant antigen in cases of
invasive candidiasis (39, 51). The fact that antibodies to SEN enhance
the phagocytosis of group A streptococci of heterologous M types (Fig.
6) lends further support to the surface location of SEN and indicates
that antibodies to surface molecules other than M protein are opsonic
for group A streptococci, although M-specific antibodies are more
effective (33). It is likely that the immune response to streptococcal
-enolase may also play an important role in the final outcome of a
streptococcal infection in view of the fact that
-enolase is also
present in several hematopoietic cells as a surface-expressed molecule
(18). However, whether such an immune response does occur in humans
during streptococcal infection is presently unknown.
The presence of -enolase on the surface of streptococci and also on
the surface of a variety of mammalian tissues including brain
(neuron-specific enolase) (8, 18) adds new insight in the role of
SEN-specific antibodies in post-streptococcal autoimmune diseases such
as glomerulonephritis and neurological disorders such as Sydenham's
chorea, a major manifestation of rheumatic fever (53). Since
anti-enolase-specific antibodies have been reported in systemic
rheumatic diseases (54), and autoimmune polyglandular syndrome (55),
the latter as a result of C. albicans-specific enolase, as
well as cell-mediated immunity to enolase in schizophrenia (56), a
possible role of SEN in post-streptococcal autoimmune diseases cannot
be ruled out.
On the basis of biochemical and biological properties, SEN together with SDH/Plr (4, 6) develop an emerging theme for a new class of bacterial surface proteins. In vitro cross-protective nature of the anti-SEN antibodies, coupled with the ubiquity of SEN on the streptococcal surface, may prove useful for an immunotherapeutic intervention against streptococcal diseases. In addition to this, its potential role in autoimmune disease suggests that SEN is an important biologically active molecule with a substantial role in streptococcal pathogenesis.
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ACKNOWLEDGEMENT |
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We thank Patricia A. Ryan for editorial help in this manuscript and Clara Eastby for technical assistance in the production of monoclonal antibodies.
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FOOTNOTES |
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* This work was supported by USPHS Grant AI11822 from the National Institutes of Health and in part by a grant from SIGA Pharmaceuticals.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8165; Fax: 212-327-7584; E-mail: panchov{at}rockvax.rockefeller.edu
1
The abbreviations used are: SDH, streptococcal
surface dehydrogenase; EACA, -aminocaproic acid; MES,
2-[N-morpholino]ethanesulfonic acid; Plr, plasmin
receptor; plasmin(ogen), plasminogen and plasmin; SEN, streptococcal
enolase; PAGE, polyacrylamide gel electrophoresis; PEP,
phosphoenolpyruvate; 2-PGE, 2-phosphoglycerate; PVDF, polyvinylidene difluoride; PMSF, phenylmethylsulfonyl fluoride; tPA, tissue-type plasminogen activator; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BSA, bovine serum albumin; cfu, colony-forming units; ASD,
azido-salicylamido]ethyl-1-3'-dithiopropionate.
2 V. Pancholi and V. A. Fischetti, unpublished data.
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REFERENCES |
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