Bacterial surface-associated plasmin formation is
believed to contribute to invasion, although the underlying molecular
mechanisms are poorly understood. To define the components necessary
for plasmin generation on group A streptococci we used strain AP53 which exposes an M-like protein ("PAM") that contains a
plasminogen-binding sequence with two 13-amino acid residues long
tandem repeats (a1 and a2). Utilizing an Escherichia
coli-streptococcal shuttle vector, we replaced a 29-residue long
sequence segment of Arp4, an M-like protein that does not bind
plasminogen, with a single (a1) or the combined a1a2 repeats of PAM.
When expressed in E. coli, the purified chimeric Arp/PAM
proteins both bound plasminogen, as well as plasmin, and when used to
transform group A streptococcal strains lacking the plasminogen-binding
ability, transformants with the Arp/PAM constructs efficiently bound
plasminogen. Moreover, when grown in the presence of plasminogen, both
Arp/PAM- and PAM-expressing streptococci acquired surface-bound
plasmin. In contrast, plasminogen activation failed to occur on PAM-
and Arp/PAM-expressing streptococci carrying an inactivated
streptokinase gene: this block was overcome by exogenous streptokinase.
Together, these results provide evidence for an unusual co-operation
between a surface-bound protein, PAM, and a secreted protein,
streptokinase, resulting in bacterial acquisition of a host protease
that is likely to spur parasite invasion of host tissues.
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INTRODUCTION |
Plasminogen is a single chain 92-kDa glycoprotein present in
plasma (~200 mg/liter) and in extracellular fluids. The protein contains five characteristic kringle domains that mediate interactions with multiple ligands, such as fibrin(ogen),
2-antiplasmin
(
2AP),1 and
cellular plasminogen receptors (1-3). In vivo, two specific activators, the tissue-type plasminogen activator and urokinase, convert the single-chain zymogen to a two-chain enzyme, plasmin, by
cleavage at a single site (4). Plasmin efficiently degrades fibrin and
probably also participates in the breakdown of soft tissue
glycoproteins (5). The plasma concentration of active plasmin is
normally low, due to its rapid inactivation by proteinase inhibitors,
primarily
2AP. Under pathophysiological conditions some
pathogens, including group A, C, and G streptococci and
Staphylococcus aureus, secrete highly specific and potent
plasminogen activators such as streptokinase and staphylokinase,
respectively (1, 2). Other bacteria, including Escherichia
coli and Yersinia pestis, express surface-bound
plasminogen activators (6, 7). Evidence suggest that plasmin generated
by bacterial plasminogen activators contributes to virulence, possibly
by facilitating bacterial penetration through host barriers (7).
It has been known for some time that many virulent bacterial species
express surface proteins capable of binding plasmin(ogen) without
causing its activation (8). Some of these can also contribute to
bacterial dissemination (9). For Streptococcus pyogenes
(group A streptococci), a major causative agent of both mucosal and
skin infection in humans, four major types of surface proteins have
been forwarded as candidate plasmin(ogen)-binding proteins. Of these
the dehydrogenase-like Plr protein (10-12) binds primarily plasmin,
whereas a 45-kDa protein (13) also binds plasminogen. Some members of
the M protein family, a well known class of surface-exposed
streptococcal virulence determinants (14), have been shown to capture
plasmin(ogen) indirectly, through fibrinogen (15, 16). Finally, a set
of group A streptococcal strains, associated with skin infection
(17-19), expose M-like proteins that bind plasminogen as well as
plasmin, directly and with high affinity (20, 21). For one of these
proteins, PAM (plasminogen-binding group A
streptococcal M-like protein) from type 53 group A
streptococci, a plasminogen-binding sequence characterized by two
13-amino acid residues long repeats (a1 and a2), has been identified in
its surface-exposed variable NH2-terminal domain. Similar
motifs are also present in M-like proteins from the other "skin"
strains (21).
Here we have set out to study the molecular requirements for the
binding and activation of plasminogen by PAM-expressing bacteria. We
demonstrate a joint role for the plasminogen-binding motif in PAM and
streptokinase in the acquisition and exposure of plasmin at the surface
of group A streptococci.
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MATERIALS AND METHODS |
Bacterial Strains and Culture Conditions--
Streptococcal
strains, their M or M-like proteins and known ligands for these
proteins are listed in Table I. AP4, a
type M4 group A streptococcal strain expressing Arp4 (22) and the PAM-expressing type 53 group A streptococcal strain AP53 were obtained
from the Institute of Hygiene and Epidemiology, Prague, Czech Republic.
JRS4, an M6 protein-expressing group A streptococcal strain (23, 24),
and JRS145 (25), an M6-deficient derivative of JRS4, were from Dr. June
Scott, Emory University, Atlanta, GA. JRS145(Arp), a JRS145 strain
transformed with the shuttle plasmid pJRS264 (26) that encodes the
arp4 gene of AP4, was a kind gift from Dr. Gunnar Lindahl.
Streptococci were grown in Todd-Hewitt broth (Difco, Detroit, MI), or
in Todd-Hewitt broth supplemented with 30% (v/v) fresh human
heparinated plasma, in 5% CO2 at 37 °C for 16 h.
Streptococci transformed with plasmids carrying resistance markers for
antibiotics were grown in medium supplemented with 15 mg
liter
1, 200 mg liter
1, or 70 mg
liter
1 erythromycin, kanamycin, or spectinomycin,
respectively. The streptococci were harvested by centrifugation, washed
twice in 0.15 M NaCl, 0.03 M phosphate, pH 7.2 (PBS), and were finally resuspended in PBS. Transformed E. coli LE392 was grown in LB medium supplemented with erythromycin
(750 mg liter
1), kanamycin (200 mg liter
1),
or spectinomycin (20 mg liter
1) depending on the
selection marker of the plasmid.
Binding of Radiolabeled Ligands to Streptococci--
Binding of
[125I]-labeled plasminogen, IgA, or fibrinogen to
bacteria was measured in a total volume of 250 µl of PBS containing 0.1% (v/v) Tween 20. Following incubation for 60 min at 20 °C, 2 ml
of PBS containing 0.1% Tween 20 was added, and the bacteria were
centrifuged at 4,000 × g for 5 min. The supernatant
was discarded and the radioactivity associated with the pellet was
measured in a
-counter. The number of plasminogen-binding proteins
expressed on streptococci was determined from experiments in which
125I-labeled plasminogen (0.02 nM) was allowed
to compete with various concentrations of unlabeled plasminogen
(50-0.025 nM) for the binding to streptococci (1 × 109 bacteria ml
1). The number of receptors
was then estimated from the intercept with the abscissa of a curve
generated by plotting bound (abscissa) versus bound/free
(ordinate) plasminogen.
Plasma Absorption Experiments--
Streptococci
(1010 cells) were incubated with 1.0 ml of fresh human
plasma for 60 min at 20 °C. Bacterial cells were pelleted and washed
three times with PBS. To elute absorbed proteins, the bacteria were
incubated with 0.1 M glycine, pH 2.0, for 10 min at
20 °C. The bacteria were removed by centrifugation (4000 × g, 5 min), and the proteins of the supernatant were analyzed
by SDS-PAGE and immunoblotting (see below).
Plasminogen Activation Assays--
Heat-killed streptococci
(1010 cells) were incubated with 1.0 ml of human plasma for
60 min at 20 °C. The bacteria were washed three times with 10 ml of
PBS containing 0.1% Tween 20. The bacteria were resuspended in
blocking buffer containing 100 units of group C streptococcal
streptokinase (Chromogenix, Mölndal, Sweden) and incubated again
for 60 min at 20 °C, followed by centrifugation and two washes with
PBS containing 0.1% Tween 20. The pellet was incubated in 2.5 ml of
PBS containing 0.4 M NaCl and 80 µM of the
chromogenic substrate H-D-Val-Leu-Lys-p-nitroanilide
(S2251; Chromogenix) for 60 min at 37 °C. The bacteria were
pelleted, and the absorbance of the supernatant was measured at 405 nm. Alternatively, 1 × 1010 cells from an overnight
culture were washed three times in PBS, and subsequently added to a
mixture of 2.8 ml of Todd-Hewitt broth and 1.2 ml of plasma (for some
experiments supplemented with 100 units of group C streptococcal
streptokinase). The cultures were incubated at 37 °C in 5%
CO2; at varying time points the cultures were terminated by
centrifugation at 4 °C. The bacterial pellets were washed three
times in ice-cold PBS containing 0.1% Tween 20 and placed on ice.
Plasmin activity associated with the bacteria was measured as described
above.
Construction of Arp/PAM Chimeric Proteins--
The Arp4-encoding
plasmid, pJRS264, contains unique SacI and BstBI
sites at positions 315 and 403 of the arp4 gene. For
construction of the Arp/a1a2 construct, a 110-base pair DNA segment
encoding the a1a2 repeat unit of PAM was generated by PCR using
chromosomal DNA from AP53 streptococci as template and the synthetic
oligonucleotides 5'-GAGGAGCTCAAAGATGATGTTGAGAAGCTTACC-3' and
5'-GCTCCGGAGATCATGTCTCTCGCTTTTAAGTCG-3' as primers, thereby
introducing SacI and HpaII recognition sites, respectively. The 110-base pair DNA fragment was ligated with SacI/BstBI-digested pJRS264 DNA to yield the
Arp/a1a2 construct. The Arp/a1 construct was generated by ligating the
annealed synthetic phosphorylated oligonucleotides
5'-CAAGGTAGATGCTGAGTTGCAACGACTTAAAAACGAGAGACATGAAGGTGAAAATCAAGATCTT-3' and
5'CGAAGATCTTGATTTTCACCTTCATGTCTCTCGTTTTTAAGTCGTTGCAACTCACATCTACCTTGAGCT-3', with SacI/BstBI-digested pJRS264 DNA.
Inactivation of the Streptokinase Gene (ska)--
Chromosomal
DNA from AP53 or JRS4 streptococci was used as templates for PCR to
amplify a 1180-base pair ska fragment with primers SK3
(5'-GACGTC GACACTTGCATCTCTGGAAAATAGTC-3') and SK4 (5'-GACGGATCCATGAGTGACGATTGAGGAGTCAC-3'), or to amplify a 950-base pair
ska fragment with primers SK6
(5'-GACGGATCCTTTCTGAGAAATATTACGTCC-3') and SK7
(5'-GACGAGCTCGGTACCTTTCTATTGATGGGAAAATTGC-3'); the relative locations
of the SK primers in the ska sequence are indicated in Fig.
1C. The 1180- and 950-base pair fragments were digested with
SalI/BamHI or BamHI/SacI,
respectively, and were then ligated with
SalI/SacI-digested pUC18 DNA. Following
transformation and amplification in E. coli, the resultant
plasmid DNA was made linear with BamHI and ligated with the
BamHI-digested kanamycin-resistance cassette
Km-2 (25).
Plasmid DNA was prepared from a kanamycin-resistant transformant and
digested with SalI and SacI. The resulting
SalI/SacI fragment, containing
Km-2 flanked by
ska fragments, was blunted and ligated with the
SalI-digested and blunted plasmid pJRS233. Plasmid pJRS233
contains an erythromycin resistance marker (erm), a
temperature-insensitive E. coli replicon, and a promiscuous replicon that is sensitive to temperatures above 35 °C in both E. coli and S. pyogenes (27). Following
transformation and subsequent propagation in E. coli at
37 °C, the resulting plasmid (pJRS233-kana-ska) was used
to electroporate S. pyogenes. Kanamycin-resistant and erythromycin-sensitive isolates were selected following sequential culturing of the transformed bacteria at 30 and 37 °C, respectively. Integration of
Km-2 into the ska genes of AP53 and
JRS145, respectively, was monitored by PCR: the oligonucleotides SK9
(5'-ACTCATCCAATCTTTATCTCC-3') and SK10 (5'-TGCAACGGGTCAAAGAGGCC-3'),
located on opposite strands immediately outside the ska
fragment defined by SK3 and SK6, were used in combination with the
Km-2-derived outwardly directed primers KM1
(5'-CTCTTCATCCTCTTCGTCTTG-3') and KM2 (5'-ATCCGCAACTGTCC ATACTCT-3')
(Fig. 1C). PCR-positive clones failed to produce active streptokinase as determined by analysis of growth supernatants and
streptococcal whole cell lysates by the S-2251 assay. No difference in
the growth rates was observed between the ska positive and the ska negative streptococcal isolates.
Recombinant DNA Techniques--
Standard ligation and DNA
isolation procedures were employed (28). Ligase, T4 DNA polymerase, and
restriction enzymes were from Promega (Madison, WI). PCR was performed
(29) using Amplitaq (Perkin-Elmer, Wilton, CT). The sequences of the SK
primers were derived from the published S. pyogenes
streptokinase gene sequence (30, 31). Chimeric Arp/PAM genes
were sequenced using the dideoxy nucleotide chain termination method
(32). Electroporation of E. coli and streptococci was
performed as described (25).
Protein Analysis and Preparation of Affinity
Matrices--
Recombinant PAM, Arp4, and Arp/PAM proteins were
purified from whole cell lysates of overnight cultures of E. coli LE392 transformed with the corresponding genes cloned in
pJRS264 or derivatives thereof. PAM and Arp/PAM hybrid proteins were
purified on plasminogen agarose, whereas Arp4 was purified on an
agarose column coupled with human IgA. Proteins bound to the affinity
columns were eluted using 0.1 M glycine-HCl, pH 2.0, and
the effluent was dialyzed against PBS. Plasminogen was purified on a
column with lysine-Sepharose 4B (Pharmacia, Uppsala, Sweden). Proteins
bound to the column were eluted with 0.2 M
-aminocaproic
acid. Fractions containing plasminogen were pooled and dialyzed against
PBS. Plasmin was generated by incubation of 0.8 mg of plasminogen with
80 µg of tissue-type plasminogen activator at 37 °C, for 45 min.
Activation was interrupted by incubation (20 °C, 10 min) with a
100-fold molar excess of aprotinin. The efficiency of plasminogen
activation was monitored by SDS-PAGE (33) and immunoblot experiments
(34), with 125I-labeled anti-plasminogen purified from
rabbit plasma by immunoaffinity chromatography. For electroblot
analysis, proteins were transferred to polyvinylidene difluoride
membranes. The membranes were blocked in PBS containing 0.25% Tween 20 and 0.25% gelatine (blocking buffer), incubated with radiolabeled
proteins in blocking buffer, and finally washed in blocking buffer
containing 0.5 M NaCl. Ligand blotting was done with
125I-labeled plasminogen, IgA, or fibrinogen. Iodination of
proteins was done using the chloramine T method (35). Coupling of
proteins to agarose columns with preactivated
N-hydroxysuccinimide ester (HiTrap) was done according to
the manufacturer's instructions (Pharmacia, Uppsala, Sweden). For
binding analysis in microtiter plates (Microtest III, Becton-Dickinson,
Cockeysville, MD) wells were coated with 100 µl of the protein
solution (10 µg ml
1) in PBS and incubated overnight at
4 °C. The following day, 100 µl of blocking buffer was added and
the plates were again incubated overnight at 4 °C. On day 3 the
plates were washed with blocking buffer. A mixture containing the
radiolabeled ligand (125I-labeled plasminogen or plasmin)
and the unlabeled competitor (plasminogen, plasmin, or
-aminocaproic
acid) was then added in a total volume of 150 µl in blocking buffer.
For experiments with plasmin, a 100-fold molar excess of aprotinin was
included in each incubation. After incubation at 20 °C for 4 h,
the plates were washed four times in blocking buffer and the
radioactivity in the wells was counted. For affinity calculations,
125I-plasminogen (0.1 nM) or
125I-plasmin (0.1 nM) was mixed with increasing
concentrations of unlabeled plasminogen or plasmin, in serial 2-fold
dilutions (0.05 to 50 nM for plasminogen and 6 nM to 0.8 µM for plasmin). Calculation of
affinity constants were made using the formula of Scatchard (36).
 |
RESULTS |
Activation of Plasminogen Bound to the Surface of S. pyogenes--
We followed the time course of plasminogen/plasmin
binding to AP53 streptococci expressing the PAM protein on their
surface. To this end the bacteria were grown in medium containing 30%
human plasma. Following centrifugation, the plasma proteins bound to the bacterial surface were eluted, separated by SDS-PAGE, and visualized by immunoblotting using a 125I-labeled specific
antibody recognizing both human plasmin and plasminogen (Fig.
1, panel A, top). The
plasmin/plasminogen ratio increased with time of incubation (Fig.
1A). Using a chromogenic substrate we were able to
demonstrate that at least part of the bacteria-associated plasmin was
present in its active form (Fig. 1A, bottom). These results
indicate that efficient plasminogen-binding and activation
mechanism(s), which are not blocked by the proteinase inhibitors
present in human plasma, must exist at the streptococcal surface.

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Fig. 1.
Acquisition of plasmin by streptokinase
producing and non-producing PAM-expressing S. pyogenes.
Protein PAM-expressing AP53 streptococci from an overnight culture were
washed three times with PBS and then used to inoculate fresh culture
medium containing 30% human plasma. The cultures were terminated at
the indicated time points and the bacteria were harvested. Following repeated washing, the bacteria were divided in two equal parts. The
bacterial-bound proteins from one aliquot were eluted with 0.1 M glycine-HCl, pH 2.0, and analyzed by Western blotting
with 125I-anti-plasminogen as the probe using
ska positive (panel A, top) and ska
negative AP53 streptococci (panel B, top). The other aliquot was resuspended in buffer containing the chromogenic substrate S2251
and incubated for 60 min at 37 °C. The bacteria were then pelleted
and the absorbance of the supernatant was measured at 405 nm
(A and B, bottom). The data are representative of
five experiments with single samples. Insertional inactivation of the ska gene in AP53 and JRS145 streptococci was accomplished
with the kanamycin-resistance cassette Km-2, flanked ska
fragments generated by PCR using the primer combinations SK3/SK4 and
SK6/SK7 (panel C). The external ska primers SK9
or SK10 were used in combination with primers KM1 or KM2 from the
Km-2 sequence to verify the chromosomal integration of the cassette
by PCR.
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The a1-repeat of PAM Is Sufficient to Endow Group A Streptococci
with Plasminogen Binding--
Initially we focused on the role of PAM
for the streptococcal capture of plasmin(ogen). However, previous
experiments with recombinant proteins and synthetic peptides (21) have
suggested that a major plasminogen-binding site is located in a
30-amino acid residue long segment containing a sequence with two
characteristic tandem repeats (a-repeats) in the
NH2-terminal variable region of PAM. To test the notion
that a-repeats confer plasminogen binding properties, we used another
M-like protein, Arp4, that lacks a plasminogen-binding site, but
exposes a well defined IgA-binding site (37), as a recipient for the
a-repeats of PAM (Fig. 2). Manipulations
were done in the E. coli-streptococcal shuttle vector pJRS264 that encodes the complete Arp4 protein. We first replaced 29 residues in the IgA-binding region of Arp4 (positions 29-57 of the
Arp4 sequence) by a segment of 36 residues from PAM which holds both a1
and a2 (positions 53-88 of the PAM sequence; see Ref. 20), or by a
segment of 13 residues that comprises only a1 (positions 62 to 74). We
then transformed E. coli with the corresponding plasmids and
isolated the chimeric constructs Arp/a1a2 and Arp/a1. Western blots of
the chimeric proteins probed by 125I-plasminogen or IgA
revealed that both Arp/a1a2 and Arp/a1 bound plasminogen, but not IgA
(Fig. 3). In competitive binding
experiments, unlabeled plasminogen inhibited the binding of
125I-plasminogen to Arp/a1a2 and Arp/a1 that had been
immobilized in microtiter plates (Fig.
4A). Similarly, unlabeled
plasmin inhibited the binding of radiolabeled plasmin to the two
chimeric proteins (Fig. 4B). The affinity constants
(Ka) for the interactions between plasminogen and
plasmin and the two chimeras were calculated by Scatchard plots from
the data obtained in the competitive binding experiments. For the
interaction with plasminogen, the Ka values were
calculated to be 6 × 108 M
1
for both chimeras (Fig. 4A, insets), a value close to that
(8 × 108 M
1) previously
determined for the interaction between radiolabeled plasminogen and
immobilized PAM. The corresponding Ka values for the
interactions between plasmin and the chimeric proteins (Fig. 4B,
insets) were 1 order of magnitude lower (3 × 107
M
1 and 6 × 107
M
1, for Arp/a1a2 and Arpa1, respectively). As
was the case for the interaction between 125I-plasminogen
and PAM (20), the binding of plasminogen to the Arp/PAM constructs
could be inhibited by the lysine analogue
-aminocaproic acid at low
concentrations, indicating that the interaction involves lysine-binding
site(s) in plasminogen (data not shown). Hence, the results show that
the a-repeat motif(s) can confer the plasminogen-binding properties of
PAM to a plasminogen non-binding member of the M protein family and
that the a1-repeat contains the major plasminogen-binding site of
PAM.

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Fig. 2.
Construction of Arp/PAM chimeric
proteins. A, SacI and BstBI DNA fragments of the
arp4 gene (open bar) were replaced with PAM gene
fragments (shaded bar) overlapping a single (Arp/a1, inserted PAM sequence: DAELQRLKNERHE) or both a-repeats (Arp/a1a2, inserted PAM sequence: LKDDVEKLTADAELQRLKNERHEEAELERLKSERHD). When
making the constructs, breaking of the heptad repeat patterns in Arp4
and PAM (39) was avoided. The arrowheads point to the IgA-
and plasminogen-binding sites. "C" denotes conserved
repeated segments that are present in many M or M-like proteins. The
bars representing the sequence segments of the M proteins
are not drawn to scale.
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Fig. 3.
Binding of plasminogen to Arp/PAM chimeric
proteins. The purified recombinant proteins PAM (lane
1), Arp4 (lane 2), Arp/a1a2 (lane 3), and
Arp/a1 (lane 4) were separated by SDS-PAGE and stained with
Coomassie Blue (Stain). The relative molecular masses of
standard marker proteins run simultaneously are given on the
left. Separated proteins were transferred to polyvinylidene difluoride membranes by electroblotting, and identical replicas were
probed with 125I-plasminogen or 125I-IgA
(Blot).
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Fig. 4.
Inhibition of the binding of radiolabeled
plasminogen or plasmin to immobilized Arp/PAM chimeric proteins.
The interactions were inhibited with various concentrations of
unlabeled plasminogen (A), or plasmin (B), at the
indicated concentrations. The affinity constants
(Ka) for the interactions between radiolabeled plasminogen (A, inset), or plasmin (B, inset),
and the immobilized Arp/a1 or Arp/a1a2 proteins were calculated from
Scatchard plots of data obtained when an equilibrium had been reached.
The data represent the mean values of three experiments with duplicate samples.
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The plasmids encoding Arp/a1a2 and Arp/a1 were then used to transform
the group A streptococcal strain JRS145, an isogenic M-protein-deficient (emm
) derivative of the
M6-expressing strain JRS4. Using radiolabeled ligands, streptococci
expressing the Arp/PAM constructs were shown to bind plasminogen but
failed to bind IgA (Fig. 5). Furthermore, heat-killed Arp/a1a2- and Arp/a1-expressing streptococci absorbed plasminogen from plasma, whereas the Arp4-expressing strain or JRS145
did not (Fig. 6). The finding that
JRS145(Arpa1) showed a reduced capacity to bind
125I-plasminogen compared with JRS145(Arpa1a2), although
the recombinant chimeric proteins have the similar affinity for the
zymogen, is likely to be attributable to different surface expression
levels. Thus, estimation of the numbers of receptors from Scatchard
analysis of experiments where 125I-plasminogen and
different concentrations of plasminogen were allowed to compete for the
binding to JRS145 transformed with the two PAM/Arp hybrid genes, showed
that JRS145(Arpa1a2) expressed 30 times as many chimeric molecules as
compared with JRS145(Arpa1) (data not shown). Because of this
difference, JRS145(Arpa1a2) was used in further experiments.

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Fig. 5.
Binding of radiolabeled protein ligands to
S. pyogenes expressing chimeric Arp/PAM proteins. The
binding of 125I-labeled purified plasminogen or IgA to
emm strain JRS145 (negative control; ), to
Arp4-expressing strain JRS145(Arp) ( ), to strains JRS145(Arp/a1a2)
() and JRS145(Arp/a1) ( ) that express Arp/PAM chimeric proteins,
and to the PAM-expressing strain AP53 (positive control; ) was
measured. The data represent mean values from three separate
experiments with duplicate samples each.
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Fig. 6.
Plasma absorption patterns of
Arp/PAM-expressing S. pyogenes. Heat-killed group A
streptococci were incubated with 1.0 ml of human plasma for 30 min at
20 °C. Following washing, the absorbed proteins were eluted with 0.1 M glycine-HCl, pH 2.0. The acid-stripped bacteria were
pelleted, and 20 µl of each supernatant containing 20-40 µg of
total protein was analyzed by SDS-PAGE under reducing conditions; for
control, 2 µg of purified human plasminogen was included (left
panel). An electroblotted replica of the gel was probed with
125I-anti-plasminogen (right panel). Elutes from
the following strains were analyzed: lane 1, JRS145(Arp/a1);
lane 2, JRS145(Arp/a1a2); lane 3, AP53;
lane 4, JRS145(Arp); lane 5, JRS145; lane
6, plasminogen.
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We also transformed strain JRS4, the parent strain of JRS145 that
expresses the fibrinogen-binding M6 protein, with the plasmid encoding
the hybrid Arp/a1a2 protein, and thereby converted the phenotype of
JRS4 from plasminogen non-binding to plasminogen binding while
retaining its fibrinogen binding capacity (Fig. 7, inset). When grown in
medium containing plasma, significant surface acquisition of plasmin
was observed with transformed JRS4, but not with the wild-type strain
(Fig. 7).

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Fig. 7.
Binding and activation of plasminogen on M
protein-expressing streptococci transformed with an Arp/PAM
construct. The M6 protein expressing wild-type group A
streptococcal strain JRS4 was transformed with the Arp/a1a2-encoding
plasmid. The 125I-plasminogen (circles) and
fibrinogen (squares) binding capacity of the parent strain
and the transformant was measured (inset). The parent and
the transformed strains were then grown in culture medium containing
30% human plasma. The cultures were terminated at the indicated time
points, and the bacteria were harvested. After repeated washes, the
bacterial pellets were resuspended in buffer containing the chromogenic
substrate S2251. Following incubation for 60 min at 37 °C the
bacteria were pelleted, and the absorbance of the supernatant was
measured at 405 nm. Parent strain, open bars and
symbols; transformed strain, solid bars and
symbols.
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Co-operation between Streptokinase and the a-repeats of PAM in the
Generation of Streptococcal-associated Plasmin--
Our initial
experiments with the AP53 strain demonstrated a conversion of
plasminogen to plasmin after >30 min of incubation of the bacteria
with plasma-containing medium. Because of the presence of a large molar
excess of proteinase inhibitors in plasma which likely inactivate any
exogenous plasminogen activators, we speculated that activators of
bacterial origin, such as streptokinase, could be involved in plasmin
generation. Indeed, streptokinase added to plasminogen-loaded
heat-inactivated AP53 cells converted plasminogen to plasmin in a
dose-dependent manner (data not shown). To rigorously test
the hypothesis that streptokinase contributes to the activation of
surface-associated streptococcal plasminogen, we inactivated the
streptokinase gene (ska) of strain AP53 by homologous
recombination (Fig. 1C). Ska
AP53
streptococci bound 125I-plasminogen at a level (65% of the
total radioactivity) comparable to the parent strain (63%), and
demonstrated a plasma absorption pattern similar to that of the
original isolate (data not shown). However, little if any conversion of
plasminogen was observed when the ska
variant
was incubated with human plasma (Fig. 1B). The inability of
ska
AP53 streptococci to acquire surface-associated
plasmin could be reversed by addition of streptokinase (data not
shown).
The notion that streptokinase might be critically involved in
plasminogen activation at the streptococcal surface was further investigated in a derivative of the M protein-deficient group A
streptococcal strain JRS145 also deleted in the streptokinase gene. As
expected, this strain failed to bind plasmin(ogen) at all (data not
shown). Transformation with the plasmid encoding Arp/a1a2 conferred
plasminogen-binding properties to
JRS145(emm
/ska
).
However, little if any activation of plasminogen was observed by
immunoblotting and by the amidase activity assay (Fig.
8). In contrast, expression of Arp/a1a2
in ska+ JRS145 bacteria allowed acquisition of
plasminogen from plasma-containing growth medium as well as
time-dependent conversion of plasminogen to plasmin (Fig.
8). As with the AP53 ska
strain, the inability
of streptokinase-deficient JRS145(Arp/a1a2) streptococci to capture
plasmin could be restored following addition of streptokinase. In
accord with these observations we found that heat-killed
JRS145(Arp/a1a2) bacteria, unable to secrete streptokinase, bound
plasminogen but failed to acquire significant amounts of plasmin (data
not shown). Hence, the co-operation of two bacterial effectors, the
surface-bound acceptor protein PAM and the secretory activator protein
streptokinase, is mandatory for generation of human plasmin at group A
streptococcal surfaces.

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Fig. 8.
Binding and conversion of plasminogen by
Arp/PAM-expressing S. pyogenes in the absence or presence
of endogenous streptokinase. JRS145 ska+ or
ska isolates that had been transformed with
the Arp/a1a2-encoding plasmid were grown overnight. The cultures were
used to inoculate fresh culture medium containing 30% human plasma and
terminated at the indicated time points. Following centrifugation and
washing, the bacterial pellet was divided in two equal parts. The
plasma proteins absorbed by the bacteria from one aliquot were eluted and analyzed by Western blot experiments using
125I-anti-plasminogen as a probe (top). The
other aliquot was dissolved in buffer containing the chromogenic
substrate S2251 for 60 min at 37 °C, the bacteria were pelleted, and
the absorbance of the supernatant was measured at 405 nm
(bottom). Left panel, streptokinase expressing
strain; right panel, streptokinase-deficient strain. The
data are representative of five experiments with single samples.
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DISCUSSION |
Group A streptococci elaborate at least two classes of
plasmin(ogen)-binding proteins, i.e. secretory proteins such
as streptokinase and streptococcal cysteine proteinase (38), and
surface attached proteins such as PAM (20), Plr (10), and a 45-kDa
protein (13). Moreover, group A streptococci can capture plasmin(ogen) indirectly through fibrinogen which in turn binds to M-like proteins (15). Such a multiplicity of interactions with the key player of the
fibrinolytic pathway suggests that the plasmin(ogen)-binding property
carries a selective advantage to streptococci, motivating an analysis
of the relative importance and biological role of the different
mechanisms.
In the present work we have focused on the role of protein PAM, a
member of the streptococcal M family of surface proteins. To assess the
contribution of the PAM a-repeats for in vitro plasmin acquisition by group A streptococci, we grafted them on the
plasmin(ogen) non-binding protein Arp4, another member of the M protein
family. We chose Arp4 as the "recipient" molecule since this
protein and PAM are similar in many ways. Thus, both proteins are
anchored to the bacterial cell wall through their highly conserved
COOH-terminal segments, they contain three conserved C-repeats in the
COOH-terminal half of the protein, and have surface-exposed variable
NH2-terminal regions. Moreover, both PAM and Arp can be
predicted to form
-helical coiled coil dimers as a consequence of
their high contents of characteristic heptad repeats (39). Finally, the
binding patterns of PAM and Arp resemble one another: (i) both proteins
bind C4bp with high affinity through a site in the
NH2-terminal hypervariable domain (40); (ii) they do not
bind fibrinogen; (iii) a unique binding property (i.e.
plasminogen-binding for PAM and IgA-binding for Arp4) is for both
proteins located in the central region of their
NH2-terminal domains. The experiments showed that
replacement of the IgA-binding site of Arp by the a-repeats of PAM was
necessary and sufficient to endow group A streptococci expressing two
(JRS145) or three (JRS4) of the other putative plasmin(ogen)-binding
surface proteins (10, 13, 15) with the capacity to bind plasmin and
plasminogen, thereby emphasizing the power of the PAM motif in relation
to other structures believed to contribute to group A streptococcal
plasmin(ogen) acquisition. Moreover, the data establish PAM as the most
well characterized cell-surface receptor for plasminogen and the a1
repeat of 13 residues as the smallest and best defined internal
plasminogen acceptor site known, making it a candidate for analyses of
structural requirements for interactions between kringles and sequences
lacking carboxyl-terminal lysines.
The binding to PAM is mediated through the lysine-binding site(s) of a
plasminogen fragment comprising its first three kringle domains (20).
The principal physiological regulator of plasmin,
2AP,
binds to related site(s) (41). Given the high affinity for the
interaction with plasmin(ogen), this protease is likely to be protected
from regulation by
2AP when bound by PAM-expressing streptococci. Our finding that absorption and activation of plasminogen on PAM- and Arp/PAM-expressing streptococci can take place in plasma
supports such a notion. Since the binding is mediated through lysine-binding site(s), the catalytic domain of plasminogen remains accessible by the major streptococcal plasminogen activator,
streptokinase. Indeed, streptokinase binds to PAM-expressing bacteria
or to PAM-coated polyacrylamide beads only when they have been
pre-absorbed with plasminogen.2 This suggests
that streptokinase might bind and activate PAM-bound plasminogen
although our present experiments do not entirely rule out the
possibility that a cycle of dissociation from and association with PAM
is required to allow plasminogen activation in solution. Unlike plasmin
in solution, neither the plasminogen-streptokinase complex nor the
plasmin-streptokinase complex can be inactivated by
2AP
(42). Therefore the finding that PAM- and Arp/PAM-expressing bacteria
acquire active plasmin even in the presence of plasma points to the
possibility that bacteria gain an unregulatable plasminogen activator
due to the deposition of plasmin(ogen)-streptokinase complexes on the
streptococcal surfaces.
The key role of plasmin is to degrade fibrin (43, 44). However, due to
its broad specificity, plasmin can serve a host of other functions,
e.g. cleavage of connective tissue proteins and basement
membrane components, and activation of latent metalloproteinases. Cells
that are able to generate plasmin on their surface may therefore use it
to degrade physiological barriers other than fibrin. Indeed, plasmin-induced matrix degradation has been shown to facilitate the
tissue penetration of tumor cells exhibiting an increased surface-associated plasminogen activation (45, 46). The present results
as well as data published by other investigators suggest that bacteria
and certain mammalian cells may use similar mechanisms to cause
pericellular proteolysis. Recent results using in vivo models suggest that surface-bound plasmin(ogen) can contribute to
bacterial invasion. Thus, expression of a surface-bound plasminogen activator vastly increased the virulence of Y. pestis by
facilitating their dissemination from the site of infection in the skin
of mice (7). Moreover, plasminogen boosted spirochtemia in mice infected with the plasminogen-binding bacterial species Borrelia burgdorferi (9). It is conceivable that plasmin bound to the streptococcal surface may also function as a means to break tissue barriers, thereby promoting the spreading of these bacteria. The results presented here emphasize the unique property of some group A
streptococci to expose a surface protein that binds plasminogen with
high affinity, and at the same time to secrete a highly potent plasminogen activator that likely activates the surface-bound plasminogen. The joint role for protein PAM and streptokinase in the
acquisition of surface-bound plasmin by group A streptococci associated
with skin infections is yet another facet of the complex interplay that
governs host-parasite relationships.
Dr. Gunnar Lindahl is gratefully acknowledged
for valuable discussions and reagents.