 |
INTRODUCTION |
Streptococcus pyogenes is an important human pathogen
that causes a variety of diseases such as pharyngitis, impetigo,
scarlatina, and erysipelas. More severe infections caused by this
organism are necrotizing fasciitis and streptococcal toxic shock-like
syndrome. S. pyogenes binds several human plasma proteins
via its surface proteins. The surface proteins studied in most detail
are the M or M-like proteins, which are responsible for the ability of S. pyogenes to resist phagocytosis (1, 2). M proteins bind several human plasma proteins like fibrinogen (3), IgG (4, 5), and
regulatory proteins of the complement system (6, 7). The binding
activities of M proteins have been proposed to be of importance for the
antiphagocytic activity of these proteins (2, 6-9).
Several different streptococcal species also bind
2M1 (10-16),
which is an abundant homotetrameric plasma protein of 718 kDa best
characterized as a proteinase inhibitor (17). During infection proteinases are released both by bacteria and damaged host cells, and
tight regulation of proteolytic activity is thus essential. S. pyogenes secretes a cysteine proteinase (SCP), which has several important functions and is regarded as a major virulence factor of this
bacterium (18-25). There is no described inhibitor of the SCP even
though it is known that essentially all proteinases from the four
classes (metallo, cysteine, aspartic, and serine proteinases) are
inhibited by
2M (26). Inhibition of proteinases by
2M is achieved by cleavage of the bait region of
2M, which induces cleavage of an internal thioester in
2M (26). The free glutamyl group of the thioester can
form amide bonds with lysyl amino groups in the proteinase (27). These
events induce conformational changes in the structure of
2M and results in an entrapment of the proteinase (28).
The trapped enzyme remains active against smaller substrates but is
sterically hindered to cleave larger ones (26). The proteinase complexed form of
2M is recognized and cleared by a
specific receptor, referred to as low density lipoprotein
receptor-related protein, present on hepatocytes, macrophages, and
fibroblasts (29).
2M has also been implicated in
immunoregulatory events by direct effects on macrophages, modulation of
the effect of growth factors and cytokines, and effects on antigen
presentation (30-33). Furthermore
2M is a major carrier
for zinc and cadmium in plasma (34).
The interaction between streptococci and the two forms of
2M is highly specific. Human pathogenic streptococci
(groups A, C, and G) only bind to the native form of
2M,
whereas bovine and equine group C streptococci interact with the
proteinase complexed form of
2M (16). The binding of
native
2M to group C and G streptococci has been
attributed to protein G (11, 12), a surface-associated molecule with
separate binding sites also for human IgG and human serum albumin
(35-37). An
2M-binding surface protein of 78 kDa from
group A streptococci has also been described, but the sequence of this
protein is not known (10). In the present work a novel
2M-binding protein in S. pyogenes is
identified and characterized. A role is demonstrated for this molecule,
called protein GRAB (protein G-related
2M-binding protein), in the regulation of proteolytic activity at the streptococcal surface. Thus,
important bacterial surface proteins and virulence determinants are
protected from proteolytic degradation by
2M bound to
protein GRAB.
 |
EXPERIMENTAL PROCEDURES |
Bacterial Strains and Growth Conditions--
S.
pyogenes strains denoted AP are from the Institute of Hygiene and
Epidemiology (Prague, Czech Republic). The KTL3 and KTL9 strains are
blood isolates, and the KTL6 is a throat isolate from the Finnish
Institute for Health. The SF370 strain is the ATCC 700294 strain. For
molecular cloning purposes the DH5
strain of Escherichia
coli was used. Streptococci were grown in Todd-Hewitt broth
(Difco, Detroit, MI) with 0.2% yeast extract (Difco) (THY) in 5%
CO2 at 37 °C. E. coli were grown in Luria
Bertoni broth (10 g of tryptone (Difco), 10 g of NaCl, and 5 g of yeast extract (Difco)/liter) supplemented with 2 g
glucose/liter when using the pMal-p2 vector. For growth on Petri dishes
15 g/liter of bacto agar (Difco) was added. When E. coli
contained plasmid either 100 µg/ml ampicillin (Sigma) or 50 µg/ml
of kanamycin (Sigma) was added to the medium.
Proteins and Peptides--
Human
2M was purified
from fresh frozen plasma, and wild-type protein G was prepared from
group G streptococcus strain G148 as described (11). The streptococcal
cysteine proteinase was purified and tested as described (22). Peptides
with sequences derived from protein GRAB were synthesized and analyzed
for purity and correct sequence as described (38). Fibrinogen, trypsin, and soybean trypsin inhibitor were from Sigma. Heparinized plasma was
prepared by centrifugation (3000 × g for 10 min) of
blood from a healthy volunteer. For radiolabeling of proteins, 20 µg of protein was suspended in PBS and labeled with IODO-BEADS (Pierce) according to the instructions from the manufacturer, using one bead for
5 min in the incubation step. Labeled proteins were separated from free
125I on a PD10 column (Amersham Pharmacia Biotech).
Binding Assays and Competition--
Bacteria were harvested in
early stationary phase or after overnight culture, washed in PBS with
0.05% Tween 20 and 0.02% azide (PBSAT), and resuspended in the same
buffer. Concentration of bacteria was determined by spectrophotometry,
and 2 × 109, 1 × 109, or 4 × 108 bacteria were incubated with radiolabeled
2M or fibrinogen in 225 µl of PBSAT for 50 min. For
competition different amounts of unlabeled inhibitor was added. After
centrifugation, the radioactivity of the pellets was determined and
expressed as percentages of the added activity, deducting the
nonspecific binding to the polypropylene tubes or, when
2M was used, the binding to the mutant strain MR4 (see
"Results").
Protein Separation and Western Blotting--
Proteins were
separated by SDS-polyacrylamide gel electrophoresis (PAGE) (39) and
transferred to a Protane nitro-cellulose filter (Schleicher & Shull)
using a Trans-blot semidry transfer cell (Bio-Rad). Alternatively
proteins were applied directly to the membrane. Filters were blocked
using PBS with 0.25% Tween 20 and 0.25% gelatin followed by
incubation of the filter with radiolabeled
2M in 5 ml of
the same buffer for 3 h at 37 °C. Membranes were washed three
times for 10 min using PBS with 0.05% Tween 20 and 0.5 M
NaCl, exposed on a BAS-III imaging plate, and scanned with a
Bio-Imaging Analyzer BAS-2000 (Fuji Photo Films Co. Ltd., Japan).
Polymerase Chain Reaction, Cloning Procedures, and
Sequencing--
Genomic DNA was prepared from S. pyogenes
as described (40) modified by adding a 60-min digestion with 1000 units/ml of mutanolysin (Sigma) and 100 mg/ml lysozyme (Sigma) in TE
buffer (10 mM Tris·Cl, 1 mM EDTA, pH 7.4),
replacing the initial incubation step. PCR was performed using
Taq polymerase (Life Technologies, Inc.) and synthetic
oligonucleotides hybridizing to grab. Primers hybridized to
the following nucleotides in Fig. 2B: primer 1, 101-125;
primer 2, 101-128; primer 3, 160-185; primer 4, 594-563; and primer
5, 627-605. Restrictions enzymes and ligase were from Life
Technologies, Inc. and standard ligation, transformation, and plasmid
isolation methods were used (41). For PCR screening and for cloning in
pGEM (Promega, Madison, WI) primers 1 and 5 were used. Sequencing of
the pGEM-grab plasmids was performed using an ABI-470 prism
and Taq dyed dideoxy terminator kit (Perkin-Elmer, Norwalk,
CT). Primers 3 and 5 had sites for EcoRI and PstI
respectively and were used in PCR to generate a fragment of
grab, which was cleaved by these enzymes and cloned into the
corresponding site of the pMal-p2 vector (New England Biolabs, Beverly,
MA) and transformed into E. coli. This resulted in
production of the fusion protein containing the maltose-binding protein
(MBP) and protein GRAB upon induction with
isopropyl-1-thio-
-D-galactopyranoside (Promega). MBP-GRAB was purified by affinity chromatography using an amylose resin
(New England Biolabs) according to the instructions of the manufacturer. To generate a mutant strain devoid of protein GRAB on its
surface, a fragment of grab lacking the part encoding the putative cell wall attachment region was generated by PCR from the KTL3
strain using primers 2 and 4. The fragment was cut with XhoI
and HindIII which exclusively cut within primers 2 and 4, respectively, and cloned into the corresponding site of the
streptococcal suicide plasmid pFW13 (33) and electroporated into
E. coli. Plasmid was purified, and 2 µg of plasmid was
electroporated into the KTL3 strain (42) for homologous recombination
to occur. Transformants were plated on THY+15 g agar/l with 150 µg of
kanamycin/ml.
Animal Experiments--
Female NMRI mice weighing approximately
25 g were injected intraperitoneally with washed bacteria from an
overnight culture in 1 ml of sterile PBS. The number of bacteria
injected was determined by spectrophotometry and verified by colony
counting. Mice were observed for 7 days after challenge, and survival
was assessed at intervals of roughly 2 h. Blood samples were drawn
from ill mice injected with MR4 and plated on THY plates with and
without kanamycin to confirm the presence of the plasmid on the
bacterial chromosome. Statistical analysis of survival time was
performed with the Wilcoxon rank sum test.
Northern Blotting--
Total RNA from S. pyogenes was
purified using a FastprepTM cell disrupter (Savant, Holbrok, NY) as
described previously (43). KTL3 or AP1 bacteria were cultured in THY
medium either for 12 h (late stationary phase) or to an
A620 of 0.4 (early logarithmic phase), 0.65 (late logarithmic phase), or 0.8 (early stationary phase), before
harvest by centrifugation at 3,800 × g for 10 min at
4 °C. Pellets were resuspended in water followed by disruption for
2 × 20 s at setting 6.0 using FastRNATM kit with glass
beads (BIO 101, Vista, CA) according to the manufacturers instructions. For Northern blot experiments, 5 µg of total RNA was separated on 1%
agarose in HEPES buffer (0.2 M Na-HEPES, pH 7.0, 50 mM NaAc, 10 mM EDTA), blotted onto Hybond-N
filters (Amersham Pharmacia Biotech), and hybridized with probe,
generated by PCR from KTL3 using primers 1 and 5. Probe was purified on
a MicroSpinTM S-200 HR column (Amersham Pharmacia Biotech) and
radiolabeled with [
-32P]dATP using MegaprimeTM
(Amersham Pharmacia Biotech). To estimate the amount of RNA loaded in
each well, a probe was constructed from 16 S ribosomal RNA sequence
from S. pyogenes. This sequence was obtained by homology
search between the Streptococcal Genome Sequencing Project sequence
data base and 16 S sequence of Enterococcus sulfureus
(accession number X55133). From this sequence two oligonucleotides
5'-ATG TTA GTA ATT TAA AAG GGG-3' and 5'-TTT AAG AGA TTA GCT TGC CGT-3'
were used to PCR amplify an ~800-base pair fragment from KTL3 DNA,
which was labeled as above. Hybridization was performed at 50 °C for
14 h. After hybridization the membranes were washed in 6× SSC + 0.1% SDS and then in 0.1× SSC + 0.1% SDS and air dried followed by
exposure and scanning (see "Protein Separation and Western
Blotting").
Analyses of Proteolysis--
2 × 109 KTL3 or
MR4 cells were incubated with 20 µg of
2M for 40 min
and carefully washed with PBS. These bacteria were either incubated
with radiolabeled trypsin for 5 min followed by the determination of
bound radioactivity as above or with 0.3 µg of unlabeled trypsin,
which was allowed to react with surface bound
2M for 5 min. Free trypsin (not in complex with
2M) was blocked by adding a 4-fold molar excess of soybean trypsin inhibitor. Cells
were pelleted by centrifugation, and the resulting pellet was washed
once in 1 ml of PBS and resuspended in 150 µl of PBS supplemented
with 40 µg of chloramphenicol/ml (Roche Molecular Biochemicals). The
remaining activity of trypsin in the supernatant and the resuspended
pellet was determined using the chromogenic substrate
N
-bensoyl-L-arginine
p-nitroanilide (Sigma) at a final concentration of 0.25 mg/ml by measuring A405 after 3 h. The
obtained value for MR4 was subtracted from that of KTL3 and compared
with a standard, where the same assay was run in parallel using
purified
2M of known concentration. For protection
assays, bacteria were preincubated with
2M or fibrinogen
(20 µg) as above and treated with 0.1 µg of trypsin in PBS with
chloramphenicol as above for 60 min at 37 °C. Bound fibrinogen was
eluted twice with 0.1 M glycine, pH 2.0, and bacteria were
diluted 10 times in PBSAT supplemented with 10 mM
benzamidine (Sigma) and chloramphenicol as above. 4 × 106 bacteria were tested for binding of radiolabeled fibrinogen.
Radiolabeled SCP was activated in activation buffer (1 mM
EDTA and 10 mM dithiothreitol in 0.1 M
NaAc-HAc, pH 5.0) for 30 min at 40 °C. Activated SCP (4 µl) was
mixed with either 4 µg of
2M or 2 µl of plasma in 20 µl of PBS. Following incubation for 15 min at 37 °C the mixture
was subjected to SDS-PAGE using nonreducing conditions followed by
autoradiography. 2 × 109 bacteria were preincubated
with 10 µg
2M, washed, and incubated with radiolabeled
and activated SCP for 15 min. Bacteria were pelleted by centrifugation,
and the pellet was washed with 2 ml of PBSAT and recentrifuged. The
radioactivity of the pellet was measured, and bound material was
released by suspension of pellet in nonreducing SDS-PAGE sample buffer.
Finally the eluate was subjected to SDS-PAGE and autoradiography.
Alternatively bacteria were pretreated with 10 µg of
2M, washed, and digested with 0.2 µg of activated SCP
in 100 µl of PBS (final concentration of dithiothreitol, 0.2 mM) with chloramphenicol as above for 2 h at 37 °C.
Bacteria were pelleted and resuspended in PBSAT with chloramphenicol
and 6 mM of iodoacetamid (Sigma), and 4 × 106 bacteria were subjected to binding assay with
radiolabeled fibrinogen in the same buffer.
Other Methods--
For precipitation of proteins the sample was
incubated with 6% trichloroacetic acid for 30 min on ice followed by
centrifugation at 15 000 × g (4 °C for 20 min).
Homology searches were performed using available sequences from the
Streptococcal Genome Sequencing Project (Department of Chemistry and
Biochemistry, the University of Oklahoma, Norman, OK) and at the
National Center for Biotechnology Information by using the BLAST
network service (Wisconsin package, version 8, Genetics Computer Group,
Madison, WI).
 |
RESULTS |
S. pyogenes Binds Native
2M via a Protein G-like
Protein--
Bacteria from different strains of S. pyogenes
were harvested in late logarithmic growth phase and tested for their
ability to bind radiolabeled native
2M (Fig.
1A). The binding ranged from 0 to 76% and differed between strains also within a given M serotype. No
strain bound the trypsin complexed form of
2M (data not
shown). The KTL3 strain of the clinically important M1 serotype, which
bound 53% of added
2M, was chosen for further studies.
The binding of radiolabeled
2M to the KTL3 strain was blocked both by nonradioactive
2M and by protein G (Fig.
1B). The Scatchard plot for the reaction between
2M and KTL3 bacteria (Fig. 1C) suggests two
interactions: a high affinity interaction, Ka = 2.0 × 108 M
1 (560 sites/bacterium), and a low affinity interaction, Ka = 5.3 × 106 M
1 (4000 sites/bacterium). Because binding of
2M to the KTL3
strain could be competed by protein G, we hypothesized that the
2M binding was mediated by a protein G-related protein.
Thus, the protein sequence of protein G from strain G148 was used in a
tBLASTn search against the Streptococcal Genome Sequencing Project data
base (44). A gene coding for a protein showing homology to the
2M-binding E domain (11), to the signal sequence, and to
the cell wall attachment of protein G, was identified. The protein,
named protein GRAB, consisted of 217 amino acids (aa) with a deduced
molecular mass of 22.8 kDa. In Fig.
2A a schematic representation
of the homology between protein GRAB and protein G is shown. Protein GRAB was found to contain the consensus sequence for Gram-positive surface proteins (LPXTGX), followed by a stretch
of 19 hydrophobic amino acids and a 7-residue-long hydrophilic COOH
terminus (Fig. 2B). The first 34 aa of protein GRAB showed
homology to the signal sequence of protein G and was followed by 35 aa
homologous to the E domain of protein G (Fig. 2B). Spacing
the regions with homology to protein G, two unique repeated regions of
28 aa were identified in protein GRAB. A BLASTp search revealed that
protein GRAB has significant homology to several surface proteins from Gram-positive bacteria. The predicted mature protein GRAB (aa 35-187
in Fig. 2B) is, however, homologous only to protein G
(accession numbers X06173, M13825, and X53324) and to an
albumin-binding protein from Streptococcus canis (accession
numbers A44801 and M95520).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Binding of
2M to S. pyogenes.
A, binding of 125I-labeled 2M to
different strains of S. pyogenes. Values are the mean of
three experiments ± S.E. B, the binding of
125I- 2M bacteria of the KTL3 strain was
competed with unlabeled 2M ( ) or protein G ( ).
Bars represent ± S.D. C, Scatchard plot for
the reaction between 2M and KTL3 bacteria suggesting two
binding sites with different affinities.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 2.
Protein GRAB: relation to protein G,
molecular organization, and sequence. A, schematic
representation of protein G (strain 148) and protein GRAB (strain
SF370). In protein G 2M binding is located in the E
domain, human serum albumin binding in the A and B domains, and IgG
binding in the C domains. Homologous regions are shaded, and
the two proteins are associated with the bacterial surface through
their COOH-terminal W and M domains. The signal sequence is denoted
Ss. In protein GRAB the R repeats are 28 amino acids long,
and 26 of the residues are identical between the two repeats.
B, complete nucleotide and amino acid sequence of
grab/protein GRAB.
|
|
Distribution and Expression of the grab Gene--
The same strains
that were used in the screening for
2M binding were
subjected to PCR using primers hybridizing with grab. A PCR
product could be generated from all strains except for the AP9 strain,
but the size of the product varied between 500 and 850 base pairs (Fig.
3A). Sequencing of the PCR
product from four strains revealed that the size polymorphism was due
to a variable number of the 28-aa repeats (Fig. 3B).
Comparing the sequences from these four strains and the one presented
in the Streptococcal Genome Sequencing Project revealed that protein
GRAB is highly conserved. Both the COOH and the NH2
terminii were close to 100% conserved, whereas the repeated region
showed 86% identity between strains (Fig. 3B). The
transcription of grab was investigated by Northern blotting
where total RNA samples from the
2M-binding KTL3 strain
and the nonbinding AP1 strain were electrophorized, blotted, and probed
with a PCR product generated from grab. Detectable amounts
of grab RNA was found in KTL3 but not in AP1 bacteria (Fig.
4). Maximum expression was found in early
logarithmic phase, whereas the expression dropped to undetectable
amounts in the late stationary phase. The same filters were probed with
a probe hybridizing with 16 S ribosomal RNA, which verified that the
same amount of RNA had been applied to each well (Fig. 4).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Analysis of the grab gene in
different strains of S. pyogenes. A, PCR analysis
of twelve S. pyogenes strains of nine different M serotypes.
B, schematic comparison of grab in five strains
of S. pyogenes.
|
|

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 4.
The grab gene is expressed
in 2M-binding KTL3 bacteria but
not in nonbinding AP1 bacteria. Total RNA from the KTL3 and AP1
strains was isolated from bacteria in early logarithmic phase
(EL), late logarithmic phase (LL), early
stationary phase (ES), or late stationary phase
(LS), subjected to Northern blotting, and probed with a
probe hybridizing with grab. In the right panel
an identical filter was probed with a probe hybridizing with 16 S RNA,
verifying that the same amount of RNA was applied to each well.
|
|
The NH2-terminal Region of Protein GRAB Interacts with
2M--
DNA encoding the predicted mature protein GRAB
(aa 34-189 in Fig. 2B) in the KTL3 strain was PCR cloned
into the pMal-p2 vector to produce a fusion protein between MBP and
protein GRAB. The fusion protein, MBP-GRAB, was purified by affinity
chromatography on an amylose resin, subjected to SDS-PAGE, and blotted
to a nitrocellulose filter. The filter was probed with radiolabeled
2M, and both protein G and the MBP-GRAB fusion were
found to bind
2M, whereas MBP did not (Fig.
5A). Similarly MBP-GRAB,
protein G, and MBP were applied in slots to a nitrocellulose membrane
and probed with
2M and again MBP-GRAB bound
2M, whereas MBP did not (Fig. 5B). Moreover
MBP-GRAB, but not MBP, was found to compete for the binding of
radiolabeled
2M to KTL3 bacteria (Fig.
6). Thus, both protein GRAB and protein G
can inhibit the binding of
2M to KTL3 bacteria,
indicating that the two proteins interact with the same region in
2M. Finally a peptide covering the extreme NH2 terminus of the mature protein GRAB (aa 34-56 Fig.
2B) inhibited the binding of
2M to KTL3
bacteria, whereas an overlapping peptide (aa 49-68 in Fig.
2B) did not affect the binding (Fig. 6). The results map the
binding of
2M to the NH2-terminal part of
protein GRAB.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 5.
Protein GRAB binds
2M. A, a fusion protein
between mature protein GRAB and a maltose-binding protein (MBP-GRAB),
streptococcal protein G, and MBP alone were separated by SDS-PAGE (10%
gel) under reducing conditions. Two identical gels were run; one was
stained with Coomassie Blue (STAIN), and one was blotted to
nitrocellulose and probed with radiolabeled 2M
(BLOT). B, various amounts of MBP-GRAB, protein
G, and MBP were applied to a nitrocellulose filter which was incubated
with radiolabeled 2M.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
2M binds to the
NH2-terminal part of protein GRAB. The binding of
radiolabeled 2M to KTL3 bacteria was inhibited by
MBP-GRAB ( ) and a synthetic peptide covering the 23 NH2-terminal amino acid residues of protein GRAB ( )
(amino acids 34-56 in Fig. 2B). MBP ( ) and peptide 51-68 ( ) did
not interfere with the interaction. Values are given as
percentages ± S.D.
|
|
A S. pyogenes Mutant Devoid of Protein GRAB on Its Surface Does Not
Bind
2M and Is Attenuated in Virulence--
To
inactivate grab a PCR-generated 468-base pair internal
fragment (nucleotides 113-580 in Fig. 2B) of
grab lacking the part encoding the cell wall anchoring
region was cloned into the pFW13 suicide vector to generate
pFW-grab (Fig. 7A).
pFW-grab was electroporated into KTL3 bacteria for
homologous recombination (Fig. 7A), and several
kanamycin-resistant transformants were obtained. Using this cloning
strategy the mutant should be devoid of surface bound protein GRAB and
instead secrete a truncated form (aa 34-174 in Fig. 2B).
One transformant called MR4 was selected, and its ability to bind
radiolabeled
2M was completely abolished (Fig.
7A). Moreover, when the supernatants from an overnight
culture of MR4 and KTL3 were precipitated with trichloroacetic acid,
subjected to SDS-PAGE, blotted to nitrocellulose, and probed with
radiolabeled
2M, the MR4 strain was found to secrete an
2M-binding protein of 32 kDa, which was not identified
in the KTL3 medium (Fig. 7B). The predicted size of the
mature protein GRAB is 14.9 kDa, but apparently it migrates much slower
in SDS-PAGE. It is a common observation that the molecular mass of
surface proteins in Gram-positive bacteria is often overestimated by
SDS-PAGE, which also explains that the MBP-GRAB fusion migrates slower
than predicted. MR4 and KTL3 bacteria had similar growth
characteristics in THY medium and showed identical binding of
fibrinogen (data not shown). The mutant survived as well as the wild
type in fresh human blood (data not shown), but when injected
intraperitoneally in NMRI mice, MR4 was found to be attenuated in
virulence. When 108 bacteria were injected a significant
(p = 0.005) increase in survival time was observed
(Fig. 7C). Using 107 bacteria in the inoculum,
none of the mice in either the KTL3 or the MR4 group died (6 animals in
each group). Colony counting of the inoculum showed that identical
numbers of KTL3 and MR4 bacteria were injected. Blood samples from mice
infected with MR4 were plated on THY agar ± kanamycin, and the
number of colonies were similar on the two plates, showing that MR4
retained pFW13 on the chromosome.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 7.
A S. pyogenes mutant devoid
of surface-associated protein GRAB shows no
2M binding and is attenuated in
virulence. A, homologous recombination was used to delete
the membrane spanning M domain and the COOH-terminal part of the W
domain in strain KTL3 to generate the non- 2M-binding
mutant MR4. B, growth media from KTL3 and MR4 were
trichloroacetic acid-treated and precipitated, and proteins were
subjected to SDS-PAGE (12% gels, reducing conditions). One gel was
stained with Coomassie Blue (Stain), and one was blotted to
nitrocellulose and probed with radiolabeled 2M (Blot).
C, NMRI mice were injected intraperitoneally with
108 KTL3 or MR4 bacteria, and mice were followed for 7 days. Mean time to death, with the range in parentheses, was
significantly longer in mice injected with MR4 (p = 0.005).
|
|
2M Is Active and Protects the M Protein from Tryptic
Digestion when Bound to Protein GRAB--
To determine whether
2M bound to protein GRAB was in its native form, KTL3 or
MR4 cells were incubated with
2M or buffer and carefully
washed. Radiolabeled trypsin was added and allowed to react with
2M followed by determination of radioactivity in the
bacterial pellet. To KTL3 bacteria preincubated with
2M, 7.5 ± 0.4% (n = 3) of added trypsin was bound,
whereas the binding of trypsin to bacteria incubated in buffer or to
MR4 bacteria preincubated with
2M was below 0.5%,
demonstrating that at least some of
2M bound to KTL3
bacteria via protein GRAB is able to trap trypsin. KTL3 or MR4 cells
were incubated with
2M and carefully washed. From
2M-pretreated bacteria, bound
2M was
eluted and subjected to SDS-PAGE. It was found that 0.5 µg of
2M was bound to 2 × 109 KTL3 bacteria,
whereas no
2M was eluted from MR4 bacteria (data not
shown). In parallel, the amount of native
2M bound to
KTL3 bacteria was estimated by calculating the amounts of trypsin
trapped by
2M. This
N
-bensoyl-L-arginine
p-nitroanilide assay showed that 2 × 109
KTL3 bacteria bound 0.27 ± 0.03 µg (n = 3) of
native
2M, which represents approximately 50% of what
could be eluted from the bacteria. These results demonstrate that a
major part of the
2M bound to the surface of KTL3 is
active as a proteinase inhibitor (Fig.
8). The complexes between trypsin and
2M were efficiently released from the KTL3 surface,
because all trypsin activity was found in the supernatant. The trypsin
treatment used in this assay destroyed
2M binding of
KTL3 bacteria (data not shown), making it impossible to determine
whether the release of the trypsin-
2M complexes from the
KTL3 surface was due to degradation of protein GRAB or to dissociation
of trypsin-
2M complexes from protein GRAB.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 8.
2M bound to protein
GRAB protects the M protein from trypsin digestion. KTL3 or MR4
bacteria were either preincubated with buffer (KTL3 and
MR4), 2M
(KTL3+ 2M and
MR4+ 2M), or fibrinogen
(KTL3+Fib and MR4+Fib). Bacteria were washed and
subjected to trypsin digestion. Following inhibition of trypsin
activity and elution of bound fibrinogen (4 µg of fibrinogen was
bound to 2 × 109 bacteria), the binding of
radiolabeled fibrinogen to the bacterial preparations was determined.
Values are ± S.D., n = 3.
|
|
A characteristic of surface-bound streptococcal M proteins is the
susceptibility to trypsin degradation (1). This lead us to investigate
whether preincubation of KTL3 bacteria with
2M could
protect the M protein and thus fibrinogen binding, from proteolytic
degradation by trypsin. It was found that the binding of fibrinogen to
KTL3 could be preserved by
2M pretreatment, whereas
binding of fibrinogen to MR4 was unaffected by preincubation with
2M (Fig. 8). Fibrinogen is susceptible to trypsin
cleavage, but preincubation of bacteria with fibrinogen did not protect the M protein from tryptic degradation (Fig. 8), showing that the
protective effect of
2M was due to inhibition of trypsin and not merely an effect of
2M being a substrate for trypsin.
2M Traps SCP and Protects the M Protein from SCP
Degradation--
Radiolabeled and activated SCP was mixed with either
purified
2M or with plasma and subjected to nonreducing
SDS-PAGE and autoradiography. Part of the radioactivity appeared as a
band with the apparent size of
2M, indicating that a
covalent complex had been formed between SCP and
2M
(Fig. 9A). Pretreatment of KTL3 and MR4 with
2M resulted in an 2.6-fold increased
binding of radiolabeled and activated SCP to KTL3 (2.6 ± 0.57%
compared with 6.85 ± 0.92%). Binding of radiolabeled, activated
SCP to MR4 bacteria was below 2.5% and was not affected by
2M pretreatment. When bound material was eluted from the
bacteria and subjected to SDS-PAGE and autoradiography, SCP was in
complex with
2M in the KTL3, but not in the MR4 material
(Fig. 9B). The supernatants were separated on the same gel,
and a small proportion of the radioactivity from the
2M
pretreated KTL3 bacteria could be seen as band with the apparent size
of
2M (data not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 9.
2M traps SCP and
protects M protein from SCP digestion. A, radiolabeled and
activated SCP was mixed with 2M or plasma. These samples
(SCP+ 2M and SCP+plasma),
radiolabeled and activated SCP (SCP), or radiolabeled
2M ( 2M) were subjected
to nonreducing SDS-PAGE (8%) followed by autoradiography.
B, KTL3 or MR4 bacteria were either preincubated with buffer
(KTL3 and MR4) or with 2M
(KTL3+ 2M and
MR4+ 2M). Bacteria were washed and
incubated with activated and radiolabeled SCP. Following washing and
elution these samples, radiolabeled and activated SCP (SCP),
or radiolabeled 2M ( 2M)
were subjected to nonreducing SDS-PAGE (8%) followed by
autoradiography. C, KTL3 or MR4 bacteria were preincubated
either with buffer (KTL3 and MR4) or with
2M (KTL3+ 2M and
MR4+ 2M), washed, and digested with
SCP. Following inhibition of SCP activity, the binding of radiolabeled
fibrinogen to the bacteria was determined. Values are ± S.D.,
n = 3.
|
|
SCP can release biologically active fragments of streptococcal surface
proteins, one of which is a fibrinogen-binding fragment of the M
protein (22). To test whether
2M could inhibit this proteolytic cleavage, KTL3 and MR4 bacteria were harvested at logarithmic growth phase. The bacteria were incubated with
2M, washed, and subjected to SCP digestion. The bacteria
were tested for their ability to bind radiolabeled fibrinogen. The
results show that
2M pretreatment preserved the
fibrinogen binding of the KTL3 strain and not of the MR4 strain (Fig.
9C). The experiments described above demonstrate that
2M in solution, or bound to S. pyogenes via
protein GRAB, can trap SCP and thereby protect M protein from SCP cleavage.
 |
DISCUSSION |
The combined work of many research groups has emphasized the
significance of proteolysis in microbial pathogenesis, and the starting
point for this study was to investigate the molecular basis for the
interaction between S. pyogenes and
2M, a
major proteinase inhibitor of human plasma. The finding that wild-type protein G of group C and G streptococci could inhibit this interaction lead to the identification of protein GRAB, a previously unknown
2M-binding surface molecule in S. pyogenes.
Protein G is widely known and used as an IgG-binding reagent (35, 36).
However, this multifunctional protein also has affinity for human serum albumin (37) and
2M (12). Whereas the interactions with
IgG and human serum albumin are mediated by separate repeated domains (45, 46),
2M-binding is located in the
NH2-terminal nonrepeated E domain of protein G (11). The
homology between the far NH2-terminal sequences of protein
G and protein GRAB and the results of inhibition experiment performed
with synthetic peptides based on the protein GRAB sequence maps the
binding of
2M to the very tip of these streptococcal
surface proteins. It is possible that the large and bulky
2M molecule requires this kind of exposed binding site, which will also allow
2M to interact simultaneously with
more than one protein GRAB molecule. This would increase the affinity of the interaction and could perhaps explain the high affinity interaction between protein GRAB and
2M. The nature of
the low affinity interaction is not known, and it is not likely to be of importance, because the amount of
2M bound to the
bacteria corresponds to the number of high affinity binding sites.
The grab gene was found in 11 of 12 tested strains of
S. pyogenes, and the NH2-terminal A domain of
protein GRAB is well conserved. Thus, in the five isolates where the
grab sequence is known, the identity was 98% at the amino
acid level, suggesting that
2M binding adds selective
advantages to streptococci. This notion is also supported by the
similar
2M binding properties of proteins GRAB and G and
by the observation that their 35 NH2-terminal amino acid
residues show 74% identity. Given the short generation times in
bacteria and the fact that the proteins are found in different bacterial species, this represents a remarkably high degree of homology. On the other hand the number of repeats in protein GRAB differs considerably between isolates, and their sequences are less
conserved as compared with the A, W, and M domains. The function of
these repeats is unknown, but they could play a role as spacers, exposing the
2M-binding A domain at the bacterial surface.
In the MR4 strain, where the cell wall anchor of protein GRAB had been
deleted to give rise to a secreted form of protein GRAB, the binding of
2M was completely lost. To investigate whether
2M binding and protein GRAB was involved in the
virulence of S. pyogenes, we used a mouse intraperitoneal
challenge model. The time from injection to death of the animals was
significantly prolonged using MR4 as compared with KTL3 bacteria,
suggesting a role for protein GRAB and proteinase inhibition in
S. pyogenes virulence. Protein GRAB is the only
2M-binding surface protein expressed by KTL3 bacteria
and the S. pyogenes
2M-binding protein reported by Chhatwal et al. (10) is larger than protein GRAB (78 kDa as compared with 23 kDa). The mode of interaction between protein GRAB and
2M is also different from that of the
78-kDa protein and
2M. When bound to the 78-kDa protein,
2M was reported to be converted to a non-native form
(10). This is in contrast to the
2M bound to KTL3 cells
via protein GRAB. In this case
2M is still active as a
proteinase inhibitor.
The cysteine proteinase produced by S. pyogenes was the
first prokaryotic cysteine proteinase to be isolated (18). This extracellular enzyme efficiently releases biologically active fragments
of S. pyogenes surface proteins, including members of the M
protein family and a C5a peptidase (22). In vivo most molecular mechanisms are tightly controlled, suggesting that the proteolytic activity of SCP at the bacterial surface could be regulated
by proteinase inhibitors. The observation that kininogens, human plasma
proteins, and cysteine proteinase inhibitors, have affinity for M
proteins (47) supported this hypothesis. However, instead of being
inactivated, SCP was found to cleave kininogens, resulting in the
release of the potent proinflammatory peptide bradykinin (24). The
demonstration here that
2M bound to protein GRAB traps
and thereby inhibits the cleavage of M proteins by SCP represents a
mechanism by which a bacterial proteinase is regulated by a host
proteinase inhibitor bound to a bacterial surface protein. Such a
mechanism has previously not been reported.
The anti-phagocytic M protein is very trypsin-sensitive (1). As shown
here
2M bound to protein GRAB protects the M protein from trypsin degradation. This implicates that the presence of
2M, a broad spectrum proteinase inhibitor, at the
bacterial surface protects the bacterium and its surface proteins from
proteolytic attack. At the site of infection proteinases are actively
produced and secreted by neutrophils, whereas damaged cells and tissues passively leak intracellular proteinases. The activity of these enzymes
is believed to facilitate spreading of the infection by tissue
degradation. Binding of proteinase inhibitors like
2M and kininogens to bacterial surfaces could therefore, apart from protecting the bacteria from proteolysis, deplete the microenvironment from proteinase inhibitors. Such a mechanism would enhance inflammation and tissue degradation. More recently it has become clear that several
bacterial species, including S. pyogenes, survive and multiply within, for instance, epithelial cells (48). It could be
speculated that
2M bound to the bacterial surface via
protein GRAB protects the microorganism from intracellular proteinases.
Apart from its function as a proteinase inhibitor, a role for
2M has also been implicated in immune regulation
(30-33). Moreover
2M is an important carrier of zinc
and other trace metals in plasma (34), and binding of
2M
to the bacterial surface could be a mean for the bacterium to capture
these essential metal ions. If protein GRAB and its
2M
binding activity is connected with important biological functions in
S. pyogenes, this would explain the highly conserved
NH2-terminal sequence in the A domain and make protein GRAB
a potentially interesting vaccine target.