From the
Streptococcus pyogenes are important pathogenic
bacteria which produce an extracellular cysteine proteinase
contributing to their virulence and pathogenicity. S. pyogenes also express surface molecules, M proteins, that are major
virulence determinants due to their antiphagocytic property. In the
present work live S. pyogenes bacteria of the M1 serotype were
incubated with purified cysteine proteinase. Several peptides were
solubilized, and analysis of their protein-binding properties and amino
acid sequences revealed two internal fibrinogen-binding fragments of M1
protein (17 and 21 kDa, respectively), and a 36-kDa IgG-binding
NH
Streptococcus pyogenes bacteria are the causative agent
of suppurative infections such as acute pharyngitis, impetigo, and
erysipelas. Scarlet fever, necrotizing fasciitis, sepsis, and a toxic
shock syndrome are also caused by these bacteria, whereas rheumatic
fever and glomerulonephritis are medically important sequelae following
acute S. pyogenes infections.
S. pyogenes expresses a number of surface proteins. Among these, the M
proteins are major virulence determinants due to their antiphagocytic
property (for M protein references, see Ref. 1). M proteins exist in
more than 80 different serotypes, and they form
S. pyogenes also produces several
extracellular proteins that are involved in virulence. One of these
proteins is a cysteine proteinase which has been shown to be identical
to the extracellular pyrogenic exotoxin B
(17) . However, the
enzyme is also active within the bacterial cell
(18) . At the
cell surface the streptococcal cysteine proteinase (SCP) is known to
cleave M protein, and the enzyme has fibrinolytic activity
(19) . It has also been reported to activate human interleukin
1
In the
present work SCP was purified from a S. pyogenes strain (AP1)
of the M1 serotype, one of the most common M protein serotypes. Apart
from M1 protein and C5a peptidase, AP1 bacteria also express an
IgG-binding surface molecule called protein H
(9, 22) .
The studies demonstrate that SCP is capable of releasing functionally
active fragments of these surface proteins. Possible implications of
this observation for S. pyogenes pathogenicity and virulence
are discussed.
The specificity and mechanism of release were tested
utilizing proteinase inhibitors. Digestions were performed at 5
µg/ml SCP, and the proteolytic activity was analyzed by SDS-PAGE
and by fibrinogen and IgG binding. The release of streptococcal surface
proteins by SCP was completely blocked by the presence of 10
µM E-64 (a cysteine proteinase inhibitor) whereas 6
mM benzamidine (a serine proteinase inhibitor) or 10
mM EDTA did not effect the solubilization of the peptides
shown in Figs. 2 and 4. Neither did inhibition of bacterial protein
synthesis by chloramphenicol during SCP digestion influence the release
of streptococcal surface proteins.
The biological properties of the
SCP-released streptococcal C5a peptidase fragment was now analyzed and
compared with an E. coli-expressed PCR fragment corresponding
to the mature C5a peptidase molecule except for the COOH-terminal
membrane spanning region (Fig. 3 B). This fragment,
covering amino acids 32-1139 and with an apparent molecular mass
of 130 kDa, was expressed in E. coli clones identified with
antibodies raised against the 116-kDa SCP-released C5a peptidase
fragment. In these clones, a dominating band around 130 kDa was seen
(compare Fig. 5, D and E). It should be
mentioned that the molecular mass determined from the sequence
32-1139 is 121.337 kDa. However, due to their proline-rich
wall-spanning regions, the molecular mass of cell surface proteins in
Gram-positive bacteria is often overestimated in SDS-PAGE
(42) .
Following the same isolation protocol as described for the 116-kDa
streptococcal fragment, the E. coli-produced material was
purified to homogeneity from E. coli lysates (Fig. 5,
E-G)
Activation of both the classical and the alternative
pathway of the complement system leads to the formation of C5a, a major
chemoattractant responsible for the rapid neutrophilic infiltration at
inflammatory loci. In an under agarose migration assay, streptococcal
C5a peptidase inhibits directed migration of PMN by cleavage of the
COOH-terminal seven amino acids of C5a
(43) . In the assay, ZAS
containing C5a is used to obtain a directed migration of PMN. As
demonstrated in Fig. 6, the streptococcal and the E.
coli-produced C5a peptidase fragments both inhibited ZAS-induced
migration of PMN. The results show that the cysteine proteinase of
S. pyogenes releases a biologically active fragment of cell
surface-bound C5a peptidase with the potential to inhibit phagocytic
cells from reaching the site of infection.
SCP was the first prokaryotic cysteine proteinase to be
isolated and characterized (for references, see Ref. 44). The enzyme is
secreted into the growth medium as an inactive zymogen which by living
S. pyogenes bacteria, or when injected into mice,
The results demonstrate that SCP also at low concentrations
efficiently removes M1 protein and protein H from the streptococcal
surface. Previous studies have shown that these fibrous hair-like
proteins have a tendency to self-associate
(2, 49) and,
in the case of protein H, participate in the formation of bacterial
aggregates.
Rheumatic fever involving
the heart and joints and glomerulonephritis involving the kidneys are
clinically important complications following suppurative S.
pyogenes infections. Both conditions are believed to be
immunological diseases in which deposits of antibodies or
antigen-antibody complexes are found in the affected organs. The large
majority of clinical S. pyogenes isolates express Ig-binding
surface proteins
(14) , and the demonstration that SCP releases
an IgG-binding fragment may have pathophysiological consequences.
Soluble complexes between Ig and Ig-binding surface proteins could
through the blood circulation reach for instance the kidney and bind to
the basal membrane, where subsequent activation of the complement
system will lead to tissue damage. This hypothetical scenario is
supported by the fact that protein H activates complement,
The host-parasite relationship represents a
delicate balance between numerous molecular interactions. The discovery
(38, 53) and elucidation of the function
(15) of the C5a peptidase demonstrates how sophisticated some of
the mechanisms are that operate in this relationship. Here we find that
SCP solubilizes a well-defined fragment of the peptidase from the
bacterial surface including its substrate binding regions
(43) .
This release of a fragment capable of inhibiting the recruitment of
phagocytes to the site of infection further underlines the complexity
of molecular mechanisms contributing to microbial pathogenicity and
virulence.
We are indebted to Britt Turesson for technical advise
and to Chun-Li Liu for excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal fragment of protein H, an IgGFc-binding surface
molecule. M protein also plays a role in streptococcal adherence, and
removal of this and other surface proteins could promote bacterial
dissemination, whereas the generation of soluble complexes between
immunoglobulins and immunoglobulin-binding streptococcal surface
proteins could be an etiological factor in the development of
glomerulonephritis and rheumatic fever. Thus, in these serious
complications to S. pyogenes infections immune complexes are
found in affected organs. The cysteine proteinase also solubilized a
116-kDa internal fragment of C5a peptidase, another streptococcal
surface protein. Activation of the complement system generates C5a, a
peptide stimulating leukocyte chemotaxis. C5a-mediated granulocyte
migration was blocked by the 116-kDa fragment. This mechanism, by which
phagocytes could be prevented from reaching the site of infection, may
also contribute to the pathogenicity and virulence of S. pyogenes.
-helical
coiled-coil hair-like structures at the bacterial surface
(2) .
M proteins of most serotypes show affinity for fibrinogen
(3) .
Many strains of S. pyogenes also express immunoglobulin
(Ig)
-binding surface proteins
(4) . Several of these
IgG- and/or IgA-binding molecules have been isolated and characterized
(5, 6, 7, 8, 9, 10, 11) .
The Ig-binding proteins are structurally related to M proteins, and in
a given strain of S. pyogenes the genes encoding the M protein
and the Ig-binding protein are closely linked
(7, 10, 11, 12, 13) . The role
for Ig-binding proteins in virulence and pathogenicity is yet unclear,
but most clinical isolates of S. pyogenes do express these
surface molecules
(14) . The C5a peptidase, another
surface-bound protein in S. pyogenes, has a more well-defined
role in virulence. This large molecule (125 kDa) cleaves complement
factor C5a and disables it as a chemoattractant for polymorphonuclear
leukocytes (PMN) (
)(15) . The gene for C5a peptidase
is closely linked to the genes encoding the M and Ig-binding proteins,
and in some strains the three genes are co-ordinately regulated
(16) .
(20) . Specific blocking of cysteine proteinase activity
suppresses growth of S. pyogenes in vitro and protects mice
infected with lethal doses of these bacteria
(21) . Taken
together these data indicate a role for SCP in virulence.
Strains
S. pyogenes strain AP1 used in
this study is the 40/58 strain from the World Health Organization
Collaborating Centre for references and Research on Streptococci,
Institute of Hygiene and Epidemiology, Prague, Czech Republic. Its
protein binding properties have been described
(9, 22, 23) .
Proteins
Human IgG and fibrinogen were purchased
from Sigma. The purification of proteins H and M1 has been described
(9, 22, 23) . SCP zymogen was purified from the
medium of AP1 bacteria. The bacteria were grown in a yeast extract
dialysate peptone medium
(17) for 20 h. Tetrathionate (Sigma)
was added to 0.03% after 4 h of growth. The bacteria were centrifuged
at 3000 g for 20 min, and the supernatant was
subjected to ammonium sulfate precipitation (80%) followed by
fractionation on S-Sepharose in a buffer gradient (5-250
mM MES, pH 6.0). The zymogen was further purified by gel
filtration on Sephadex G-200. Fractions were monitored for absorbance
at 280 nm, by SDS-PAGE and by dot immunobinding using anti-SCP
antibodies.
(
)
Incubation of the zymogen in 10
mM dithiothreitol (Sigma) leads to a complete removal of the
NH
-terminal 118 amino acids
(24, 25) , and
the resulting SCP appeared as a single band when applied to SDS-PAGE
(Fig. 4, left lane). The gel was blotted to
polyvinylidene difluoride (PVDF), and the SCP band was subjected to
NH
-terminal amino acid sequencing. The sequence obtained,
QPVVKSLLDS, is identical to a previously published
NH
-terminal amino acid sequence of SCP
(24) . The
gene encoding SCP in the AP1 strain also translates into this
NH
-terminal sequence.
The purity of the
preparation was further tested by using an azocasein assay
(26) with the modification that the assay was performed in 0.15
M NaCl, 0.03 M phosphate, pH 7.2 (PBS). The enzymatic
activity of the SCP preparation was completely blocked by E-64 (Sigma),
a specific cyseine proteinase inhibitor, but was not affected by
benzamidine (Sigma), phenylmethylsulfonyl fluoride (Sigma), or EDTA.
The chromogenic substrate H-D-Ile-Pro-Arg-pNA (S-2288,
Chromogenix, Mölndal, Sweden) is a substrate for a broad spectrum
of serine proteinases but was not susceptible to cleavage by the SCP
preparation when used according to the producer. IgG and fibrinogen
were labeled with
I using the chloramine T-method
(27) and proteins H and M1 according to Bolton and Hunter
(28
Figure 4:
Release of streptococcal surface proteins
by SCP digestion. AP1 bacteria were digested at different SCP
concentrations, and the solubilized peptides were analyzed by SDS-PAGE
(10% gel). Two-fold dilutions of SCP from 12 to 0.1 µg/ml bacterial
suspension and an undigested control are shown. The molecular mass of
the activated SCP is 28 kDa. The SCP preparation used for the digestion
was run in the far left lane marked SCP (approximately 20
µg/ml).
Enzymatic Treatment of
Streptococci
Streptococci were grown in Todd-Hewitt broth
(Difco, Detroit, MI) at 37 °C for 16 h and harvested by
centrifugation at 3000 g for 20 min. The bacteria were
washed twice in PBS and resuspended in PBS to 10% (v/v) (2
10
cells/ml). In some experiments chloramphenicol (Sigma)
was added to 170 µg/ml. The purified SCP zymogen was activated by
incubation in 10 mM dithiothreitol for 3 h at 37 °C. The
activation causes a reduction of the thiol group at the active site and
the autocatalytical removal of the NH
-terminal propeptide
(24, 25) . Various amounts of the activated proteinase
were added to bacterial suspensions followed by incubation for 3 h.
Digestions were terminated by addition of iodo-acetic acid (Sigma) to a
final concentration of 6 mM. Bacteria were pelleted at 3000
g for 20 min, and the resulting pellets and
supernatants were saved. Prior to further analysis, supernatants were
dialyzed against the buffers used in the competitive binding assay and
for affinity chromatography (see below). Starting materials for the
purification of fibrinogen- and IgG-binding peptides were obtained by
incubating 10 ml of 10% AP1 suspensions with 0.2 and 0.4 µg SCP/ml,
respectively.
Binding of Radiolabeled Proteins to SCP-treated AP1
Streptococci
AP1 bacteria incubated with different
concentrations of activated SCP (see above) were resuspended in PBS
containing 0.02% NaN and 0.25% Tween-20. The binding of
radiolabeled fibrinogen or IgG to the bacteria was determined
(9) .
Competitive Binding Assays
Polyclonal IgG was
coupled to polyacrylamide beads (Immunobeads, Bio-Rad).
I-Labeled protein H in 0.1 ml of veronal buffer, pH 7.35,
containing 0.15 M NaCl and 0.1% gelatine, 0.1 ml of
Immunobeads-coupled IgG in the same buffer, and 0.2 ml of supernatant
from proteinase digestions diluted 1:1 in the same buffer, were mixed
and incubated overnight at 20 °C. Two ml of the same buffer
containing 0.01 M EDTA were added, beads were centrifuged,
washed, and the radioactivity of the pellets was measured.
Corresponding experiments were done with fibrinogen coupled to beads
and radiolabeled M1 protein as the ligand. To increase the sensitivity
of the assays, a concentration of beads resulting in submaximal binding
of radiolabeled ligand was chosen.
Electrophoresis, Western Blots, Gel Filtration, and
Affinity Chromatography
SDS-polyacrylamide gel electrophoresis
(PAGE) was performed as described by Neville
(29) . Before
loading, samples were boiled for 3 min in sample buffer containing 2%
SDS and 5% -mercaptoethanol. For Western blot analysis, proteins
were transferred to PVDF membranes as described by Towbin et al.(30) . Membranes were blocked, incubated with radiolabeled
fibrinogen or IgG, and subsequently washed as described
(31) .
Gel filtration was performed on a Pharmacia fast protein liquid
chromatography system. The samples were applied to a Superose 12 HP
10/30 column (Pharmacia, Uppsala, Sweden) equilibrated with 0.1
M Tris-HCl, pH 8.0, containing 0.15 M NaCl and 5
mM EDTA, and eluted in 0.5 ml fractions at 6.0 ml/h.
NHS-activated Sepharose (HiTrap, Pharmacia) was coupled with fibrinogen
or IgG and used for affinity chromatography. Samples in PBS were
applied, and the columns were rinsed with five volumes of PBS. Bound
material was eluted with 0.1 M glycine, pH 2.0. The pH was
adjusted to 7.0 with 1 M Tris. Eluted material was
concentrated 10 times.
Cloning Procedures
PCR was performed using Taq DNA polymerase (Promega, Madison, WI) with oligonucleotides
constructed from sequences in the C5a peptidase gene
(32) using
AP1 chromosomal DNA as the template. PCR products resulting from
amplification with these primers were ligated into the high expression
vector pHD389
(33) as described
(34) . Genomic DNA
preparation, ligation, and transformation procedures were as described
(35) . Plasmids were transformed into Escherichia coli strain JM109
(35) .
Migration Assay
PMN were prepared from blood of
healthy human volunteers by dextran (Pharmacia) sedimentation,
Lymphoprep (Nycomed, Oslo, Norway) density separation, and 0.87%
ammonium chloride lysis. Cells were washed in Hanks' balanced
salt solution , and the concentration was set to
50 ,000 cells/µl. An under agarose assay system for the
measurement of chemotaxis was used
(36, 37) . The
studies were done using gelatine (Difco), agarose (Indubiose A37,
Gallard-Schlesinger Chemical Co., New York), and Medium 199 (Flow,
Irvine, CA) with Earl's salts buffered with HEPES. All values of
directed migration are expressed in terms of chemotactic indices
defined as directed migration ( A) minus the spontaneous
migration ( B), and divided by the spontaneous migration
according to the formula (( A-B)/ B). The directed
migration control consisted of zymosan-activated serum (ZAS) diluted in
buffer. Spontaneous migration was measured to wells containing
Hanks' balanced salt solution. Data points represent the mean of
triplicate determinations of a single experiment. All experiments were
performed at least twice. For the preparation of ZAS
(37) , 10
mg of zymosan (Sigma) was washed twice in PBS, mixed with 1 ml of
normal human serum, and rotated at 37 °C for 30 min. The zymosan
was removed by centrifugation at 750 g for 10 min, and
the supernatant (ZAS) was heated at 56 °C for 30 min and stored at
20 °C in aliquots. Hanks' balanced salt solution was
used to dilute ZAS.
Generation and Purification of C5a Peptidase
Fragments
Fragments of the C5a peptidase were obtained either by
digestion of AP1 bacteria with SCP (see above) or by expression in
E. coli of a PCR-generated DNA fragment covering the mature
C5a peptidase except for the hydrophobic membrane spanning region.
Thus, PCR primers were synthesized from nucleotides 940-963 and
4263-4234 in the published sequence
(32) , and the PCR
product was cloned into the high expression vector pHD389
(33) .
Positive clones were identified with PCR and by insert size, and
selected clones were verified by partial DNA sequencing. C5a peptidase
corresponding to the correct 3.3-kilobase insert was purified from
E. coli lysates, whereas C5a peptidase fragments from AP1
streptococci were isolated from SCP digests of the bacteria. In both
cases, the purification protocol included ammonium sulfate
precipitation under conditions described
(38) followed by gel
filtration on a Superose 12B column. The presence of C5a peptidase
fragments in various fractions was analyzed by SDS-PAGE and Western
blots using rabbit antibodies to the enzyme.
Other Methods
Amino-terminal amino acid sequence
analysis was performed on a Applied Biosystems gas-phase sequenator
model 470A equipped with an on-line phenylthiohydantoin analyzer model
120A. Samples were first separated by SDS-PAGE, blotted to PVDF
membranes, and stained with Coomassie Blue. Finally, bands were cut out
from the membranes and subjected to amino acid sequencing.
Double-stranded DNA sequencing was performed using the dideoxy chain
termination method
(39) with Sequenase version 2.0 (U. S.
Biochemical Corp.). Ammonium sulfate precipitation was performed as
described
(40) . Antibodies to the purified SCP zymogen and a
116-kDa C5a peptidase fragment, cut out of SDS-PAGE gels (Fig. 5,
lane C), were raised in rabbits. As jugded by Western blot
experiments, they were specific.
Figure 5:
Purification of C5a peptidase fragments.
AP1 streptococci were digested with 5 µg SCP/ml bacterial
suspension. The resulting supernatant ( A) was precipitated
with ammonium sulfate ( B), followed by gel filtration on a
Superose 12 column ( C). A lysate of E. coli containing the plasmid pHD389 is shown in D. In
E, the E. coli lysate represents a clone expressing a
pHD389 insert covering the C5a peptidase gene except its 3`-end. This
fragment was also purified by ammonium sulfate precipitation
( F) followed by gel filtration ( G). Samples were
separated by SDS-PAGE (8% gel) and stained with Coomassie
Blue.
SCP Solubilizes IgG- and Fibrinogen-binding Peptides
from the Streptococcal Surface
AP1 bacteria were incubated with
activated SCP at different concentrations. Following digestion, the
bacterial cells were tested for IgG- and fibrinogen-binding activity.
Starting at 0.1-1 µg SCP/ml bacterial suspension, both
binding activities gradually decreased, fibrinogen-binding being
slightly more resistant to the proteinase (Fig. 1 A). At
the bacterial surface, protein H is predominantly responsible for
IgG-binding whereas M1 protein binds fibrinogen. Competitive binding
assays were established for these protein-protein interactions (see
``Experimental Procedures''), and the supernatants obtained
after SCP digestions were analyzed for inhibitory activity
(Fig. 1 B). In the case of M1 protein, maximum inhibition
of the binding to fibrinogen was seen with 0.2 µg SCP/ml which in
the standard curve of the competitive binding assay is equivalent to
0.1 µg of M1 protein/ml. Maximum inhibition of the binding of
protein H to IgG was obtained when the bacteria were digested with 0.4
µg SCP/ml bacterial suspension. These supernatants contained
inhibitory material corresponding to 0.2-0.3 µg protein H/ml.
Inhibition decreased at SCP concentrations above 0.4 µg/ml,
suggesting that solubilized fibrinogen- and IgG-binding peptides were
degraded and inactivated at this and higher SCP concentrations.
Figure 1:
IgG- and fibrinogen-binding
streptococcal proteins are solubilized by SCP. A, AP1 bacteria
were digested with SCP at different concentrations. Bacteria were
centrifuged, washed, resuspended, and tested for binding of
radiolabeled IgG () or fibrinogen (
). B, the
supernatants from the SCP digestions above were tested for inhibition
of the binding of radiolabeled protein H to immobilized IgG (
) or
inhibition of the interaction between radiolabeled M1 protein to
fibrinogen (
).
Identification of Protein H and M1 Protein Fragments
Released by SCP
To obtain material for the identification of
IgG- and fibrinogen-binding peptides released by SCP, larger amounts of
AP1 bacteria were treated with 0.4 and 0.2 µg SCP/ml bacterial
suspension, respectively. After centrifugation the resulting
supernatants were subjected to affinity chromatography on IgG- or
fibrinogen-Sepharose. Fig. 2, left, demonstrates that a
35-kDa IgG-binding peptide was eluted from IgG-Sepharose. The
NH-terminal amino acid sequence of this material was
determined as Glu-Gly-Ala-Lys-Ile, which is identical to the 5
NH
-terminal amino acid residues of intact protein H
(9) . As depicted in Fig. 3 A, this fragment of
protein H (called fragment I) covers the entire extracellular region of
the molecule.
Figure 2:
Identification and purification of IgG-
and fibrinogen-binding peptides. AP1 bacteria (10% v/v) were digested
with 0.4 µg SCP/ml, and the resulting supernatant was separated on
12% SDS-PAGE. This material ( lane A, left) was
subjected to affinity chromatography on IgG-Sepharose. Lane B,
run-through material. Lane C, material eluted with 0.1
M glycine, pH 2.0 ( Fragment I). AP1 surface proteins
solubilized with 0.2 µg SCP/ml bacterial suspension were analyzed
by SDS-PAGE (13.6% gel) and subjected to fibrinogen-Sepharose ( lane
A, Stain). The run-through material is shown in lane
B. Coomassie Blue did not stain any material eluted from
fibrinogen-Sepharose with 0.1 M glycine, pH 2.0 ( lane
C, Stain). The peptides of an identical gel were
transferred to a PVDF filter which was probed with
I-labeled fibrinogen and autoradiographed
( Blot). Fibrinogen-binding peptides were visualized (denoted
II and III in lane A,
Stain)
Figure 3:
Schematic representations of protein H, M1
protein, and the C5a peptidase. Fragments generated by SCP or produced
in E. coli are indicated. The three proteins are all
associated with the streptococcal cell surface through their
COOH-terminal regions. The NH-teminal signal sequences
( Ss) are indicated. A, in protein H, IgG binding is
in the A-B domains whereas fibrinogen binding in M1 protein is found in
the A-B3 region. M1 protein also has a weak IgG binding activity which
is located in the S domain. The sequences of the Ss, C, and D domains
in protein H and M1 protein are homologous. B, in the C5a
peptidase, the amino acids ( D130, H193, and
S512) that constitute the charge relay system crucial for the
enzyme activity (32) are indicated.
The surface proteins released with 0.2 µg SCP/ml
bacterial suspension were applied to a fibrinogen-Sepharose column
(Fig. 2, right). None of the peptides inhibiting the
binding of radiolabeled M1 protein to immobilized fibrinogen in the
competitive binding assay could be purified by affinity chromatography.
However, when the material was transferred from SDS-PAGE to PVDF
filters and incubated with radiolabeled fibrinogen two bands (denoted
II and III in Fig. 2) reacted with the probe
(Fig. 2, Blot), suggesting that the affinity is not high
enough to allow purification of the corresponding peptides by affinity
chromatography. The bands, 17 and 21 kDa, respectively, were cut out of
PVDF membranes and sequenced. Both had the NH-terminal
amino acid sequence Leu-Arg-His-Glu-Asn, which is found in the M1
protein of the AP1 strain starting at position 66
(23) . In M1
protein, fibrinogen binding has been mapped to a region between amino
acid residues 66 and 195
(23, 41) . Thus, fragments II
and III both span this region (Fig. 3 A). Fragment II
also contains the IgG-binding S region
(23) but still showed no
affinity for IgG-Sepharose (Fig. 2, left). This is not
surprising as the affinity between similar M1 fragments and IgG is much
lower (>100-fold) as compared to intact M1 protein, which binds IgG
with an association constant of 3.4
10
M
(23) .
SCP Solubilizes a C5a Peptidase Fragment Inhibiting
Granulocyte Migration
It was noted that at SCP concentrations
above 0.1 µg/ml bacterial concentration a band with an apparent
molecular mass of 116 kDa appeared in the digests (Fig. 4).
Compared to the IgG- and fibrinogen-binding peptides this material was
more resistant to SCP, and the intensity of the band increased up to
SCP concentrations around 20-30 µg/ml AP1 suspension. At
higher concentrations, the 116-kDa peptide was gradually degraded, but
even at 200 µg/ml the band was clearly visible (not shown). A
larger batch, 10 ml, of AP1 bacteria was digested with 5 µg SCP/ml,
and the supernatant was subjected to ammonium sulfate precipitation
followed by gel filtration. A highly purified 116-kDa peptide was
obtained (Fig. 5, lane C), and its
NH-terminal sequence was determined to be
Lys-Thr-Ala-Asp-Thr-Pro-Ala-Thr, a sequence which was identified also
in streptococcal C5a peptidase starting at position 90. Thus, the
molecular mass of this internal C5a peptidase fragment, cleaved and
solubilized by SCP, suggests that it apart from the 58
NH
-terminal amino acids
(32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89) ,
covers the cell surface-exposed part of the streptococcal C5a peptidase
(Fig. 3 B).
Figure 6:
The C5a
peptidase fragments generated by SCP or produced in E. coli both block leukocyte migration. The migration of PMN was measured
in an under agarose migration assay. A, migration to
Hanks' buffer. B, migration to zymosan-activated serum
diluted to 60% in Hanks' buffer. C, as in B plus 25 µg/ml of the purified SCP-released streptococcal C5a
peptidase fragment; D, as in B plus 25 µg/ml of
the purified E. coli produced truncated C5a peptidase;
E, as in B plus 25 µg/ml of human serum
albumin.
is readily activated. SCP is also found inside the streptococcal
cell where it appears almost entirely in its activated form
(18) . In eukaryotic cells, cysteine proteinases play important
roles in the intracellular catabolism of peptides and proteins, and
they are also involved in the proteolytic processing of, for instance,
proenzymes. Although nothing is known about the intracellular
activities of SCP, it appears likely that the enzyme has similar
functions in the streptococcal cell. Outside the bacterium, SCP
activates human interleukin 1
(20) and cleaves fibronectin
and vitronectin
(45) . In relation to the present work, it is
noteworthy that SCP was originally connected with proteolytic activity
destroying the serological reactivity of the type-specific M protein of
S. pyogenes(19) . Apart from M protein, S.
pyogenes expresses additional surface proteins. In the present
study it was investigated whether SCP could release functional
fragments of M protein and/or other surface proteins from a strain of
the M1 serotype. A world-wide increase in serious systemic S.
pyogenes infections during the last decade has particularly been
associated with this M serotype
(46, 47, 48) .
(
)
By the action of SCP, these
bacterial cell-cell interactions, and perhaps also interactions with
epithelial cells, could be regulated. S. pyogenes organisms
expressing M protein have indeed been shown to adhere better to human
pharyngeal, buccal, and tongue epithelial cells in vitro than
M-deficient bacteria
(50, 51) . The cleavage of M1
protein and protein H by SCP could promote the spreading of the
infection, for instance when nutrients are scarce and pH is low as in
the center of suppurative streptococcal infection
(52) . The
level of secretion of SCP is dependent on environmental factors like pH
(44) , and when grown at pH 5.5-6.0 most strains produce
considerable amounts of SCP (10-150 mg/liter growth medium). The
concentrations used in this study to solubilize surface proteins were
much lower. Therefore, it is also feasible that in vivo the
concentration of SCP at the site of infection will reach levels that
are high enough to remove surface proteins from the bacteria, thereby
changing their physicochemical and biological properties. The
demonstration that various surface proteins show different
susceptibility for SCP suggests that the bacterium through expression
of SCP can modify its composition of these proteins in response to
environmental conditions. Such a mechanism will facilitate the
adaptation of the bacterium to its host.
(
)
and we are currently investigating whether there are human
tissue-specific proteins interacting with protein H and/or protein
H
IgG complexes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.