©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Streptococcal Cysteine Proteinase Releases Biologically Active Fragments of Streptococcal Surface Proteins (*)

Andreas Berge , Lars Björck (§)

From the (1) Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, P. O. Box 94, S-22100 Lund, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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 -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) .

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 (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.

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.


EXPERIMENTAL PROCEDURES

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.

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.

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.




RESULTS

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).

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.


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.




DISCUSSION

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, 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) .

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.() 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.

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,() and we are currently investigating whether there are human tissue-specific proteins interacting with protein H and/or protein HIgG complexes.

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.


FOOTNOTES

*
This work was supported by the Swedish Medical Research Council Projects 7480 and 10434, King Gustav V:s 80-years foundation, the Medical Faculty, Lund University, the Foundations of Kock, Schyberg, and sterlund, High Tech Receptor AB, and the Swedish Research Council for Engineering Sciences Project 123. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, P. O. Box 94, S-221 00 Lund, Sweden. Tel.: +46-46-104492; Fax: +46-46-157756; E mail: Andreas.Berge@medkem.lu.se.

The abbreviations used are: Ig, immunoglobulin; PMN, polymorphonuclear leukocytes; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; ZAS, zymosan-activated serum; SCP, streptococcal cysteine proteinase; MES, 4-morpholinepropanesulfonic acid; PCR, polymerase chain reaction.

J. Cooney, C.-L. Liu, and L. Björck, manuscript in preparation.

I.-M. Frick and L. Björck, in preparation.

A. Berge, A. G. Sjöholm, and L. Björck, manuscript in preparation.


ACKNOWLEDGEMENTS

We are indebted to Britt Turesson for technical advise and to Chun-Li Liu for excellent technical assistance.


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