Protein GRAB of Streptococcus pyogenes Regulates Proteolysis at the Bacterial Surface by Binding alpha 2-Macroglobulin*

Magnus RasmussenDagger , Hans-Peter Müller§, and Lars BjörckDagger

From the Dagger  Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, S-221 00 Lund, Sweden and the § Department of Immunology and Transfusion Medicine, Greifswald University, D-17487 Greifswald, Germany

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In the molecular interplay between pathogenic microorganisms and their host, proteolytic mechanisms are believed to play a crucial role. Here we find that the important human pathogen Streptococcus pyogenes (group A Streptococcus) expresses a surface protein with high affinity (Ka = 2.0 × 108 M-1) for alpha 2-macroglobulin (alpha 2M), the dominating proteinase inhibitor of human plasma. The immunoglobulin-binding protein G of group C and G streptococci also contains an alpha 2M-binding domain and a gene encoding protein GRAB (protein G-related alpha 2M-binding protein) was identified in the S. pyogenes Genome Sequencing data base. The grab gene is present in most S. pyogenes strains and is well conserved. Protein GRAB has typical features of a surface-attached protein of Gram-positive bacteria. It also contains a region homologous to parts of the alpha 2M-binding domain of protein G and a variable number of a unique 28-amino acid-long repeat. Using Escherichia coli-produced protein GRAB and synthetic GRAB peptides, the alpha 2M-binding region was mapped to the NH2-terminal part of protein GRAB, which is the region with homology to protein G. An isogenic S. pyogenes mutant lacking surface-associated protein GRAB showed no alpha 2M binding activity and was attenuated in virulence when injected intraperitoneally in mice. Finally, alpha 2M bound to the bacterial surface via protein GRAB was found to entrap and inhibit the activity of both S. pyogenes and host proteinases, thereby protecting important virulence determinants from proteolytic degradation. This regulation of proteolytic activity at the bacterial surface should affect the host-microbe relation during S. pyogenes infections.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 2M (26). Inhibition of proteinases by alpha 2M is achieved by cleavage of the bait region of alpha 2M, which induces cleavage of an internal thioester in alpha 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 alpha 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 alpha 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). alpha 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 alpha 2M is a major carrier for zinc and cadmium in plasma (34).

The interaction between streptococci and the two forms of alpha 2M is highly specific. Human pathogenic streptococci (groups A, C, and G) only bind to the native form of alpha 2M, whereas bovine and equine group C streptococci interact with the proteinase complexed form of alpha 2M (16). The binding of native alpha 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 alpha 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 alpha 2M-binding protein in S. pyogenes is identified and characterized. A role is demonstrated for this molecule, called protein GRAB (protein G-related alpha 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 alpha 2M bound to protein GRAB.

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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 DH5alpha 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 alpha 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 alpha 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 alpha 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 alpha 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-beta -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 [alpha -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 alpha 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 alpha 2M for 5 min. Free trypsin (not in complex with alpha 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 Nalpha -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 alpha 2M of known concentration. For protection assays, bacteria were preincubated with alpha 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 alpha 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 alpha 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 alpha 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).

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S. pyogenes Binds Native alpha 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 alpha 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 alpha 2M (data not shown). The KTL3 strain of the clinically important M1 serotype, which bound 53% of added alpha 2M, was chosen for further studies. The binding of radiolabeled alpha 2M to the KTL3 strain was blocked both by nonradioactive alpha 2M and by protein G (Fig. 1B). The Scatchard plot for the reaction between alpha 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 alpha 2M to the KTL3 strain could be competed by protein G, we hypothesized that the alpha 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 alpha 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).


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Fig. 1.   Binding of alpha 2M to S. pyogenes. A, binding of 125I-labeled alpha 2M to different strains of S. pyogenes. Values are the mean of three experiments ± S.E. B, the binding of 125I-alpha 2M bacteria of the KTL3 strain was competed with unlabeled alpha 2M () or protein G (open circle ). Bars represent ± S.D. C, Scatchard plot for the reaction between alpha 2M and KTL3 bacteria suggesting two binding sites with different affinities.


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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 alpha 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 alpha 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 alpha 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).


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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.


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Fig. 4.   The grab gene is expressed in alpha 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 alpha 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 alpha 2M, and both protein G and the MBP-GRAB fusion were found to bind alpha 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 alpha 2M and again MBP-GRAB bound alpha 2M, whereas MBP did not (Fig. 5B). Moreover MBP-GRAB, but not MBP, was found to compete for the binding of radiolabeled alpha 2M to KTL3 bacteria (Fig. 6). Thus, both protein GRAB and protein G can inhibit the binding of alpha 2M to KTL3 bacteria, indicating that the two proteins interact with the same region in alpha 2M. Finally a peptide covering the extreme NH2 terminus of the mature protein GRAB (aa 34-56 Fig. 2B) inhibited the binding of alpha 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 alpha 2M to the NH2-terminal part of protein GRAB.


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Fig. 5.   Protein GRAB binds alpha 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 alpha 2M (BLOT). B, various amounts of MBP-GRAB, protein G, and MBP were applied to a nitrocellulose filter which was incubated with radiolabeled alpha 2M.


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Fig. 6.   alpha 2M binds to the NH2-terminal part of protein GRAB. The binding of radiolabeled alpha 2M to KTL3 bacteria was inhibited by MBP-GRAB () and a synthetic peptide covering the 23 NH2-terminal amino acid residues of protein GRAB (black-square) (amino acids 34-56 in Fig. 2B). MBP (open circle ) 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 alpha 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 alpha 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 alpha 2M, the MR4 strain was found to secrete an alpha 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.


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Fig. 7.   A S. pyogenes mutant devoid of surface-associated protein GRAB shows no alpha 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-alpha 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 alpha 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).

alpha 2M Is Active and Protects the M Protein from Tryptic Digestion when Bound to Protein GRAB-- To determine whether alpha 2M bound to protein GRAB was in its native form, KTL3 or MR4 cells were incubated with alpha 2M or buffer and carefully washed. Radiolabeled trypsin was added and allowed to react with alpha 2M followed by determination of radioactivity in the bacterial pellet. To KTL3 bacteria preincubated with alpha 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 alpha 2M was below 0.5%, demonstrating that at least some of alpha 2M bound to KTL3 bacteria via protein GRAB is able to trap trypsin. KTL3 or MR4 cells were incubated with alpha 2M and carefully washed. From alpha 2M-pretreated bacteria, bound alpha 2M was eluted and subjected to SDS-PAGE. It was found that 0.5 µg of alpha 2M was bound to 2 × 109 KTL3 bacteria, whereas no alpha 2M was eluted from MR4 bacteria (data not shown). In parallel, the amount of native alpha 2M bound to KTL3 bacteria was estimated by calculating the amounts of trypsin trapped by alpha 2M. This Nalpha -bensoyl-L-arginine p-nitroanilide assay showed that 2 × 109 KTL3 bacteria bound 0.27 ± 0.03 µg (n = 3) of native alpha 2M, which represents approximately 50% of what could be eluted from the bacteria. These results demonstrate that a major part of the alpha 2M bound to the surface of KTL3 is active as a proteinase inhibitor (Fig. 8). The complexes between trypsin and alpha 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 alpha 2M binding of KTL3 bacteria (data not shown), making it impossible to determine whether the release of the trypsin-alpha 2M complexes from the KTL3 surface was due to degradation of protein GRAB or to dissociation of trypsin-alpha 2M complexes from protein GRAB.


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Fig. 8.   alpha 2M bound to protein GRAB protects the M protein from trypsin digestion. KTL3 or MR4 bacteria were either preincubated with buffer (KTL3 and MR4), alpha 2M (KTL3+alpha 2M and MR4+alpha 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 alpha 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 alpha 2M pretreatment, whereas binding of fibrinogen to MR4 was unaffected by preincubation with alpha 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 alpha 2M was due to inhibition of trypsin and not merely an effect of alpha 2M being a substrate for trypsin.

alpha 2M Traps SCP and Protects the M Protein from SCP Degradation-- Radiolabeled and activated SCP was mixed with either purified alpha 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 alpha 2M, indicating that a covalent complex had been formed between SCP and alpha 2M (Fig. 9A). Pretreatment of KTL3 and MR4 with alpha 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 alpha 2M pretreatment. When bound material was eluted from the bacteria and subjected to SDS-PAGE and autoradiography, SCP was in complex with alpha 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 alpha 2M pretreated KTL3 bacteria could be seen as band with the apparent size of alpha 2M (data not shown).


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Fig. 9.   alpha 2M traps SCP and protects M protein from SCP digestion. A, radiolabeled and activated SCP was mixed with alpha 2M or plasma. These samples (SCP+alpha 2M and SCP+plasma), radiolabeled and activated SCP (SCP), or radiolabeled alpha 2M (alpha 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 alpha 2M (KTL3+alpha 2M and MR4+alpha 2M). Bacteria were washed and incubated with activated and radiolabeled SCP. Following washing and elution these samples, radiolabeled and activated SCP (SCP), or radiolabeled alpha 2M (alpha 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 alpha 2M (KTL3+alpha 2M and MR4+alpha 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 alpha 2M could inhibit this proteolytic cleavage, KTL3 and MR4 bacteria were harvested at logarithmic growth phase. The bacteria were incubated with alpha 2M, washed, and subjected to SCP digestion. The bacteria were tested for their ability to bind radiolabeled fibrinogen. The results show that alpha 2M pretreatment preserved the fibrinogen binding of the KTL3 strain and not of the MR4 strain (Fig. 9C). The experiments described above demonstrate that alpha 2M in solution, or bound to S. pyogenes via protein GRAB, can trap SCP and thereby protect M protein from SCP cleavage.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 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 alpha 2M (12). Whereas the interactions with IgG and human serum albumin are mediated by separate repeated domains (45, 46), alpha 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 alpha 2M to the very tip of these streptococcal surface proteins. It is possible that the large and bulky alpha 2M molecule requires this kind of exposed binding site, which will also allow alpha 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 alpha 2M. The nature of the low affinity interaction is not known, and it is not likely to be of importance, because the amount of alpha 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 alpha 2M binding adds selective advantages to streptococci. This notion is also supported by the similar alpha 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 alpha 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 alpha 2M was completely lost. To investigate whether alpha 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 alpha 2M-binding surface protein expressed by KTL3 bacteria and the S. pyogenes alpha 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 alpha 2M is also different from that of the 78-kDa protein and alpha 2M. When bound to the 78-kDa protein, alpha 2M was reported to be converted to a non-native form (10). This is in contrast to the alpha 2M bound to KTL3 cells via protein GRAB. In this case alpha 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 alpha 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 alpha 2M bound to protein GRAB protects the M protein from trypsin degradation. This implicates that the presence of alpha 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 alpha 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 alpha 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 alpha 2M has also been implicated in immune regulation (30-33). Moreover alpha 2M is an important carrier of zinc and other trace metals in plasma (34), and binding of alpha 2M to the bacterial surface could be a mean for the bacterium to capture these essential metal ions. If protein GRAB and its alpha 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.

    ACKNOWLEDGEMENTS

We acknowledge the Streptococcal Genome Sequencing Project, which is funded by U. S. Public Health Service/National Institutes of Health Grant AI38406, and B. A. Roe, S. P. Linn, L. Song, X. Yuan, S. Clifton, M. McShan, and J. Ferretti. We are indebted to the referees for helpful and constructive comments on this manuscript.

    FOOTNOTES

* This work was supported by grants from Swedish Medical Research Council Project 7480; the Medical Faculty of Lund University, the Foundations of Kock, Lundberg, and Österlund; the Göran Gustavsson Foundation for Research in Natural Sciences and Medicine; and Actinova Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, P.O. Box 94, S-221 00 Lund, Sweden. Tel.: 46-46-2224492; Fax: 46-6-157756; E-mail: lars.bjorck{at}medkem.lu.se.

    ABBREVIATIONS

The abbreviations used are: alpha 2M, alpha 2-macroglobulin; SCP, streptococcal cysteine proteinase; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; MBP, maltose-binding protein; aa, amino acid(s).

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