Title: Group A Streptococcal Cysteine Protease Degrades C3 (C3b) and Contributes to Evasion of Innate Immunity
Abstract: A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic ΔspeB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the ΔspeB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the ΔspeB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections. A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic ΔspeB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the ΔspeB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the ΔspeB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections. Streptococcus pyogenes is a Gram-positive bacterium that often causes throat and skin infections such as pharyngitis and impetigo. During the past decade, it was reported that S. pyogenes causes severe infectious diseases, including acute rheumatic fever, necrotizing fasciitis, and streptococcal toxic shock syndrome (STSS) 2The abbreviations used are:STSSstreptococcal toxic shock syndromePBSphosphate-buffered salinecfucolony forming unitsHEhematoxylin and eosin.2The abbreviations used are:STSSstreptococcal toxic shock syndromePBSphosphate-buffered salinecfucolony forming unitsHEhematoxylin and eosin. (1Stevens D.L. Tanner M.H. Winship J. Swarts R. Ries K.M. Schlievert P.M. Kaplan E. N. Engl. J. Med. 1989; 321: 1-7Crossref PubMed Scopus (969) Google Scholar, 2Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. Lancet Infect. Dis. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (1906) Google Scholar). The death toll from severe infections was estimated to be at least 500,000 each year (2Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. Lancet Infect. Dis. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (1906) Google Scholar). Therefore, many researchers have focused on S. pyogenes, and some aspects of the infection have been elucidated.The initial step of S. pyogenes infection is bacterial adhesion to host epithelial cells through extracellular matrix proteins, for example, fibronectin (3Hanski E. Caparon M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6172-6176Crossref PubMed Scopus (261) Google Scholar, 4Terao Y. Kawabata S. Kunitomo E. Murakami J. Nakagawa I. Hamada S. Mol. Microbiol. 2001; 42: 75-86Crossref PubMed Scopus (147) Google Scholar, 5Terao Y. Kawabata S. Nakata M. Nakagawa I. Hamada S. J. Biol. Chem. 2002; 277: 47428-47435Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Fibronectin-binding proteins of S. pyogenes have been identified as adhesins and invasins, and their role in the invasion of epithelial cells by S. pyogenes has been examined in several studies (6Cue D. Southern S.O. Southern P.J. Prabhakar J. Lorelli W. Smallheer J.M. Mousa S.A. Cleary P.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2858-2863Crossref PubMed Scopus (88) Google Scholar, 7Terao Y. Okamoto S. Kataoka K. Hamada S. Kawabata S. J. Infect. Dis. 2005; 192: 2081-2091Crossref PubMed Scopus (38) Google Scholar); however, how the invading bacteria escape the immune system and grow in host tissues is not understood. To cause systemic and septic infections, S. pyogenes must evade the immune system of the host and survive in plasma, after which the organisms may spread into various organs via the blood stream. Histopathological studies have demonstrated that there are few or no inflammatory cells (e.g. neutrophils) at the site of infection in patients with STSS and severe streptococcal infections (8Cockerill III, F.R. Thompson R.L. Musser J.M. Schlievert P.M. Talbot J. Holley K.E. Harmsen W.S. Ilstrup D.M. Kohner P.C. Kim M.H. Frankfort B. Manahan J.M. Steckelberg J.M. Roberson F. Wilson W.R. Clin. Infect. Dis. 1998; 26: 1448-1458Crossref PubMed Scopus (51) Google Scholar, 9Hidalgo-Grass C. Dan-Goor M. Maly A. Eran Y. Kwinn L.A. Nizet V. Ravins M. Jaffe J. Peyser A. Moses A.E. Hanski E. Lancet. 2004; 363: 696-703Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).The complement system plays an important role in innate immunity, which acts as a protective shield at early phases of infection. In addition, the complement system is also an effector in the acquired immune system. Complement fragments C3a, C3b, and C5a are produced during activation of the complement system (10Müller-Eberhard H.J. Schreiber R.D. Adv. Immunol. 1980; 29: 1-53Crossref PubMed Scopus (281) Google Scholar). C3a and C5a work as anaphylatoxins that attract neutrophils. C5a and interleukin-8 are key neutrophil chemoattractants. It has been reported that C5a and interleukin-8 are neutralized by S. pyogenes (9Hidalgo-Grass C. Dan-Goor M. Maly A. Eran Y. Kwinn L.A. Nizet V. Ravins M. Jaffe J. Peyser A. Moses A.E. Hanski E. Lancet. 2004; 363: 696-703Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Chen C.C. Cleary P.P. J. Biol. Chem. 1990; 265: 3161-3167Abstract Full Text PDF PubMed Google Scholar, 12Edwards R.J. Taylor G.W. Ferguson M. Murray S. Rendell N. Wrigley A. Bai Z. Boyle J. Finney S.J. Jones A. Russell H.H. Turner C. Cohen J. Faulkner L. Sriskandan S. J. Infect. Dis. 2005; 192: 783-790Crossref PubMed Scopus (138) Google Scholar). C3b binds strongly to the surface of bacteria, mediating their opsonization (10Müller-Eberhard H.J. Schreiber R.D. Adv. Immunol. 1980; 29: 1-53Crossref PubMed Scopus (281) Google Scholar, 13Pangburn M.K. Morrison D.C. Schreiber R.D. Müller-Eberhard H.J. J. Immunol. 1980; 124: 977-982PubMed Google Scholar, 14Foster T.J. Nat. Rev. Microbiol. 2005; 3: 948-958Crossref PubMed Scopus (863) Google Scholar). In addition, surface-bound C3b forms a complex with C2a and C4b and functions as a C5a convertase, generating the most powerful anaphylatoxin, C5a (10Müller-Eberhard H.J. Schreiber R.D. Adv. Immunol. 1980; 29: 1-53Crossref PubMed Scopus (281) Google Scholar, 13Pangburn M.K. Morrison D.C. Schreiber R.D. Müller-Eberhard H.J. J. Immunol. 1980; 124: 977-982PubMed Google Scholar, 14Foster T.J. Nat. Rev. Microbiol. 2005; 3: 948-958Crossref PubMed Scopus (863) Google Scholar).Pandiripally et al. (15Pandiripally V. Gregory E. Cue D. Infect. Immun. 2002; 70: 6206-6214Crossref PubMed Scopus (81) Google Scholar) reported that the surface protein FbaA of S. pyogenes recruits complement regulatory protein factor H (FH) and factor H-like protein 1 (FHL-1Stevens D.L. Tanner M.H. Winship J. Swarts R. Ries K.M. Schlievert P.M. Kaplan E. N. Engl. J. Med. 1989; 321: 1-7Crossref PubMed Scopus (969) Google Scholar). Based on this, the we postulated that C3b is inactivated on the surface of S. pyogenes. To date, however, there have been few reports on C3 (C3b)-degrading molecules in S. pyogenes or other bacteria.We recently found that C3 (C3b) is degraded in sera from infected areas in patients with STSS but not in sera from healthy volunteers. Additionally, genetic inactivation of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease, in S. pyogenes decreases the bacterium resistance to phagocytosis and reduces its ability to spread into various organs in mice (16Lukomski S. Burns Jr., E.H. Wyde P.R. Podbielski A. Rurangirwa J. Moore-Poveda D.K. Musser J.M. Infect. Immun. 1998; 66: 771-776Crossref PubMed Google Scholar, 17Kuo C.F. Luo Y.H. Lin H.Y. Huang K.J. Wu J.J. Lei H.Y. Lin M.T. Chuang W.J. Liu C.C. Jin Y.T. Lin Y.S. Microb. Pathog. 2004; 36: 273-285Crossref PubMed Scopus (25) Google Scholar). These studies, however, did not examine how SpeB contributes to bacterial propagation in vivo. On the basis of the previous findings, we hypothesized that S. pyogenes produces one or more C3 (C3b)-degrading proteases, one of which is SpeB, allowing the bacterium to evade the host immune system.EXPERIMENTAL PROCEDURESHuman Sera and Streptococcal Strains—All of the studies on patients and volunteers were performed with approval of the ethics committee Osaka University Graduate School of Dentistry and Asahi General Hospital, and all patients gave informed consent. Human blood samples were collected from the veins of eight healthy volunteers (age 31.3 ± 4.9 years) and during surgery in 13 patients with STSS (age 35.2 ± 23.8 years) from an infected area in the acute phase at the surgical site (supplemental Fig. S1). S. pyogenes strain SSI-9 (serotype M1) was isolated from a Japanese patient with STSS (4Terao Y. Kawabata S. Kunitomo E. Murakami J. Nakagawa I. Hamada S. Mol. Microbiol. 2001; 42: 75-86Crossref PubMed Scopus (147) Google Scholar), and its isogenic ΔspeB mutant was constructed with a suicide vector as described previously (18Okamoto S. Kawabata S. Terao Y. Fujitaka H. Okuno Y. Hamada S. Infect. Immun. 2004; 72: 6068-6075Crossref PubMed Scopus (39) Google Scholar). Briefly, we amplified an internal portion of the speB gene by PCR and ligated it into pSF151 (19Tao L. LeBlanc D.J. Ferretti J.J. Gene. 1992; 120: 105-110Crossref PubMed Scopus (131) Google Scholar). Strain SSI-9 was transformed with the resulting plasmid by electroporation, and the transformant was selected on a spectinomycin-containing agar plate. Following verification of targeted mutagenesis by colony-directed PCR (4Terao Y. Kawabata S. Kunitomo E. Murakami J. Nakagawa I. Hamada S. Mol. Microbiol. 2001; 42: 75-86Crossref PubMed Scopus (147) Google Scholar), we designated the ΔspeB mutant as strain TR-11. There was no growth difference on laboratory medium between strains SSI-9 and TR-11 (18Okamoto S. Kawabata S. Terao Y. Fujitaka H. Okuno Y. Hamada S. Infect. Immun. 2004; 72: 6068-6075Crossref PubMed Scopus (39) Google Scholar).Construction of Recombinant SpeB—rSpeB was prepared as follows. Chromosomal DNA was extracted from S. pyogenes strain SSI-9 as a template for PCR. The entire length of the speB gene was amplified by PCR using primers 5′-CATGCCATGGATAAAAAGAAATTAGGTATCAG-3′ (sense, containing an NcoI recognition site) and 5′-CCCCCGGGAGGTTTGATGCCTACAACAGCACTTTGG-3′ (antisense, containing a SmaI recognition site). To produce rSpeB with a C-terminal histidine tag, the PCR fragment was digested with NcoI and SmaI and then cloned into NcoI/SmaI-digested pIVEX2.3-MCS vector (Roche Applied Science). The scpA (streptococcal C5a peptidase) gene was provided by Dr. P. P. Cleary (University of Minnesota) (11Chen C.C. Cleary P.P. J. Biol. Chem. 1990; 265: 3161-3167Abstract Full Text PDF PubMed Google Scholar) and was ligated into expression vector pGEX-6P-1 (GE Healthcare Bio-Sciences KK). The resulting plasmids, which harbored the speB or the scpA gene, were introduced into Escherichia coli strain BL21 (DE3) pLysE (Novagen), and the transformant was induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside at 20 °C for 24 h. The rSpeB protein was produced as a 43-kDa zymogen and converted to 28-kDa active form by self-processing during purification by nickel-nitrilotriacetic acid-agarose (Qiagen) and diethylaminoethyl column chromatography (Bio-Rad). The rScpA protein was purified by glutathione-Sepharose 4B (GE Healthcare Bio-Sciences KK) and diethylaminoethyl column chromatography. The purified rSpeB and rScpA (C5a peptidase) proteins were dialyzed against PBS.Western Blot Analysis—Western blotting was performed as described previously (5Terao Y. Kawabata S. Nakata M. Nakagawa I. Hamada S. J. Biol. Chem. 2002; 277: 47428-47435Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Sera from patients with STSS or healthy volunteers were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking with a solution of 5% skim milk and 10% goat serum (Tissue Culture Biologicals), the membrane was incubated with rabbit anti-human C3b serum (Nordic Immunology). Immunoreactive bands were detected with alkaline phosphatase-labeled goat anti-rabbit IgG (Cell Signaling) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium alkaline phosphatase substrate solution (Moss). To examine the proteolytic activity of SpeB, 5 μl of C3b (Calbiochem, 200 μg/ml) or human sera were incubated with an equal volume of rSpeB (0–10 μm), wild-type strain SSI-9 (2 × 106 cfu/ml), or the ΔspeB mutant strain TR-11 (2 × 106 cfu/ml). Following a 1-h incubation at 37 °C, each mixture was examined by Western blot analysis with anti-human C3b serum.N-terminal Amino Acid Sequencing—The targeted protein was separated using 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was stained with 0.1% Coomassie Brilliant Blue R-250 (CBB) for 1 h and then destained with 7.5% acetic acid containing 40% methanol and washed with distilled water for 24 h. N-terminal amino acid sequencing was performed using the Edman degradation method with an ABI protein sequencer model 491HT (Applied Biosystems).Biomolecular Interaction Analysis (Biacore)—The direct surface plasmon resonance was measured using the Biacore X biomolecular interaction monitoring system (Biacore International AB). Biacore analysis was performed as described previously (20Terao Y. Yamaguchi M. Hamada S. Kawabata S. J. Biol. Chem. 2006; 281: 14215-14223Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Human C3b was diluted and adjusted to 100 μg/ml in 10 mm sodium acetate (pH 4.0) and then immobilized on the surface of a sensor chip CM5 (Biacore International AB) using an Amine Coupling Kit (Biacore International AB). Lyophilized rSpeB was suspended in HBSP buffer (10 mm HEPES, 150 mm NaCl, 3.4 mm EDTA, 0.005% Tween 20, pH 7.4) and adjusted to 2.5, 5, and 10 μm. For binding analysis, rSpeB was injected at a flow rate of 5 μl/min at 37 °C. C3b degrading activity was quantified as the decrease in resonance units on the sensorgram and analyzed with BIA evaluation software version 4.02 (Biacore International AB) as described previously (21Steinrücke P. Aldinger U. Hill O. Hillisch A. Basch R. Diekmann S. Anal. Biochem. 2000; 286: 26-34Crossref PubMed Scopus (23) Google Scholar).Bactericidal Test—In vitro whole blood bactericidal assays were performed with a modified version of the bactericidal test described by Lancefield (22Lancefield R.C. J. Exp. Med. 1957; 107: 525-544Crossref Scopus (173) Google Scholar). S. pyogenes strains from patients were cultured until mid-log phase (optical density at 600 nm = 0.5–0.6) and adjusted with PBS or culture supernatant to 2–5 × 103 cfu/ml. Human whole blood was collected from a healthy donor, who was asked to confirm that the donor did not have an infection. The blood was also examined by ASO-latex (Seiken) to confirm that it was not infected with S. pyogenes. The blood was heparinized, and 90-μl samples of it were mixed with 10 μl of each bacterial suspension. After a 3-h incubation at 37 °C on a rotary mixer, the mixtures were grown on blood-agar plates, and the number of colonies were counted. For the C3b inhibition assay, rabbit anti-C3b or preimmune F(ab′)2 fragment was prepared using an ImmunoPure F(ab′)2 preparation kit (Pierce), and, prior to the bactericidal assay, 10 μg of the F(ab′)2 fragment was incubated with 100 μl of human blood at 37 °C for 1 h.Infection of Mice—All of the animal procedures complied with the Osaka University Graduate School of Dentistry guidelines and were approved by the institutional animal care and ethics committee. BALB/c mice (6-week-old, female) were purchased from Charles River Japan. S. pyogenes strains were grown at 37 °C to mid-log phase and washed twice with PBS. The bacteria were suspended in PBS, and the suspensions were adjusted to 2 × 106 cfu/ml. The mice were anesthetized and injected subcutaneously or intramuscularly in the quadriceps with 0.1 ml of bacterial suspension followed by 0.9 ml of air to form an air pouch (17Kuo C.F. Luo Y.H. Lin H.Y. Huang K.J. Wu J.J. Lei H.Y. Lin M.T. Chuang W.J. Liu C.C. Jin Y.T. Lin Y.S. Microb. Pathog. 2004; 36: 273-285Crossref PubMed Scopus (25) Google Scholar, 22Lancefield R.C. J. Exp. Med. 1957; 107: 525-544Crossref Scopus (173) Google Scholar). The mice were anesthetized and blood was collected every 24 h for 7 days, and 100 μl of the blood were spread on blood-agar plates and incubated at 37 °C. All of theβ-hemolytic colonies were examined for the presence of S. pyogenes by colony-directed PCR with fbaA-specific primers and counted. In addition, muscle samples were obtained under anesthesia from mice that received an intramuscular injection. The samples were taken so that a wide margin around the abscess or the injection site was collected. The excised tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (HE) solution (Fluka) or were immunostained with rabbit anti-FbaA serum (4Terao Y. Kawabata S. Kunitomo E. Murakami J. Nakagawa I. Hamada S. Mol. Microbiol. 2001; 42: 75-86Crossref PubMed Scopus (147) Google Scholar) and an EnVision kit (Dako).C3 (C3b) Degradation and Measurement of Complement Activity in Vivo—BALB/c mice were infected with strain SSI-9 or TR-11 in the quadriceps, and blood samples were collected on days 0, 2, 7, and 14. Whole blood was centrifuged, and the serum was analyzed using a complement activity measuring kit, CH50 (Seiken). Two weeks after infection, each quadriceps section was incised, shredded, and frozen at -80 °C. After 10 min, the tissues were centrifuged at 4 °C, and the supernatants were immediately applied to a 7.5% SDS-PAGE gel. C3 (C3b) degradation at the site of infection was examined by Western blotting using anti-C3b serum.Statistical Analysis—The significance of differences between the means of groups was evaluated using a nonparametric Mann-Whitney U test. All of the tests were analyzed using Stat-View J-5.0 software (SAS Institute Inc.). Differences were considered significant at p < 0.05.RESULTSC3 (C3b) Was Not Detected in Sera Collected from Infected Areas of STSS Patients—In most cases of STSS, histopathological examination showed little or no infiltration of neutrophils around S. pyogenes at early stages of infection, and the bacterial organisms are found along the fascia (Fig. 1). To investigate the interaction between the host and S. pyogenes in STSS, during operations, sera were collected from the infected area of STSS patients. We then examined whether S. pyogenes isolated from an STSS patient can degrade complement C3 (C3b), a complement fragment that plays an important role in innate immunity by activating the alternative pathway and by binding to the bacterial surfaces to mediate opsonization. In the alternative pathway, C3b acts with factor B to form the alternative pathway C3 convertase. Subsequently, the C3 convertase may act with an additional C3b molecule to form the alternative pathway C5 convertase. During activation via classical pathway, C4 acts with C2 to form the classical pathway C5 convertase upon addition of a C3b molecule, and then the C5 convertase generates C5a. Therefore, elimination of C3 (C3b) by S. pyogenes is expected to prevent the accumulation of C5a, the most powerful anaphylatoxin, reducing neutrophil migration and bacterial opsonization. As shown in Fig. 2, Western blotting with anti-C3b showed that C3b (C3) was not present in sera from patients with STSS collected from the site of infection, whereas C3 (C3b) was clearly detected in sera from all of the healthy volunteers. In contrast, C5 in blood were very low level. There was not a significant difference among all samples from the healthy volunteers and patients with STSS by Western blotting (supplemental Fig. S2). Therefore, we speculated that, in STSS, C3 (C3b) is degraded into fragments at sites of infection by S. pyogenes proteases.FIGURE 2Analysis of C3 (C3b) in human serum samples. C3 (C3b) was examined by Western blot analysis using anti-C3b serum. A, blood samples were collected from eight healthy volunteers (age 31.3 ± 4.9 years). Purified C3b (lane C3b) and control healthy serum (lane C; Sigma) were used as positive controls. B, blood samples were collected from 13 patients with STSS (age 35.2 ± 23.8 years). In both panels, lane M contains molecular mass markers. The blood samples from patients with STSS were from infected areas (see Fig. S1), which means that the healthy donor bloods from veins might not be an exact control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Streptococcal Cysteine Protease SpeB Degrades Complement C3 (C3b)—S. pyogenes expresses two major proteases, one of which is an extracellular cysteine protease named SpeB (24Bohach G.A. Hauser A.R. Schlievert P.M. Infect. Immun. 1988; 56: 1665-1667Crossref PubMed Google Scholar), and the other is a cell-associated serine protease named C5a peptidase (11Chen C.C. Cleary P.P. J. Biol. Chem. 1990; 265: 3161-3167Abstract Full Text PDF PubMed Google Scholar). We tested the ability of recombinant SpeB (rSpeB) and C5a peptidase to degrade purified C3b in healthy human serum. Fig. 3A shows that the purified human C3b (>116-kDa bands; black arrows) was degraded by rSpeB into ∼85-kDa fragments (gray arrowheads). In contrast, recombinant C5a peptidase, ScpA, could not degrade C3b (data not shown). Furthermore, rSpeB degraded C3b in serum, even though it contains a variety of ions and inhibitors (Fig. 3A, lanes 6 and 7).FIGURE 3Degradation of C3b by SpeB protease. A, purified C3b or healthy human serum was incubated with rSpeB or S. pyogenes for 1 h at 37 °C. The reactions were analyzed by Western blotting with anti-C3b serum. The black arrows indicate intact C3b or alternatively processed forms, and the red arrowheads show the C3b degradation products. Lane 1, molecular mass markers; lane 2, 1 μg of purified C3b; lane 3, 10 μm purified rSpeB; lanes 4–6, 1 μg of C3b with 0.1–10 μm rSpeB; lane 7, healthy human serum; lane 8, healthy human serum with 10 μm rSpeB; lane 9, healthy human serum and wild-type strain SSI-9 (1 × 104 cfu/well); lane 10, healthy human serum with the ΔspeB strain TR-11 (1 × 104 cfu/well). B, 100 μl of 1 μm rSpeB or S. pyogenes strains SSI-9 or TR-11 (1 × 104 cfu/well) were added to 5% skim milk agar. After a 1-h incubation at 37 °C, the proteolytic activities were observed under the light. C, reverse transcription-PCR analysis of S. pyogenes strain SSI-9 and TR-11. Total RNA was extracted from each strain at the mid-log phase and subjected to reverse transcription-PCR with targeted gene-specific primer pairs, as described previously (4Terao Y. Kawabata S. Kunitomo E. Murakami J. Nakagawa I. Hamada S. Mol. Microbiol. 2001; 42: 75-86Crossref PubMed Scopus (147) Google Scholar, 18Okamoto S. Kawabata S. Terao Y. Fujitaka H. Okuno Y. Hamada S. Infect. Immun. 2004; 72: 6068-6075Crossref PubMed Scopus (39) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further investigate the role of native SpeB in C3b degradation, we compared its degradation in serum by wild-type S. pyogenes strain SSI-9, which was isolated from a patient with STSS, and its isogenic ΔspeB mutant strain TR-11, which was constructed by integrating the spectinomycin resistance gene into the speB gene. Homologous recombination in TR-11 was confirmed by colony-directed PCR (data not shown) and by the loss of the proteolytic activity on skim milk agar (Fig. 3B). Western blotting showed that SSI-9 but not TR-11 could cleave serum C3 (C3b) (Fig. 3A, lanes 8 and 9). Reverse transcription-PCR analysis was used to investigate the transcriptional levels of other virulence genes in strain TR-11, which confirmed that the major virulence factors were not affected by deletion of the speB gene, as compared with the wild-type strain SSI-9 (Fig. 3C). We also extracted the cleaved fragment of C3b from a 15% SDS-PAGE gel and determined the N-terminal amino acid sequence to be GQREVVAD, which was identical to the N terminus of the β-chain (25Bruijn de M.H. Fey G.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 708-712Crossref PubMed Scopus (306) Google Scholar). In addition, the other SpeB-cleavage sites were determined by using a partial cleavage condition (15 min, 37 °C). Identical sites were used, as follows: LLPVG (156 amino acid residues from the N terminus of C3), SNLDEDIIA (749 amino acid residues), DPERLGREG (948 amino acid residues), and IQPGA (1472 amino acid residues). These results showed that SpeB was able to cleave one or more sites of the C3 (C3b) molecule.Recombinant SpeB Rapidly Degrades C3b—These results might support the hypothesis that SpeB contributes to the inhibition of the complement system by degrading C3b; however, there was a problem that required further investigation. To evade opsonization or inhibit other immunological functions of C3b, it is necessary that S. pyogenes cannot only degrade C3b but also carry out the degradation rapidly when the two proteins come into contact. Biacore analysis confirmed that rSpeB can rapidly cleave immobilized C3b (Fig. 4A). The arrow in Fig. 4A indicates that the C3b in contact with SpeB shifted to a hydrolysis product. The initial rates of hydrolysis of C3b can be obtained from the initial slopes (first 30 s) in the sensorgram (Fig. 4A, lines a–c). In this system, 1000 resonance units correspond to a mass shift of 1 ng/mm2 (26Sternberg E. Persson B. Roos H. Urbaniczky C. Science. 1991; 143: 513-526Google Scholar). A plot of the initial rate of C3b cleavage as a function of the rSpeB concentration was linear (Fig. 4B). Biacore analysis further demonstrated that 10 μm rSpeB degrades C3b at an initial rate of 12 pg/s (Fig. 4B).FIGURE 4Biomolecular interactions of human C3b and rSpeB. A, Biacore analysis was performed to examine the proteolysis of human C3b by rSpeB. C3b (100 μg/ml) was immobilized on a sensor chip CM5. HBSP was used as a running buffer and was delivered at a flow rate of 5 μl/min at 37 °C. The concentration of rSpeB was adjusted to 2.5–10 μm, and a 60-μl injection volume was used. The reduction in resonance unit values indicates the extent of proteolytic cleavage. The vertical arrow indicates that the C3b in contact with SpeB shifted to a hydrolysis product. B, initial rate of C3b degradation as a function of the rSpeB concentration. The initial rate of proteolysis (first 30 s) was determined from the initial slope in the sensorgram (lines a–c in panel A). The correlation coefficient was 0.9978.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Survival Rate in Human Blood Was Lower for the ΔspeB Mutant than the Wild-type Strain—We next examined the survival of wild-type strain SSI-9 and ΔspeB strain TR-11 in the blood of healthy, uninfected volunteers. A 3-h bactericidal test (Fig. 5A) showed that strain SSI-9 survived significantly at a higher rate than strain TR-11 (p < 0.05). Interestingly, the wild-type strain survived at a higher rate when the suspension was made in culture supernatant than when it was made in PBS (p < 0.005), but this difference was not observed for the mutant strain. Furthermore, as shown in Fig. 5B, inhibi