SIC, a Secreted Protein of Streptococcus pyogenesThat Inactivates Antibacterial Peptides

Abstract

Some isolates of the significant human pathogenStreptococcus pyogenes, including virulent strains of the M1 serotype, secrete protein SIC. This molecule, secreted in large quantities, interferes with complement function. As a result of natural selection, SIC shows a high degree of variation. Here we provide a plausible explanation for this variation and the fact that strains of the M1 serotype are the most frequent cause of severe invasive S. pyogenes infections. Thus, protein SIC was found to inactivate human neutrophil α-defensin and LL-37, two major antibacterial peptides involved in bacterial clearance. This inactivation protectedS. pyogenes against the antibacterial effect of the peptides. Moreover, SIC isolated from S. pyogenes of the M1 serotype was more powerful in this respect than SIC variants from strains of M serotypes 12 and 55, serotypes rarely connected with invasive infections. Some isolates of the significant human pathogenStreptococcus pyogenes, including virulent strains of the M1 serotype, secrete protein SIC. This molecule, secreted in large quantities, interferes with complement function. As a result of natural selection, SIC shows a high degree of variation. Here we provide a plausible explanation for this variation and the fact that strains of the M1 serotype are the most frequent cause of severe invasive S. pyogenes infections. Thus, protein SIC was found to inactivate human neutrophil α-defensin and LL-37, two major antibacterial peptides involved in bacterial clearance. This inactivation protectedS. pyogenes against the antibacterial effect of the peptides. Moreover, SIC isolated from S. pyogenes of the M1 serotype was more powerful in this respect than SIC variants from strains of M serotypes 12 and 55, serotypes rarely connected with invasive infections. Todd Hewitt broth phosphate-buffered saline colony forming units PBS Tween enzyme-linked immunosorbent assay polyvinylidene difluoride Streptococcus pyogenes is one of the most common and important human bacterial pathogens. It causes relatively mild infections such as pharyngitis (strep throat) and impetigo but also serious clinical conditions like rheumatic fever, poststreptococcal glomerulonephritis, necrotizing fasciitis, septicemia, and a toxic-shock syndrome (1Bisno A.L. Stevens D.L. New Engl. J. Med. 1996; 334: 240-245Crossref PubMed Scopus (843) Google Scholar, 2Cunningham M.W. Clin. Microbiol. Rev. 2000; 13: 470-511Crossref PubMed Scopus (1769) Google Scholar). Increases in the number of life-threatening systemic S. pyogenes infections have been reported world-wide since the late 1980s and have attracted considerable attention and concern (3Nowak R. Science. 1994; 264: 1665Crossref PubMed Scopus (62) Google Scholar, 4Stevens D.L. Annu. Rev. Med. 2000; 51: 271-288Crossref PubMed Scopus (163) Google Scholar). Based on the highly polymorphic M protein, a surface protein of S. pyogenes (for references see Ref. 5Fischetti V.A. Clin. Microbiol. Rev. 1989; 2: 285-314Crossref PubMed Scopus (638) Google Scholar), isolates are divided into more than 100 serological subtypes, and systemic infections are most frequently caused by organisms of the M1 serotype (6Musser J.M. Krause R.M. Krause R.M. Emerging Infections. Academic Press, New York1998: 185-218Google Scholar). Protein SIC was originally isolated from the growth medium of an M1 strain (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). All strains of the M1 serotype secrete SIC and so do M57 organisms, whereas strains of 53 other serotypes were found to lack thesic gene (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Subsequent work has identified distantly related sic variants also in M12 and M55 strains (8Hartas J. Sriprakash K.S. Microb. Pathog. 1999; 26: 25-33Crossref PubMed Scopus (36) Google Scholar). SIC stands for streptococcal inhibitor ofcomplement, as the protein incorporates into the membrane attack complex of complement and inhibits complement-mediated lysis of sensitized erythrocytes (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). This inhibition of membrane attack complex was recently shown to be the result of SIC preventing uptake of C567 onto cell membranes (9Fernie-King B.A. Seilly D.J. Willers C. Wurzner R. Davies A. Lachmann P.J. Immunology. 2001; 103: 390-398Crossref PubMed Scopus (84) Google Scholar). A remarkable property of SIC was reported by Stockbauer et al. (10Stockbauer K.E. Grigsby D. Pan X. Fu Y.X. Mejia L.M. Cravioto A. Musser J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3128-3133Crossref PubMed Scopus (52) Google Scholar). They found that the sequences of a large number of sic genes from different strains of the M1 serotype showed a unique degree of variation, which is in striking contrast to the lack of M1 protein variation. Moreover, in a mouse model of infection, Hoe et al. (11Hoe N.P. Nakashima K. Lukomski S. Grigsby D. Liu M. Kordari P. Dou S.J. Pan X. Vuopio-Varkila J. Salmelinna S. McGeer A. Low D.E. Schwartz B. Schuchat A. Naidich S. De Lorenzo D. Fu Y.X. Musser J.M. Nat. Med. 1999; 5: 924-929Crossref PubMed Scopus (69) Google Scholar) discovered that SIC variants arise rapidly on mucosal surfaces by natural selection. They also reported that the inhibition of complement-mediated lysis by SIC was not affected in the new SIC variants arising from natural selection, suggesting that complement inhibition is not the only function of SIC. Complement belongs to the innate immune system, and antibacterial peptides represent another important part of this defense system. These peptides, originally described in silk worms (12Steiner H. Hultmark D. Engstrom A. Bennich H. Boman H.G. Nature. 1981; 292: 246-248Crossref PubMed Scopus (1122) Google Scholar), play important roles in the clearance of bacteria at biological boundaries susceptible for infection (for references see reviews in Refs. 13Boman H.G. Immunol. Rev. 2000; 173: 5-16Crossref PubMed Scopus (293) Google Scholar, 14Lehrer R.I. Ganz T. Curr. Opin. Immunol. 1999; 11: 23-27Crossref PubMed Scopus (660) Google Scholar, 15Schröder J.-M. Harder J. Int. J. Biochem. Cell Biol. 1999; 31: 645-651Crossref PubMed Scopus (394) Google Scholar, 16Selsted M.E. Ouellette A.J. Trends Cell Biol. 1995; 5: 114-119Abstract Full Text PDF PubMed Scopus (117) Google Scholar), and the starting point for this investigation was the hypothesis that SIC, secreted in substantial amounts by S. pyogenes (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), could interfere with the activity of antibacterial peptides. Such a mechanism could help explain the variation of SIC and the high frequency of S. pyogenesinfections caused by strains of the M1 serotype. TheS. pyogenes strains AP1 (40/58) of serotype M1 and AP12 (1/71) of serotype M12 were from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. The S. pyogenes strain W38 (GT 71–154) of serotype M55 was from the late Dr. L. W. Wannamaker, and the S. pyogenes strains U15 and U17 of serotype M1 were kindly provided by Dr. Stig Holm, Umeå University, Umeå, Sweden. Bacteria were grown in Todd-Hewitt broth (TH1; Difco) at 37 °C. Protein SIC was purified from the S. pyogenes strains AP1, U15, U17, AP12, and W38 as described (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) by precipitation of the culture medium with 307 ammonium sulfate, followed by ion-exchange chromatography on Mono Q (Amersham Biosciences). Fractions containing protein SIC were combined, dialyzed against 2 mmNH4HCO3 and freeze-dried. For the antimicrobial assay (see below) protein SIC was dissolved in 10 mmTris-HCl, pH 7.5, containing 5 mm glucose. PCR was used to amplify the sicgenes from the S. pyogenes strains U15, U17, AP12, and W38. For serotype M1 strains (U15 and U17), primers were constructed corresponding to the nucleotide sequence starting 72 base pairs upstream (ACCTTTACTAATAATCGTCTTTGTTTTATAATGA) and 179 base pairs downstream (ATCTTTCTCGGACTCAGATAGTCCATAGC) of the coding sequence forsic in the AP1 strain (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). For serotype M12 (AP12) and M55 (W38) strains, a forward primer (CATTAACGAAATAATTTATTAAGGAGAG) corresponding to a region upstream of the coding sequence ofsic from AP1 and a reverse primer (CCAATGATAGTCACCAGCAATTCAGG) corresponding to a region downstream of the coding sequence of the distantly related sic from M12 and 55 strains (8Hartas J. Sriprakash K.S. Microb. Pathog. 1999; 26: 25-33Crossref PubMed Scopus (36) Google Scholar). The PCR products were purified with a High Pure PCR purification kit (Roche Applied Science) and used as templates in sequencing reactions using an ABI PRISM®BigDyeTM dideoxy terminator kit (BigDye terminator version 3.0 cycle sequencing Ready Reaction, Applied Biosystems), according to the manufacturer's instructions. Recombinant M1 protein was prepared as described previously (17Åkesson P. Schmidt K.-H. Cooney J. Björck L. Biochem. J. 1994; 300: 877-886Crossref PubMed Scopus (136) Google Scholar), α-defensin (HNP-1), ACYCRIPACIAGERRYGTCIYQGRLWAFCC (Mr 3442) was purchased from Sigma, and LL-37, LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (Mr4492) was synthesized by Innovagen AB, Lund, Sweden. The peptide GCP28, GCPRDIPTNSPELEETLTHTITKLNAEN, based on a sequence in human kininogen has been described previously (18Herwald H. Hasan A.A.K. Godovac-Zimmermann J. Schmaier A.H. Müller-Esterl W. J. Biol. Chem. 1995; 270: 14634-14642Abstract Full Text Full Text PDF PubMed Google Scholar) and was a kind gift from Dr. Heiko Herwald, Lund University, Lund, Sweden. Mouse monoclonal α-defensin antibodies were from Bachem AG, and rabbit polyclonal LL-37 antibodies were from Innovagen AB. Antiserum against protein SIC was raised in rabbits. Protein SIC was labeled with 125I using the chloramine T method (19Greenwood F.C. Hunter W.M. Glover J.S. Biochem. J. 1963; 89: 114-123Crossref PubMed Scopus (6742) Google Scholar). To inactivate the gene encoding protein SIC, AP1 bacteria were subjected to an allelic replacement mutagenesis strategy. A fragment of the up-stream sph and of the down-stream IS1562 was amplified from AP1 by PCR as described (20Rasmussen M. Müller H.-P. Björck L. J. Biol. Chem. 1999; 274: 15336-15344Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) using synthetic oligonucleotides hybridizing with nucleotides 486–512 and 1559–1533 in (21Gomi H. Hozumi T. Hattori S. Tagawa C. Kishimoto F. Björck L. J. Immunol. 1990; 144: 4046-4052PubMed Google Scholar) and 516–539 and 1614–1641 in (22Berge A. Rasmussen M. Björck L. Infect. Immun. 1998; 66: 3449-3453Crossref PubMed Google Scholar). The oligonucleotides had sites for restriction enzymes that were used to clone the sph product into multiple cloning site I of plasmid pFW13 (23Podbielski A. Spellerberg B. Woischnik M. Pohl B. Lüttiken R. Gene. 1996; 177: 137-147Crossref PubMed Scopus (176) Google Scholar) and the IS1562 product into multiple cloning site II to generate pFW13sic−. 20 ॖg of pFW13sic− was electroporated as described (24Hanski E. Fogg G. Tovi A. Okada N. Burstein I. Caparon M. Methods Enzymol. 1995; 253: 269-305Crossref PubMed Scopus (19) Google Scholar) into AP1 bacteria. Recombinants were selected on plates containing 150 ॖg/ml kanamycin. One transformant (SIC−) was obtained, and growth media from this strain was incubated with 67 trichloroacetic acid for 30 min on ice followed by centrifugation at 15,000 × g (4 °C for 20 min). Precipitated material was analyzed for SIC content using polyclonal anti-SIC antibodies in Western blot experiments. Total RNA from the AP1 and SIC− strains was isolated at early logarithmic (A620 0.3), late logarithmic (A620 0.6), or early stationary phase (A620 0.8) as described previously (20Rasmussen M. Müller H.-P. Björck L. J. Biol. Chem. 1999; 274: 15336-15344Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Northern blotting was performed as described (20Rasmussen M. Müller H.-P. Björck L. J. Biol. Chem. 1999; 274: 15336-15344Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) using a probe generated with primers hybridizing to sic (1–28, 912–889 in Ref. 7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Female NMRI mice weighing ∼25 g were injected subcutaneously with 104 mutant or wild-type bacteria in 100 ॖl of PBS followed by 900 ॖl of air. Mice were observed for 10 days. Statistical analysis of survival time was performed with the Wilcoxin rank sum test. AP1 bacteria were grown to mid-log phase in TH broth, washed, and diluted in 10 mm Tris-HCl, pH 7.5, containing 5 mm glucose. 50 ॖl of bacteria (2 × 106 colony forming units (cfu)/ml) were incubated together with α-defensin or LL-37 at various concentrations for 2 h at 37 °C. bacteria were incubated with α-defensin or LL-37 at a of together with different concentrations of protein SIC or M1 protein and the reactions were for 2 h or h To the activity of the were on TH incubated at 37 and the number of were AP1 and SIC− bacteria were grown to stationary phase in TH ॖl of TH was with 5 ॖl of the bacterial in plates at 37 °C. early logarithmic phase various amounts of LL-37 was and growth was followed by the at a The of protein SIC in the growth medium was by (see were to polyvinylidene difluoride membranes using a were in Tris-HCl, pH 7.5, containing 37 incubated protein SIC for and with containing Tween was using and was performed as described by Nature. PubMed Scopus Google Scholar) using a of or and were for min in containing and were with or subjected to Western blot were to membranes using a cell were with PBS containing Tween 20 and incubated with antibodies against protein SIC in for 30 min at 37 with and incubated with a against rabbit for 30 min at 37 °C. The membranes were washed, and of antibodies was performed using the was performed by plates with protein SIC or M1 protein at a of The plates were in in containing for 30 min and incubated with α-defensin or LL-37 for antibacterial peptides were with antibodies against α-defensin or LL-37 and was by a against mouse or rabbit All were performed at 37 °C for h followed by a in 100 pH was and the in at was 5 To the of SIC in growth medium plates with AP1 or SIC− growth medium were incubated with antibodies against protein SIC followed by a against rabbit of was as and was major and human antibacterial peptides, α-defensin and LL-37, were used in this These peptides have antibacterial activity against and bacteria. α-defensin is found in the of human but to neutrophil are also by A.J. M.E. J. 1996; PubMed Scopus Google Scholar, T. P. H. S.J. Ouellette A.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). LL-37 is by and α-defensin and LL-37 are found in including and the two peptides on bacteria I. S. S. A. M. 2000; PubMed Scopus Google Scholar). pyogenes, the sic gene is part of the and like the other genes of this sic is at an early growth phase (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), suggesting that SIC be secreted as as S. the sic gene to on an To α-defensin and LL-37 have for SIC, peptides and a peptide based on a sequence in (18Herwald H. Hasan A.A.K. Godovac-Zimmermann J. Schmaier A.H. Müller-Esterl W. J. Biol. Chem. 1995; 270: 14634-14642Abstract Full Text Full Text PDF PubMed Google Scholar), were to that were with SIC not SIC is from the M1 strain shows that SIC with the antibacterial peptides, an that was also by SIC and M1 protein were to followed by the of α-defensin or LL-37 and antibodies to the peptides. M1 protein was as a It was isolated from the strain of S. pyogenes as SIC (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), and the protein is with the cell is also from the bacterial surface by A. Björck L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). the in α-defensin and LL-37 showed for SIC, whereas the with M1 protein was at we the antibacterial effect of α-defensin and LL-37 on the AP1 the bacteria were and in to the of the peptides to that SIC was the As shown in of the peptides the bacteria. The for was for peptides. The effect of SIC was at a of the peptides and the (see that SIC the antibacterial activity of α-defensin and The inhibition that SIC is 10 more in LL-37 than M1 protein was also a of but showed grown to stationary the growth medium of S. pyogenes of the M1 serotype large of protein SIC (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). To inactivate the gene encoding protein SIC, AP1 bacteria were subjected to an allelic replacement mutagenesis strategy. One was obtained, and secreted amounts of protein SIC to the growth medium as with wild-type AP1 bacteria The ofsic from the AP1 and SIC− strains was at different of growth using Northern As shown in the of sic at late logarithmic growth and was at early stationary The was with a that the of RNA was to not the bacteria were the of the antibacterial peptide LL-37, the mutant strain SIC− was found to be as to LL-37 as the wild-type strain AP1 (see a of we SIC by M1 bacteria could the organisms against LL-37 substantial amounts of antibacterial and only LL-37 was The wild-type AP1 strain and the mutant strain SIC− were grown to early logarithmic growth phase the of SIC by AP1 bacteria was ॖg/ml growth medium as by The of protein SIC by the mutant SIC− was the this point different amounts of LL-37 were As shown in were more to LL-37 as with the strain The of LL-37 to of SIC− bacteria in was at this only of the wild-type bacteria were concentrations of LL-37 of SIC− but the AP1 strain was The of protein SIC was at different time the of SIC by AP1 bacteria in this system, at early stationary phase the of protein SIC in the growth medium of the mutant strain SIC− was the of The that SIC against antibacterial peptides at an of As SIC variants are by S. of M serotypes 55, and the as for SIC from M1 bacteria (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), SIC was purified from the growth medium of M12 and M55 to the M1 strain (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), the M12 and M55 strains ॖg of of growth medium grown to stationary Protein SIC was also purified from two strains of the M1 serotype, U15 and The purified variants shown in were for to interfere with the antibacterial activity of α-defensin and As shown in SIC but not M1 protein, the activity of the peptides. The inhibition that is more in this respect than the protein SIC variants from the M12 and M55 To a for this in sequencing of the genes encoding the SIC variants was As is a high degree of variation sicgenes from different strains of the M1 serotype (10Stockbauer K.E. Grigsby D. Pan X. Fu Y.X. Mejia L.M. Cravioto A. Musser J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3128-3133Crossref PubMed Scopus (52) Google Scholar), and are The acid sequences from the sequences were with that of protein SIC from the M1 strain AP1 number P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google A of a region of acid in SIC from U15 and and acid were observed the M1 variants thesic genes of the M12 and M55 strains are more as with the sic genes of the M1 strains The sequence of was found to be to a recently sequence number F. Spellerberg B. R. R. G. J. Infect. 2001; PubMed Scopus Google and as in a high degree of was found the acid sequence of was with that of of the acid of the SIC variants in the M1 variants have a with and This could explain the of and in the activity of α-defensin and It is that the M serotypes 55, and are to be with poststreptococcal glomerulonephritis, suggesting a for SIC in this (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, J. Sriprakash K.S. Microb. Pathog. 1999; 26: 25-33Crossref PubMed Scopus (36) Google Scholar). Moreover, the fact that M1 strains in of invasive (6Musser J.M. Krause R.M. Krause R.M. Emerging Infections. Academic Press, New York1998: 185-218Google Scholar) and that is the most inhibitor of α-defensin and LL-37, the that the of SIC with antibacterial peptides could S. pyogenes mucosal and of the SIC and acid in the and acid sequences are shown as with the sequence (7Åkesson P. Sjöholm A.G. Björck L. J. Biol. Chem. 1996; 271: 1081-1088Abstract Full Text Full Text PDF PubMed Scopus (204) Google represent and acid represent at in the acid the and the sequences are shown by acid and at in the the in has the by antibacterial peptides in the clearance of bacteria. The that SIC not only interferes with complement function but also antibacterial peptides the of the innate immune system. The of the also the highly complex S. pyogenes and human and are the that SIC could represent an important A investigation S. Hoe N.P. I. J. Kordari P. Liu M. Dou S.J. Musser J.M. Infect. Immun. 2000; PubMed Scopus Google Scholar) showed that an M1 mutant strain in which the sic gene had been was in the of mice as with the wild-type Here we using a mouse model of infection, that the mutant strain SIC− is in with the wild-type AP1 strain that SIC early of infection by antibacterial peptides. The of the work also explain the unique of the sic gene and the fact that the M1 serotype is the serotype most frequently connected with invasive S. pyogenes of mice with S. pyogenes strains AP1 and to to was in mice injected with SIC− as to AP1 using the Wilcoxin rank sum mice were injected subcutaneously with 104 AP1 or SIC− and mice were followed for 10 to was in mice injected with SIC− as to AP1 using the Wilcoxin rank sum in a new NMRI mice were injected subcutaneously with 104 AP1 or SIC− and mice were followed for 10 days. and are for to the work is that protein SIC also inhibits the antibacterial of and that are part of the mucosal innate immune system. These were reported by et al. B.A. Seilly D.J. Davies A. Lachmann P.J. Infect. Immun. PubMed Scopus Google Scholar) the of this