Streptococcus pneumoniae Sheds Syndecan-1 Ectodomains through ZmpC, a Metalloproteinase Virulence Factor
2006; Elsevier BV; Volume: 282; Issue: 1 Linguagem: Inglês
10.1074/jbc.m608542200
ISSN1083-351X
AutoresYe Chen, Atsuko Hayashida, Allison E. Bennett, Susan K. Hollingshead, Pyong Woo Park,
Tópico(s)Microbial infections and disease research
ResumoSeveral microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC. Several microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC. Numerous proteins can be released from host cell surfaces by a proteolytic cleavage mechanism known as ectodomain shedding (1Arribas J. Borroto A. Chem. Rev. 2002; 102: 4627-4637Crossref PubMed Scopus (205) Google Scholar, 2Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 3Hooper N.M. Karran E.H. Turner A.J. Biochem. 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J. 1997; 321: 265-279Crossref PubMed Scopus (556) Google Scholar, 4Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar). Thus, ectodomain shedding is increasingly recognized as an important post-translational mechanism that regulates both infectious and noninfectious inflammatory processes. Recent studies have demonstrated that microbial pathogens can stimulate the ectodomain shedding of several proteins from host cell surfaces to promote their pathogenesis (5Lemjabbar H. Basbaum C. Nat. Med. 2002; 8: 41-46Crossref PubMed Scopus (291) Google Scholar, 6Schmidtchen A. Frick I. Björck L. Mol. Microbiol. 2001; 39: 708-713Crossref PubMed Scopus (125) Google Scholar, 7Vollmer P. Walev I. Rose-John S. Bhakdi S. Infect. Immun. 1996; 64: 3646-3651Crossref PubMed Google Scholar, 8Walev I. Tappe D. Glubins E. Bhakdi S. J. Leukocyte Biol. 2000; 68: 865-872PubMed Google Scholar, 9Walev I. Vollmer P. Palmer M. Bhakdi S. Rose-John S. Proc. Natl. Acad. Sci. 1996; 93: 7882-7887Crossref PubMed Scopus (101) Google Scholar). For example, lipoteichoic acid of Staphylococcus aureus binds to platelet-activating factor receptor and activates a signaling mechanism that results in a disintegrin and metalloproteinase-10-mediated shedding of heparin-binding epidermal growth factor in epithelial cells. Shed heparin-binding epidermal growth factor then activates the heparin-binding epidermal growth factor receptor to induce mucin overexpression, which promotes lung infection by obstructing airflow and inhibiting antibacterial agents (5Lemjabbar H. Basbaum C. Nat. Med. 2002; 8: 41-46Crossref PubMed Scopus (291) Google Scholar). Extracellular proteinases secreted by Pseudomonas aeruginosa, Enterococcus faecalis, and Streptococcus pyogenes shed dermatan sulfate proteoglycans, which bind to and inactivate neutrophil-derived α-defensins (6Schmidtchen A. Frick I. Björck L. Mol. Microbiol. 2001; 39: 708-713Crossref PubMed Scopus (125) Google Scholar). Streptolysin O, a toxin virulence factor secreted by S. pyogenes, stimulates the shedding of L-selectin, interleukin-6 receptor, and CD14, and this is thought to dysregulate the host inflammatory response to promote streptococcal pathogenesis (8Walev I. Tappe D. Glubins E. Bhakdi S. J. Leukocyte Biol. 2000; 68: 865-872PubMed Google Scholar, 9Walev I. Vollmer P. Palmer M. Bhakdi S. Rose-John S. Proc. Natl. Acad. Sci. 1996; 93: 7882-7887Crossref PubMed Scopus (101) Google Scholar). Similarly, secreted products of S. aureus, P. aeruginosa, Listeria monocytogenes, and Serratia marcescens activate interleukin-6 receptor shedding (7Vollmer P. Walev I. Rose-John S. Bhakdi S. Infect. Immun. 1996; 64: 3646-3651Crossref PubMed Google Scholar), and those of S. epidermidis stimulate tumor necrosis factor-α shedding (10Mattson E. Van Dijk H. Verhoef J. Norrby R. Rollof J. Infect. Immun. 1996; 64: 4351-4355Crossref PubMed Google Scholar). Collectively, these observations suggest that various microbial pathogens activate the ectodomain shedding of inflammatory factors to dysregulate the host response to infection and promote their pathogenesis. Syndecan-1 is a type I transmembrane heparan sulfate proteoglycan predominantly expressed by epithelial cells and plasma cells, although it is also expressed by other cell types (e.g. endothelial cells, macrophages, fibroblasts) to a lesser degree (11Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2281) Google Scholar, 12Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (956) Google Scholar, 13Couchman J.R. Nat. Rev. Mol. Cell. Biol. 2003; 4: 926-937Crossref PubMed Scopus (332) Google Scholar, 14Park P.W. Reizes O. Bernfield M. J. Biol. 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Chem. 1997; 272: 14713-14720Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), and shedding is also activated under certain pathological conditions in vivo (20Haynes 3rd, A. Park P.W. Ruda F. Oliver J. Hamood A.N. Griswold J.A. Rumbaugh K.P. Infect. Immun. 2005; 73: 7914-7921Crossref PubMed Scopus (55) Google Scholar, 21Li Q. Park P.W. Wilson C.L. Parks W.C. Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 22Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (205) Google Scholar, 23Xu J. Park P.W. Kheradmand F. Corry D.B. J. Immunol. 2005; 174: 5758-5765Crossref PubMed Scopus (87) Google Scholar), suggesting that syndecan-1 shedding is one of the general host responses to tissue injury and inflammation (11Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2281) Google Scholar, 14Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). We reported previously that P. aeruginosa (18Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3062Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and S. aureus (17Park P.W. Foster T.J. Nishi E. Duncan S.J. Klagsbrun M. Chen Y. J. Biol. Chem. 2004; 279: 251-258Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) specifically activate the ectodomain shedding of syndecan-1. P. aeruginosa stimulates syndecan-1 shedding through LasA, a virulence factor for its lung infection, whereas S. aureus augments shedding through α- and β-toxins, two cytolytic toxins implicated in several staphylococcal infections. Interestingly, both P. aeruginosa LasA and S. aureus α- and β-toxins do not directly cause syndecan-1 shedding. Instead, they activate a protein-tyrosine kinase (PTK) 2The abbreviations used are: PTK, protein-tyrosine kinase; HS, heparan sulfate; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; NMuMG, normal murine mammary gland; TAPI, tumor necrosis factor-α protease inhibitor. 2The abbreviations used are: PTK, protein-tyrosine kinase; HS, heparan sulfate; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; NMuMG, normal murine mammary gland; TAPI, tumor necrosis factor-α protease inhibitor.-dependent intracellular signaling mechanism that stimulates endogenous syndecan-1 shedding at the cell surface (14Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 17Park P.W. Foster T.J. Nishi E. Duncan S.J. Klagsbrun M. Chen Y. J. Biol. Chem. 2004; 279: 251-258Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). There are several indications that syndecan-1 shedding enhances bacterial virulence. Syndecan-1 shedding occurs in vivo when mice are infected with P. aeruginosa (20Haynes 3rd, A. Park P.W. Ruda F. Oliver J. Hamood A.N. Griswold J.A. Rumbaugh K.P. Infect. Immun. 2005; 73: 7914-7921Crossref PubMed Scopus (55) Google Scholar, 22Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (205) Google Scholar), and inhibition of syndecan-1 shedding with peptide hydroxamates reduces the virulence of P. aeruginosa in lung infections in mice (22Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (205) Google Scholar). Further, syndecan-1 null mice that are incapable of shedding their syndecan-1 ectodomains markedly resist P. aeruginosa and S. aureus lung infection relative to wild type mice (20Haynes 3rd, A. Park P.W. Ruda F. Oliver J. Hamood A.N. Griswold J.A. Rumbaugh K.P. Infect. Immun. 2005; 73: 7914-7921Crossref PubMed Scopus (55) Google Scholar, 22Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (205) Google Scholar). How syndecan-1 shedding promotes bacterial pathogenesis is not completely understood, but syndecan-1 ectodomains bind to and inhibit various host defense factors, such as antimicrobial peptides, in a heparan sulfate (HS)-dependent manner (22Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (205) Google Scholar). Moreover, because soluble HS can inhibit several cytokines (23Xu J. Park P.W. Kheradmand F. Corry D.B. J. Immunol. 2005; 174: 5758-5765Crossref PubMed Scopus (87) Google Scholar, 24Fritchley S.J. Kirby J.A. Ali S. Clin. Exp. Immunol. 2000; 120: 247-252Crossref PubMed Scopus (35) Google Scholar, 25Kuschert G.S.V. Coulin F. Power C.A. Proudfoot A.E.I. Hubbard R.E. Hoogewerf A.J. Wells T.N.C. Biochemistry. 1999; 38: 12959-12968Crossref PubMed Scopus (483) Google Scholar, 26Sarrazin S. Bonnaffe D. Lubineau A. Lortat-Jacob H. J. Biol. Chem. 2005; 280: 37558-37564Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), syndecan-1 ectodomains might similarly inhibit cytokines through their HS moiety. These observations suggest that activation of syndecan-1 shedding is a broadly used pathogenic strategy to enhance microbial virulence. In this study, we investigated whether Streptococcus pneumoniae enhances syndecan-1 shedding. S. pneumoniae, a Gram-positive bacterium, is a major human pathogen that can cause both invasive and noninvasive infections, such as pneumonia, meningitis, sepsis, and otitis media (27Hollingshead S.K. Briles D.E. Curr. Opin. Microbiol. 2001; 4: 71-77Crossref PubMed Scopus (34) Google Scholar, 28Tuomanen E.I. Austrian R. Masure H.R. N. Engl. J. Med. 1995; 332: 1280-1284Crossref PubMed Scopus (311) Google Scholar). Our results show that S. pneumoniae can activate syndecan-1 shedding in a metalloproteinase-dependent but intracellular signaling-independent manner. Consistent with this activity, we also show that (i) biochemically purified fractions containing the S. pneumoniae shedding activity are composed of zinc metalloproteinase B (ZmpB) and ZmpC, (ii) purified S. pneumoniae zinc metalloproteinases directly shed syndecan-1 ectodomains, and (iii) S. pneumoniae metalloproteinases cleave syndecan-1 ectodomains at a site distinct from that cleaved by endogenous sheddases. Moreover, the deletion of zmpC was found to abrogate syndecan-1 shedding activity, whereas deletion of zmpB had no effect. These data indicate that S. pneumoniae directly sheds syndecan-1 ectodomains through ZmpC in a manner distinct from previously described mechanisms of syndecan-1 shedding. Materials—Todd-Hewitt broth was purchased from Difco, and 5% sheep blood agar plates were from Remel (Lenexa, KS). Tissue culture medium and supplements were purchased from Mediatech (Herndon, VA). GM6001 and TAPI-1 were from Calbiochem. HiTrap DEAE FF and HiPrep 16/60 Sephacryl S-300 resins, prepacked PD-10 columns, and ECL Western blotting detection reagents were from Amersham Biosciences. Molecular weight cut-off spin tubes were from Pall Life Science (Northborough, MA). IODOGEN, Sulfolink coupling resin, and protein A- and protein G-agarose beads were purchased from Pierce. The cationic nylon membrane, Immobilon Ny+, was from Millipore (Bedford, MA), and the ProBlot polyvinylidene difluoride membrane was from Applied Biosystems (Foster City, CA). Heparinase III and chondroitinase ABC were from Seikagaku (Cape Cod, MA). Normal murine mammary gland (NMuMG) epithelial cells were from our culture collection and cultured as described previously (17Park P.W. Foster T.J. Nishi E. Duncan S.J. Klagsbrun M. Chen Y. J. Biol. Chem. 2004; 279: 251-258Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 18Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3062Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). All other materials were purchased from VWR, Fisher, or Sigma. Immunochemicals—The rat monoclonal anti-mouse syndecan-1 ectodomain (281-2) and anti-mouse syndecan-4 ectodomain (Ky8.2) antibodies were purified from the conditioned medium of hybridoma cultures by protein G-agarose affinity chromatography. The rabbit anti-ZmpB antibody was generated by immunizing rabbits with the synthetic peptide C1604KTLKTREDINRYM1617K. A Cys residue was added to the N terminus of the ZmpB sequence for coupling to the Sulfolink resin. Affinity-purified anti-ZmpB antibodies were generated by consecutive protein A-agarose and synthetic peptide affinity chromatography. Horseradish peroxidase-conjugated donkey anti-rat and horseradish peroxidase-conjugated goat anti-rabbit antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Bacterial Strains and Growth Conditions—S. pneumoniae strains used in this study are listed in Table 1. Strains were grown on 5% sheep blood agar plates or in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY broth) at 37 °C without agitation. In addition to strains constructed specifically for this study, S. pneumoniae mutant strains lacking pneumolysin (Ply–), autolysin (Lyt–), hyaluronidase (Hyl–), neuraminidase (NanA–), sortases (SrtBCD–), serine protease (SP0664- and SP2239-deficient), and adhesin PspC (PspC–) were all screened after growth in THY broth supplemented with 0.2 μg/ml erythromycin (29Balachandran P. Brooks-Walter A. Virolainen-Julkunen A. Hollingshead S.K. Briles D.E. Infect. Immun. 2002; 70: 2526-2534Crossref PubMed Scopus (137) Google Scholar, 30Balachandran P. Hollingshead S.K. Paton J.C. Briles D.E. J. Bacteriol. 2001; 183: 3108-3116Crossref PubMed Scopus (93) Google Scholar).TABLE 1S. pneumoniae strains and primers usedStrain/PrimerDescription or sequenceaPrimers were based on complete genome sequence of S. pneumoniae TIGR4 (35). Lowercase letters represent mismatches used to incorporate restriction enzyme sites.Resistance/RESource/ReferenceS. pneumoniae strains TIGR4Wild type, capsule type 4Ref. 35Tettelin H. Nelson K.E. Paulsen I.T. Eisen J.A. Read T.D. Peterson S. Heidelberg J. DeBoy R.T. Haft D.H. Dodson R.J. Durkin A.S. Gwinn M. Kolonay J.F. Nelson W.C. Peterson J.D. Umayam L.A. White O. Salzberg S.L. Lewis M.R. Radune D. Holtzapple E. Khouri H. Wolf A.M. Utterback T.R. Hansen C.L. McDonald L.A. Feldblyum T.V. Angiuoli S. Dickinson T. Hickey E.K. Holt I.E. Loftus B.J. Yang F. Smith H.O. Venter J.C. Dougherty B.A. Morrison D.A. Hollingshead S.K. Fraser C.M. Science. 2001; 293: 498-506Crossref PubMed Scopus (1099) Google Scholar MA_M11Wild type, capsule type 14 MB_M41Wild type, capsule type 23F MJ_V-012Wild type, capsule type 35 MK_V-142Wild type, capsule type 22 L82016Wild type, capsule type 6BRef. 43Berry A.M. Paton J.C. Infect. Immun. 2000; 68: 133-140Crossref PubMed Scopus (196) Google Scholar EF3030Wild type, capsule type 19F D39Wild type, capsule type 2 WU2Wild type, capsule type 3 ΔigaTIGR4 Δiga::Janus, lacking IgA1 proteaseKanRThis study ΔzmpBTIGR4 ΔzmpB::Janus, lacking ZmpBKanRThis study ΔzmpCTIGR4 ΔzmpC::Janus, lacking ZmpCKanRThis study TIGR4 srtBCDsrtBCD KO mutant in strain TIGR4ErmR PLN-Aply KO mutant in strain D39ErmRRefs. 44Berry A.M. Yother J. Briles D.E. Hansman D. Paton J.C. Infect Immun. 1989; 57: 2037-2042Crossref PubMed Google Scholar and 45Briles D.E. Hollingshead S.K. Paton J.C. Ades E.W. Novak L. van Ginkel F.W. Benjamin Jr., W.H. J. Infect. Dis. 2003; 188: 339-348Crossref PubMed Scopus (193) Google Scholar AL-2lytA KO mutant in strain D39ErmRRefs. 43Berry A.M. Paton J.C. Infect. Immun. 2000; 68: 133-140Crossref PubMed Scopus (196) Google Scholar and 45Briles D.E. Hollingshead S.K. Paton J.C. Ades E.W. Novak L. van Ginkel F.W. Benjamin Jr., W.H. J. Infect. Dis. 2003; 188: 339-348Crossref PubMed Scopus (193) Google Scholar L82016 nanAnanA KO mutant in strain L82016ErmRRef. 43Berry A.M. Paton J.C. Infect. Immun. 2000; 68: 133-140Crossref PubMed Scopus (196) Google Scholar L82016 hylhyl KO mutant in strain L82016ErmRRef. 43Berry A.M. Paton J.C. Infect. Immun. 2000; 68: 133-140Crossref PubMed Scopus (196) Google Scholar WU2 SP2239SP2239 KO mutant in strain WU2ErmR WU2 SP0641SP0641 KO mutant in strain WU2ErmR EF3030 SP0641SP0641 KO mutant in strain EF3030ErmR EF3030 pspCpspC KO mutant in strain EF3030ErmRPrimers AEB-65FGACTCTTATTAGAATATAGAAAAAGNoneUpstream flank-iga AEB-66RctagtctagaCTTTTCCATTATTCCTCCTTGXbaIUpstream flank-iga AEB-67FcgcggattccTAGTGTCTATTAGGAAATAAAGBamHIDownstream flank-iga AEB-68RCCAGTCAATGCCAACTTAAGTGCAACNoneDownstream flank-iga AEB-69FGACCCCAAAAAGGGACGAAAGTTGNoneUpstream flank-zmpB AEB-70RctagtctagaCAAAAGGCTTCCAAGAAATACXbaIUpstream flank-zmpB AEB-71FcgcggattccTACGGATACGGAAATGGGTTCBamHIDownstream flank-zmpB AEB-72RCTTGTGAACTGCTAATTTTTCCTCAAAAGNoneDownstream flank-zmpB AEB-73FCAGAGGAATTGGCTGGTAGATATGGNoneUpstream flank-zmpC AEB-74RctagtctagaCATATTAACCTCGCTTTTTCXbaIUpstream flank-zmpC AEB-75FcgcggattccTAAGATTGTAGAGTTTCATTGBamHIDownstream flank-zmpC AEB-76RGACCTTTTCCTCCCCATTCGTTGACNoneDownstream flank-zmpC AEB-35FctagtctagaGTTTGATTTTTAATGGXbaIJanus 5′-end AEB-31RcgcggattccGGGCCCCTTTCCTTATGCTTTTGGBamHIJanus 3′-enda Primers were based on complete genome sequence of S. pneumoniae TIGR4 (35Tettelin H. Nelson K.E. Paulsen I.T. Eisen J.A. Read T.D. Peterson S. Heidelberg J. DeBoy R.T. Haft D.H. Dodson R.J. Durkin A.S. Gwinn M. Kolonay J.F. Nelson W.C. Peterson J.D. Umayam L.A. White O. Salzberg S.L. Lewis M.R. Radune D. Holtzapple E. Khouri H. Wolf A.M. Utterback T.R. Hansen C.L. McDonald L.A. Feldblyum T.V. Angiuoli S. Dickinson T. Hickey E.K. Holt I.E. Loftus B.J. Yang F. Smith H.O. Venter J.C. Dougherty B.A. Morrison D.A. Hollingshead S.K. Fraser C.M. Science. 2001; 293: 498-506Crossref PubMed Scopus (1099) Google Scholar). Lowercase letters represent mismatches used to incorporate restriction enzyme sites. Open table in a new tab Syndecan Shedding Assays—The cell culture-based syndecan shedding assay was performed as described previously (17Park P.W. Foster T.J. Nishi E. Duncan S.J. Klagsbrun M. Chen Y. J. Biol. Chem. 2004; 279: 251-258Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 18Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3062Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Briefly, confluent NMuMG cells in 96-well plates were incubated with test samples diluted in the NMuMG culture medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 10 μg/ml insulin) for the indicated time periods at 37 °C. The conditioned medium was collected and acidified by the addition of NaOAc (pH 4.5), NaCl, and Tween 20 to final concentrations of 50 mm, 150 mm, and 0.1% (v/v), respectively. Various volumes of acidified samples were dot-blotted onto the Immobilon Ny+ membrane, probed with 281-2 or Ky8.2 anti-syndecan-1 or -4 antibodies, and developed by ECL. Blots were scanned, and the intensity of dots was quantified by NIH Image software. Purification of the S. pneumoniae Syndecan-1 Shedding Enhancer—The TIGR4 strain was grown in 4 liters of THY broth for 8 h at 37 °Cto late log growth phase, and the culture supernatant was subjected to 40% ammonium sulfate precipitation. The precipitate was resuspended in 50 ml of autoclaved deionized H2O, dialyzed three times against 4 liters of autoclaved deionized H2O, and lyophilized. The lyophilized sample was resuspended in 10 ml of DEAE binding buffer (20 mm Tris, pH 7.4, 20 mm NaCl) and applied to a HiTrap DEAE FF 5-ml column pre-equilibrated with DEAE binding buffer. The applied sample was eluted with a 0.1–1.0 m NaCl gradient, and fractions were dialyzed against autoclaved deionized H2O and then tested for their capacity to shed syndecan-1 ectodomains by the cell culture-based shedding assay. Active fractions that eluted at ∼0.3 m NaCl were pooled, lyophilized, resuspended in 2 ml of TBS (50 mm Tris, pH 7.4, 150 mm NaCl), and fractionated by Sephacryl S-300 size exclusion chromatography. TBS was applied to the column at a flow rate of 0.4 ml/min, and 10-min fractions (4 ml) were collected. Aliquots (200 μl) of each fraction were Speed Vacdried and resuspended in NMuMG culture medium, filtersterilized, and tested for their syndecan-1 shedding activity. This purification scheme typically yielded 0.8–1 mg of the purified 170-kDa syndecan-1 shedding enhancer from 4 liters of bacterial culture supernatant. Radioiodination and Autoradiography—The purified 170-kDa syndecan-1 shedding-enhancing protein (10 μg) was incubated with 20 μg of immobilized IODOGEN and 500 μCi of Na125I in 50 μl of TBS for 10 min at room temperature. Free 125I was separated from iodinated protein by PD-10 chromatography. To estimate the purity of the 170-kDa fraction, 3 ng of the radioiodinated sample was separated by 12% SDS-PAGE. The gel was vacuum-dried and exposed to Eastman Kodak Co. BioMax film with an intensifying screen at –80 °C. The film was scanned, and the intensity of radioactive protein bands was quantified using the NIH Image software. Mass Spectrometry and N-terminal Microsequencing—The purified 170-kDa protein fraction containing the syndecan-1 shedding activity was subjected to in-gel trypsin digestion after reduction with dithiothreitol and alkylation with iodoacetamide. Tryptic digests were analyzed by MALDI-TOF mass spectrometry (MS) (ABI 4700 MALDI-TOF system) at the Baylor College of Medicine Protein Chemistry Core Laboratory (Houston, TX). MALDI-TOF MS peptide fingerprints were searched against public domain databases using MS-FIT software. One of the peptides with an m/z value of 1128.6 was further analyzed by MS/MS (ABI 4000 Q-TRAP LC MS/MS system). For N-terminal sequencing, the 170-kDa protein was transferred to a polyvinylidene difluoride membrane, excised, and analyzed by the ABI 477A peptide sequencer. Alternatively, the 170-kDa protein was digested with trypsin, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride, and the 50- and 30-kDa tryptic fragments were excised from the membrane and loaded onto the ABI 477A peptide sequencer. The deduced N-terminal sequence of the 50-kDa tryptic digest was searched by BLAST. Generation of Zinc Metalloproteinase Mutant Strains–Mutant strains devoid of the three zinc metalloproteinases were generated in the TIGR4 strain (Table 1). The strains were made by PCR ligation mutagenesis, creating gene replacements of the metalloproteinase genes with the Janus cassette containing the selectable marker aphIII, encoding kanamycin resistance (31Lau P.C. Sung C.K. Lee J.H. Morrison D.A. Cvitkovitch D.G. J. Microbiol. Methods. 2002; 49: 193-205Crossref PubMed Scopus (253) Google Scholar, 32Sung C.K. Li H. Claverys J.P. Morrison D.A. Appl. Environ. Microbiol. 2001; 67: 5190-5196Crossref PubMed Google Scholar). Primers were designed to amplify the 5′- and 3′-flanking regions of target genes zmpC, zmpB, and iga with genomic DNA from strain TIGR4 as template. Flanking amplicons were 500–1200 bp in length. Primers AEB-35F and AEB-31R were also used to amplify the 2903-bp Janus cassette. Restriction sites were integrated into the two primers nearest each target gene and into the Janus primers to allow for directional ligation as described (31Lau P.C. Sung C.K. Lee J.H. Morrison D.A. Cvitkovitch D.G. J. Microbiol. Methods. 2002; 49: 193-205Crossref PubMed Scopus (253) Google Scholar). After digestion of the amplicons, tripartite ligation of the flanking amplicons and the Janus cassette was carried out, and the ligation mix was used to transform competent S. pneumoniae TIGR4 as previously described (31Lau P.C. Sung C.K. Lee J.H. Morrison D.A. Cvitkovitch D.G. J. Microbiol. Methods. 2002; 49: 193-205Crossref PubMed Scopus (253) Google Scholar). The expected replacement of each target gene with the aphIII gene by reciprocal recombination was confirmed in kanamycin-resistant transformants by PCR. Statistical Analysis—Data are expressed as mean ± S.D. Statistical analyses were performed using STATVIEW 4.51 software (Abacus Concepts, Berkeley, CA). Differences between experimental groups and respective controls were examined by Student's t test. p values of <0.05 were deemed statistically significant. S. pneumoniae Activates Syndecan-1 Shedding—To determine whether S. pneumoniae strains possess the capacity to activate syndecan-1 shedding, nine S. pneumoniae strains with different capsular serotypes were examined for their shedding activity. Various concentrations of 8-h culture supernatants collected from S. pneumoniae strains
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