1887

Abstract

is an anaerobic Gram-positive bacterium and commensal, which is also associated with clinically important conditions such as skin and soft tissue infections. This study describes a novel subtilisin-like extracellular serine proteinase of denoted SufA (btilase of ), which is believed to be the first subtilase described among Gram-positive anaerobic cocci. SufA is associated with the bacterial cell surface, but is also released in substantial amounts during bacterial growth. Papain was used to release SufA from the surface of and the enzyme was purified by ion-exchange chromatography and gel filtration. A protein band on SDS-PAGE corresponding to the dominating proteolytic activity on gelatin zymography was analysed by MS/MS. Based on the peptide sequences obtained, the gene was sequenced. The gene comprises 3466 bp corresponding to a preprotein of 127 kDa. Like other members of the subtilase family, SufA contains the catalytic triad of aspartic acid, histidine and serine with surrounding conserved residues. A SufA homologue was identified in 33 of 34 investigated isolates of as revealed by PCR and immunoprinting. The enzyme forms dimers, which are more proteolytically active than the monomeric protein. SufA was found to efficiently cleave and inactivate the antibacterial peptide LL-37 and the CXC chemokine MIG/CXCL9, indicating that the enzyme promotes survival and colonization.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/010322-0
2007-12-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/153/12/4208.html?itemId=/content/journal/micro/10.1099/mic.0.2007/010322-0&mimeType=html&fmt=ahah

References

  1. Bals R., Wilson J. M. 2003; Cathelicidins – a family of multifunctional antimicrobial peptides. Cell Mol Life Sci 60:711–720
    [Google Scholar]
  2. Bendtsen J. D., Nielsen H., von Heijne G., Brunak S. 2004; Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795
    [Google Scholar]
  3. Björck L. 1988; Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J Immunol 140:1194–1197
    [Google Scholar]
  4. Bowler P. G., Davies B. J. 1999; The microbiology of infected and noninfected leg ulcers. Int J Dermatol 38:573–578
    [Google Scholar]
  5. Chen C. C., Cleary P. P. 1990; Complete nucleotide sequence of the streptococcal C5a peptidase gene of Streptococcus pyogenes . J Biol Chem 265:3161–3167
    [Google Scholar]
  6. Choih S., Smith Q. T., Schachtele C. F. 1979; Modification of human parotid saliva proteins by oral Streptococcus sanguis . J Dent Res 58:516–524
    [Google Scholar]
  7. Cole A. M., Ganz T., Liese A. M., Burdick M. D., Liu L., Strieter R. M. 2001; Cutting edge: IFN-inducible ELR-CXC chemokines display defensin-like antimicrobial activity. J Immunol 167:623–627
    [Google Scholar]
  8. de Château M., Björck L. 1994; Protein PAB, a mosaic albumin-binding bacterial protein representing the first contemporary example of module shuffling. J Biol Chem 269:12147–12151
    [Google Scholar]
  9. Dhople V., Krukemeyer A., Ramamoorthy A. 2006; The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochim Biophys Acta 17581499–1512
    [Google Scholar]
  10. Egesten A., Eliasson M., Johansson H. M., Olin A. I., Mörgelin M., Mueller A., Pease J. E., Frick I. M., Björck L. 2007; The CXC chemokine MIG/CXCL9 is important in innate immunity against Streptococcus pyogenes . J Infect Dis 195:684–693
    [Google Scholar]
  11. Frick I. M., Åkesson P., Herwald H., Mörgelin M., Malmsten M., Nägler D. K., Björck L. 2006; The contact system – a novel branch of innate immunity generating antibacterial peptides. EMBO J 25:5569–5578
    [Google Scholar]
  12. Ganz T. 2003; Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 3:710–720
    [Google Scholar]
  13. Goto T., Todo K., Miyamoto K., Akimoto S. 2003; Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics. Microbiol Immunol 47:1005–1016
    [Google Scholar]
  14. Hansson C., Hoborn J., Moller A., Swanbeck G. 1995; The microbial flora in venous leg ulcers without clinical signs of infection. Repeated culture using a validated standardised microbiological technique. Acta Derm Venereol 75:24–30
    [Google Scholar]
  15. Harder J., Bartels J., Christophers E., Schröder J. M. 2001; Isolation and characterization of human beta-defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 276:5707–5713
    [Google Scholar]
  16. Harrington D. J. 1996; Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun 64:1885–1891
    [Google Scholar]
  17. Harris T. O., Shelver D. W., Bohnsack J. F., Rubens C. E. 2003; A novel streptococcal surface protease promotes virulence, resistance to opsonophagocytosis, and cleavage of human fibrinogen. J Clin Invest 111:61–70
    [Google Scholar]
  18. Hayashi T., Matsubara M., Nohara D., Kojima S., Miura K., Sakai T. 1994; Renaturation of the mature subtilisin BPN′ immobilized on agarose beads. FEBS Lett 350:109–112
    [Google Scholar]
  19. Herwald H., Collin M., Müller-Esterl W., Björck L. 1996; Streptococcal cysteine proteinase releases kinins: a virulence mechanism. J Exp Med 184:665–673
    [Google Scholar]
  20. Hidalgo-Grass C., Mishalian I., Dan-Goor M., Belotserkovsky I., Eran Y., Nizet V., Peled A., Hanski E. 2006; A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues. EMBO J 25:4628–4637
    [Google Scholar]
  21. Imamura T., Potempa J., Travis J. 2004; Activation of the kallikrein-kinin system and release of new kinins through alternative cleavage of kininogens by microbial and human cell proteinases. Biol Chem 385:989–996
    [Google Scholar]
  22. Imamura T., Tanase S., Szmyd G., Kozik A., Travis J., Potempa J. 2005; Induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from Staphylococcus aureus . J Exp Med 201:1669–1676
    [Google Scholar]
  23. Kluskens L. D., Voorhorst W. G., Siezen R. J., Schwerdtfeger R. M., Antranikian G., van der Oost J., de Vos W. M. 2002; Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans . Extremophiles 6:185–194
    [Google Scholar]
  24. Knoth K., Roberds S., Poteet C., Tamkun M. 1988; Highly degenerate, inosine-containing primers specifically amplify rare cDNA using the polymerase chain reaction. Nucleic Acids Res 16:10932
    [Google Scholar]
  25. Krepel C. J., Gohr C. M., Walker A. P., Farmer S. G., Edmiston C. E. 1992; Enzymatically active Peptostreptococcus magnus : association with site of infection. J Clin Microbiol 30:2330–2334
    [Google Scholar]
  26. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  27. Lardelli M. 2002; Nonspecific, nested suppression PCR method for isolation of unknown flanking DNA (“cold-start method”. Methods Mol Biol 192:285–291
    [Google Scholar]
  28. Liotta L. A., Stetler-Stevenson W. G. 1990; Metalloproteinases and cancer invasion. Semin Cancer Biol 1:99–106
    [Google Scholar]
  29. Lo C. S., Hughes C. V. 1996; Identification and characterization of a protease from Streptococcus oralis C104. Oral Microbiol Immunol 11:181–187
    [Google Scholar]
  30. Ma B., Zhang K., Hendrie C., Liang C., Li M., Doherty-Kirby A., Lajoie G. 2003; PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun Mass Spectrom 17:2337–2342
    [Google Scholar]
  31. Matsubara M., Kurimoto E., Kojima S., Miura K., Sakai T. 1994; Achievement of renaturation of subtilisin BPN′ by a novel procedure using organic salts and a digestible mutant of Streptomyces subtilisin inhibitor. FEBS Lett 342:193–196
    [Google Scholar]
  32. Moser M., Menz G., Blaser K., Crameri R. 1994; Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus . Infect Immun 62:936–942
    [Google Scholar]
  33. Murdoch D. A. 1998; Gram-positive anaerobic cocci. Clin Microbiol Rev 11:81–120
    [Google Scholar]
  34. Nakamura Y., Gojobori T., Ikemura T. 2000; Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res 28:292
    [Google Scholar]
  35. Nohara D., Senga Y., Matsubara M., Sakai T. 2000; Media selection for refolding of thermolysin by use of immobilized preparation. J Biosci Bioeng 89:188–192
    [Google Scholar]
  36. Pagni M., Ioannidis V., Cerutti L., Zahn-Zabal M., Jongeneel C. V., Falquet L. 2004; MyHits: a new interactive resource for protein annotation and domain identification. Nucleic Acids Res 32:W332–W335
    [Google Scholar]
  37. Plaut A. G., Genco R. J., Tomasi T. B. Jr 1974; Isolation of an enzyme from Streptococcus sanguis which specifically cleaves IgA. J Immunol 113:589–591
    [Google Scholar]
  38. Potempa J., Banbula A., Travis J. 2000; Role of bacterial proteinases in matrix destruction and modulation of host responses. Periodontol 2000; 24:153–192
    [Google Scholar]
  39. Rasmussen M., Björck L. 2002; Proteolysis and its regulation at the surface of Streptococcus pyogenes . Mol Microbiol 43:537–544
    [Google Scholar]
  40. Rawlings N. D., Barrett A. J. 1994; Families of serine peptidases. Methods Enzymol 244:19–61
    [Google Scholar]
  41. Schägger H., von Jagow G. 1987; Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379
    [Google Scholar]
  42. Schmidtchen A., Frick I. M., Andersson E., Tapper H., Björck L. 2002; Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37. Mol Microbiol 46:157–168
    [Google Scholar]
  43. Scott C. F., Whitaker E. J., Hammond B. F., Colman R. W. 1993; Purification and characterization of a potent 70-kDa thiol lysylproteinase (Lys-gingivain) from Porphyromonas gingivalis that cleaves kininogens and fibrinogen. J Biol Chem 268:7935–7942
    [Google Scholar]
  44. Shinde U., Inouye M. 1995a; Folding pathway mediated by an intramolecular chaperone: characterization of the structural changes in pro-subtilisin E coincident with autoprocessing. J Mol Biol 252:25–30
    [Google Scholar]
  45. Shinde U., Inouye M. 1995b; Folding mediated by an intramolecular chaperone: autoprocessing pathway of the precursor resolved via a substrate assisted catalysis mechanism. J Mol Biol 247:390–395
    [Google Scholar]
  46. Sieprawska-Lupa M., Mydel P., Krawczyk K., Wójcik K., Puklo M., Lupa B., Suder P., Silberring J., Reed M. other authors 2004; Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus -derived proteinases. Antimicrob Agents Chemother 48:4673–4679
    [Google Scholar]
  47. Siezen R. J., Leunissen J. A. 1997; Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6:501–523
    [Google Scholar]
  48. Siezen R. J., de Vos W. M., Leunissen J. A., Dijkstra B. W. 1991; Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. Protein Eng 4:719–737
    [Google Scholar]
  49. Sörensen O. E., Thapa D. R., Roupe K. M., Valore E. V., Sjöbring U., Roberts A. A., Schmidtchen A., Ganz T. 2006; Injury-induced innate immune response in human skin mediated by transactivation of the epidermal growth factor receptor. J Clin Invest 116:1878–1885
    [Google Scholar]
  50. Steffen E. K., Hentges D. J. 1981; Hydrolytic enzymes of anaerobic bacteria isolated from human infections. J Clin Microbiol 14:153–156
    [Google Scholar]
  51. Stephens P., Wall I. B., Wilson M. J., Hill K. E., Davies C. E., Hill C. M., Harding K. G., Thomas D. W. 2003; Anaerobic cocci populating the deep tissues of chronic wounds impair cellular wound healing responses in vitro. Br J Dermatol 148:456–466
    [Google Scholar]
  52. Takagi H., Takahashi M. 2003; A new approach for alteration of protease functions: pro-sequence engineering. Appl Microbiol Biotechnol 63:1–9
    [Google Scholar]
  53. Thwaite J. E., Hibbs S., Titball R. W., Atkins T. P. 2006; Proteolytic degradation of human antimicrobial peptide LL-37 by Bacillus anthracis may contribute to virulence. Antimicrob Agents Chemother 50:2316–2322
    [Google Scholar]
  54. Travis J., Potempa J., Maeda H. 1995; Are bacterial proteinases pathogenic factors?. Trends Microbiol 3:405–407
    [Google Scholar]
  55. von Pawel-Rammingen U., Björck L. 2003; IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes . Curr Opin Microbiol 6:50–55
    [Google Scholar]
  56. Wang J. J., Swaisgood H. E., Shih J. C. 2003; Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems. Biotechnol Bioeng 81:421–429
    [Google Scholar]
  57. Zhu X. L., Ohta Y., Jordan F., Inouye M. 1989; Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature 339:483–484
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/010322-0
Loading
/content/journal/micro/10.1099/mic.0.2007/010322-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error