1887

Abstract

has several extracellular proteases with proposed roles in virulence. SspA (serine protease), SspB (cysteine protease) and Aur (metalloprotease) have been characterized previously and SspA and SspB were found to be cotranscribed. The coding region for the cysteine protease ScpA has been identified and characterized. It is in a probable bi-cistronic operon with located immediately upstream of a coding region for a 108 aa protein that is a specific inhibitor of ScpA. Using primer extension analysis promoters have been mapped and it was found that is the only sigma factor involved in the transcription of , and . The transcription of all the genes occurs maximally at post-exponential phase, being positively regulated by (accessory gene regulator) and negatively regulated by (staphylococcal accessory regulator). Furthermore represses transcription from the and operons similarly to the previously shown effect on [ Horsburgh, M., Aish, J., White, I., Shaw, L., Lithgow, J. & Foster, S. (2002). , 5457–5467 ]. Using mutations in each protease gene the proteolytic cascade of activation has been analysed. Aur, SspA, SspB and ScpA are all produced as zymogens, activated by proteolytic cleavage. Although the metalloprotease, Aur, does catalyse activation of the SspA zymogen, it is not the sole agent capable of conducting this process. Site-directed mutagenesis revealed that Aur is not capable of undergoing auto-proteolysis to achieve activation. The cysteine protease, ScpA, appears to reside outside this cascade of activation, as mature ScpA was observed in the , and mutant strains. Using a mouse abscess model, it has been shown that insertional inactivation of or results in significant attenuation of virulence, whilst mutations in or do not. It is likely the attenuation observed in the strain is due to polarity on the gene.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26634-0
2004-01-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/1/mic1500217.html?itemId=/content/journal/micro/10.1099/mic.0.26634-0&mimeType=html&fmt=ahah

References

  1. Abdelinour A., Arvidson S., Bremell T., Ryden C., Tarkowski A. 1993; The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect Immun 61:3879–3885
    [Google Scholar]
  2. Arvidson S. 2000; Extracellular enzymes. In Gram-Positive Pathogens pp. 379–385Edited by Fischetti V. A. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  3. Arnau J. K. I., Sorensen K. F., Appel F. K., Vogensen K., Hammer. 1996; Analysis of heat shock gene expression in Lactococcus lactis MG1363. Microbiology 142:1685–1691 [CrossRef]
    [Google Scholar]
  4. Banbula A., Potempa J., Travis J., Fernandez-Catalan C., Mann K., Huber R., Bode W., Medrano F. J. 1998; Amino-acid sequence and three-dimensional structure of the Staphylococcus aureus metalloprotease at 1·72 Å resolution. Structure 6:1185–1193 [CrossRef]
    [Google Scholar]
  5. Birktoft J., Breddam K. 1994; Glutamyl endopeptidases. Methods Enzymol 244:114–126
    [Google Scholar]
  6. Bischoff M., Entenza J. M., Giachino P. 2001; Influence of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus. J Bacteriol 183:5171–5179 [CrossRef]
    [Google Scholar]
  7. Bjoorklind A., Jornvall H. 1974; Substrate specificity of three different extracellular proteolytic enzymes from Staphylococcus aureus. Biochim Biophys Acta 370:524–529 [CrossRef]
    [Google Scholar]
  8. Bordo D., Argos P. 1991; Suggestions for ‘safe’ residue substitutions in site-directed mutagenesis. J Mol Biol 217:721–729 [CrossRef]
    [Google Scholar]
  9. Braun P., de Groot A., Bitter W., Tommassen J. 1998; Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa. J Bacteriol 180:3467–3469
    [Google Scholar]
  10. Chakraborty T., Leimeister-Wachter M., Domann E., Hartl M., Goebel W., Nichterlein T., Notermans S. 1992; Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. . J Bacteriol 174:568–574
    [Google Scholar]
  11. Chan P., Foster S. J. 1998; The role of environmental factors in the regulation of virulence determinants expression in Staphylococcus aureus8325-4. Microbiology 144:2469–2479 [CrossRef]
    [Google Scholar]
  12. Chan P., Foster S., Ingham E., Clements M. 1998; The Staphylococcus aureus alternative sigma factor sigmaB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J Bacteriol 180:6082–6089
    [Google Scholar]
  13. Cheung A., Ying P. 1994; Regulation of alpha- and beta-hemolysins by the sar locus of Staphylococcus aureus. J Bacteriol 176:580–585
    [Google Scholar]
  14. Cheung A., Coomey J., Butler C., Projan S., Fischetti V. 1992; Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proc Natl Acad Sci U S A 89:6462–6466 [CrossRef]
    [Google Scholar]
  15. Cheung A., Eberhardt K., Heinrichs J. 1997; Regulation of protein A synthesis by the sar and agr loci of Staphylococcus aureus. Infect Immun 65:2243–2249
    [Google Scholar]
  16. Cheung A., Chien Y., Bayer A. 1999; Hyperproduction of alpha-haemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus. Infect Immun 67:1331–1337
    [Google Scholar]
  17. Chien Y. A. C., Manna S. J., Cheung A. L, Projan. 1999; sarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation. J Biol Chem 274:37169–37176 [CrossRef]
    [Google Scholar]
  18. Coulter S., Scwan W., Ng E.7 other authors 1998; Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Mol Microbiol 30:393–404 [CrossRef]
    [Google Scholar]
  19. Deora R., Misra T. K. 1996; Characterization of the primary σ factor of Staphylococcus aureus. J Biol Chem 271:21828–21834 [CrossRef]
    [Google Scholar]
  20. Drapeau G. R. 1978; Role of a metalloprotease in activation of the precursor of staphylococcal protease. J Bacteriol 136:607–613
    [Google Scholar]
  21. Dunman P., Murphy E., Haney S.7 other authors 2001; Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. . J Bacteriol 183:7341–7353 [CrossRef]
    [Google Scholar]
  22. Elliott S. D. 1945; A proteolytic enzyme produced by group A streptococci with special reference to its effect on the type-specific M antigen. J Exp Med 81:573–592 [CrossRef]
    [Google Scholar]
  23. Filipek R., Rzychon M., Oleksy A., Gruca M., Dubin A., Potempa J., Bochtler M. 2003; The staphostatin–staphopain complex: a forward binding inhibitor in complex with its target cysteine protease. J Biol Chem 278:40959–40966 [CrossRef]
    [Google Scholar]
  24. Giachino P., Engelmann S., Bischoff M. 2001; Sigma (B) activity depends on RsbU in Staphylococcus aureus. J Bacteriol 183:1843–1852 [CrossRef]
    [Google Scholar]
  25. Gillaspy A., Lee C., Sau S., Cheung A., Smeltzer M. 1998; Factors affecting the collagen binding capacity of Staphylococcus aureus. Infect Immun 66:3170–3178
    [Google Scholar]
  26. Greenbaum D. K. F., Medzihradszky A. L., Bogyo M, Burlingame. 2000; Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem Biol 7:569–581 [CrossRef]
    [Google Scholar]
  27. Higuchi R., Krummel B., Saiki R. 1988; A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16:7351–7367 [CrossRef]
    [Google Scholar]
  28. Hofmann D., Schomburg D., Hecht H. 1993; Crystal structure of a thiol proteinase from Staphylococcus aureus V-8 in the E-64 inhibitor complex. Acta Crystallogr A Suppl 49:102
    [Google Scholar]
  29. Horsburgh M. J., Moir A. 1999; σM, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt. Mol Microbiol 32:41–50 [CrossRef]
    [Google Scholar]
  30. Horsburgh M. J., Clements M. O., Crossley H., Ingham E., Foster S. J. 2001a; PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus. Infect Immun 69:3744–3754 [CrossRef]
    [Google Scholar]
  31. Horsburgh M. J., Ingham E., Foster S. J. 2001b; In Staphylococcus aureus, Fur is an interactive regulator with PerR, contributes to virulence, and is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis. J Bacteriol 183:468–475 [CrossRef]
    [Google Scholar]
  32. Horsburgh M., Aish J., White I., Shaw L., Lithgow J., Foster S. 2002; SigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J Bacteriol 184:5457–5467 [CrossRef]
    [Google Scholar]
  33. Janzon L., Arvidson S. 1990; The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. EMBO J 9:1391–1399
    [Google Scholar]
  34. Karlsson A., Saravia-Otten P., Tegmark K., Morfeldt E., Arvidson S. 2001; Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases. Infect Immun 69:4742–4748 [CrossRef]
    [Google Scholar]
  35. Kemp E., Sammons R., Moir A., Sun D., Setlow P. 1991; Analysis of transcriptional control of the gerD spore germination gene of Bacillus subtilis 168. J Bacteriol 173:4646–4652
    [Google Scholar]
  36. Lindsay J., Foster S. 1999; Interactive regulatory pathways control virulence determinant production and stability in response to the environment in Staphylococcus aureus. Mol Gen Genet 262:323–331 [CrossRef]
    [Google Scholar]
  37. Liu T.-Y., Elliott S. 1965; Streptococcal proteinase: the zymogen to enzyme transformation. J Biol Chem 240:1138–1142
    [Google Scholar]
  38. Lowy F. D. 1998; Staphylococcus aureus infections. N Engl J Med 339:520–532 [CrossRef]
    [Google Scholar]
  39. McAleese F., Walsh E., Sieprawska M., Potempa J., Foster T. 2001; Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transcription, shedding and cleavage by metalloprotease. J Biol Chem 276:29969–29978 [CrossRef]
    [Google Scholar]
  40. McGavin M., Zahradka C., Rice K., Scott J. 1997; Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease. Infect Immun 65:2621–2628
    [Google Scholar]
  41. Miyoshi S., Shinoda S. 2000; Microbial metalloproteases and pathogenesis. Microbes Infect 2:91–98 [CrossRef]
    [Google Scholar]
  42. Miyoshi S., Wakae H., Tomochika K., Shinoda S. 1997; Functional domains of a zinc metalloprotease from Vibrio vulnificus. J Bacteriol 179:7606–7609
    [Google Scholar]
  43. Novick R. P. 2000; Pathogenicity factors and their regulation. In Gram-Positive Pathogens pp. 392–407Edited by Fischetti V. A. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  44. Novick R., Ross H., Projan S., Kornblum J., Kreiswirth B., Moghazeh S. 1993; Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J 12:3967–3975
    [Google Scholar]
  45. Patel A. H., Kornblum J., Kreiswirth B., Novick R., Foster T. J. 1992; Regulation of the protein A-encoding gene in Staphylococcus aureus. Gene 114:25–34 [CrossRef]
    [Google Scholar]
  46. Potempa J., Watorek W., Travis J. 1986; The inactivation of human plasma alpha 1-proteinase inhibitor by proteinases from Staphylococcus aureus. J Biol Chem 261:14330–14334
    [Google Scholar]
  47. Potempa J., Dubin A., Korzus G., Travis J. 1988; Degradation of elastin by a cysteine proteinase from Staphylococcus aureus. J Biol Chem 263:2664–2667
    [Google Scholar]
  48. Potempa J., Banbula A., Travis J. 2000; Role of bacterial proteinases in matrix destruction and modulation of host responses. Periodontology 24:153–192 [CrossRef]
    [Google Scholar]
  49. Prokesova L., Porwit-Bobr Z., Baran K., Potempa J., Pospisil M., John C. 1991; Effect of metalloproteinase from Staphylococcus aureus on in vitro stimulation of human lymphocytes. . Immunol Lett 27:225–230 [CrossRef]
    [Google Scholar]
  50. Prokesova L., Potuznikova B., Potempa J., Zikan J., Radl J., Hachova L., Baran K., Porwit-Bobr Z., John C. 1992; Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus. Immunol Lett 31:259–265 [CrossRef]
    [Google Scholar]
  51. Rapala-Kozik M., Potempa J., Nelson D., Kozik A., Travis J. 1999; Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases. Biol Chem 380:1211–1216
    [Google Scholar]
  52. Rasmussen M., Bjorck L. 2002; Proteolysis and its regulation at the surface of Streptococcus pyogenes. Mol Microbiol 43:537–544 [CrossRef]
    [Google Scholar]
  53. Rechtin T., Gillaspy A., Schumacher M., Brennan R., Smeltzer M., Hurlburt B. 1999; Characterization of the SarA virulence gene regulator of Staphylococcus aureus. Mol Microbiol 33:307–316 [CrossRef]
    [Google Scholar]
  54. Reed S., Wesson C., Liou L., Trumble W., Schlievert P., Bohach G., Bayles K. 2001; Molecular characterization of a novel Staphylococcus aureus serine protease operon. Infect Immun 69:1521–1527 [CrossRef]
    [Google Scholar]
  55. Rice K., Peralta R., Bast D., de Azavedo J., McGavin M. 2001; Description of staphylococcus serine protease (ssp) operon in Staphylococcus aureus and nonpolar inactivation of sspA-encoded serine protease. Infect Immun 69:159–169 [CrossRef]
    [Google Scholar]
  56. Rzychon M., Sabat A., Kosowska K., Potempa J., Dubin A. 2003; Staphostatins: an expanding new group of proteinase inhibitors with a unique specificity for the regulation of staphopains, Staphylococcus spp. cysteine proteinases. . Mol Microbiol 49:1051–1066 [CrossRef]
    [Google Scholar]
  57. Rzychon M., Filipek R., Sabat A., Kosowska K., Dubin A., Potempa J., Bochtler M. 2003; Staphostatins resemble lipocalins, not cystatins in fold. Protein Sci 12:2252–2256
    [Google Scholar]
  58. Sabat A., Kosowska K., Poulsen K., Kasprowicz A., Sekowska A., Van Ben Burg B., Travis J., Potempa J. 2000; Two allelic forms of the aureolysin gene (aur) within Staphylococcus aureus. Infect Immun 68:973–976 [CrossRef]
    [Google Scholar]
  59. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  60. Sullivan M., Yasbin R., Young F. 1984; New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene 29:21–26 [CrossRef]
    [Google Scholar]
  61. Tegmark K., Morfeldt E., Arvidson S. 1998; Regulation of agr-dependent virulence genes in Staphylococcus aureus by RNAIII from coagulase-negative staphylococci. J Bacteriol 180:3181–3186
    [Google Scholar]
  62. Ziebandt A., Weber H., Rudolph J., Schmid R., Hoper D., Engelmann S., Hecker M. 2001; Extracellular proteins of Staphylococcus aureus and the role of SarA and sigma B. Proteomics 1:480–493 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26634-0
Loading
/content/journal/micro/10.1099/mic.0.26634-0
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