Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1997 Jun 1;99(11):2574–2580. doi: 10.1172/JCI119445

Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

S Lukomski 1, S Sreevatsan 1, C Amberg 1, W Reichardt 1, M Woischnik 1, A Podbielski 1, J M Musser 1
PMCID: PMC508102  PMID: 9169486

Abstract

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.

Full Text

The Full Text of this article is available as a PDF (209.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berge A., Björck L. Streptococcal cysteine proteinase releases biologically active fragments of streptococcal surface proteins. J Biol Chem. 1995 Apr 28;270(17):9862–9867. doi: 10.1074/jbc.270.17.9862. [DOI] [PubMed] [Google Scholar]
  2. Björck L., Akesson P., Bohus M., Trojnar J., Abrahamson M., Olafsson I., Grubb A. Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor. Nature. 1989 Jan 26;337(6205):385–386. doi: 10.1038/337385a0. [DOI] [PubMed] [Google Scholar]
  3. Burns E. H., Jr, Marciel A. M., Musser J. M. Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease. Infect Immun. 1996 Nov;64(11):4744–4750. doi: 10.1128/iai.64.11.4744-4750.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chaussee M. S., Gerlach D., Yu C. E., Ferretti J. J. Inactivation of the streptococcal erythrogenic toxin B gene (speB) in Streptococcus pyogenes. Infect Immun. 1993 Sep;61(9):3719–3723. doi: 10.1128/iai.61.9.3719-3723.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cockerill F. R., 3rd, MacDonald K. L., Thompson R. L., Roberson F., Kohner P. C., Besser-Wiek J., Manahan J. M., Musser J. M., Schlievert P. M., Talbot J. An outbreak of invasive group A streptococcal disease associated with high carriage rates of the invasive clone among school-aged children. JAMA. 1997 Jan 1;277(1):38–43. [PubMed] [Google Scholar]
  6. Dewerchin M., Nuffelen A. V., Wallays G., Bouché A., Moons L., Carmeliet P., Mulligan R. C., Collen D. Generation and characterization of urokinase receptor-deficient mice. J Clin Invest. 1996 Feb 1;97(3):870–878. doi: 10.1172/JCI118489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DiPersio J. R., File T. M., Jr, Stevens D. L., Gardner W. G., Petropoulos G., Dinsa K. Spread of serious disease-producing M3 clones of group A streptococcus among family members and health care workers. Clin Infect Dis. 1996 Mar;22(3):490–495. doi: 10.1093/clinids/22.3.490. [DOI] [PubMed] [Google Scholar]
  8. Fletcher H. M., Schenkein H. A., Morgan R. M., Bailey K. A., Berry C. R., Macrina F. L. Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene. Infect Immun. 1995 Apr;63(4):1521–1528. doi: 10.1128/iai.63.4.1521-1528.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gerlach D., Knöll H., Köhler W., Ozegowski J. H., Hríbalova V. Isolation and characterization of erythrogenic toxins. V. Communication: identity of erythrogenic toxin type B and streptococcal proteinase precursor. Zentralbl Bakteriol Mikrobiol Hyg A. 1983 Sep;255(2-3):221–233. [PubMed] [Google Scholar]
  10. Hauser A. R., Schlievert P. M. Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor. J Bacteriol. 1990 Aug;172(8):4536–4542. doi: 10.1128/jb.172.8.4536-4542.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holm S. E., Norrby A., Bergholm A. M., Norgren M. Aspects of pathogenesis of serious group A streptococcal infections in Sweden, 1988-1989. J Infect Dis. 1992 Jul;166(1):31–37. doi: 10.1093/infdis/166.1.31. [DOI] [PubMed] [Google Scholar]
  12. Johnson D. R., Stevens D. L., Kaplan E. L. Epidemiologic analysis of group A streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis. J Infect Dis. 1992 Aug;166(2):374–382. doi: 10.1093/infdis/166.2.374. [DOI] [PubMed] [Google Scholar]
  13. KELLNER A., ROBERTSON T. Myocardial necrosis produced in animals by means of crystalline streptococcal proteinase. J Exp Med. 1954 May 1;99(5):495–503. doi: 10.1084/jem.99.5.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kapur V., Maffei J. T., Greer R. S., Li L. L., Adams G. J., Musser J. M. Vaccination with streptococcal extracellular cysteine protease (interleukin-1 beta convertase) protects mice against challenge with heterologous group A streptococci. Microb Pathog. 1994 Jun;16(6):443–450. doi: 10.1006/mpat.1994.1044. [DOI] [PubMed] [Google Scholar]
  15. Kapur V., Majesky M. W., Li L. L., Black R. A., Musser J. M. Cleavage of interleukin 1 beta (IL-1 beta) precursor to produce active IL-1 beta by a conserved extracellular cysteine protease from Streptococcus pyogenes. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7676–7680. doi: 10.1073/pnas.90.16.7676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kapur V., Topouzis S., Majesky M. W., Li L. L., Hamrick M. R., Hamill R. J., Patti J. M., Musser J. M. A conserved Streptococcus pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin. Microb Pathog. 1993 Nov;15(5):327–346. doi: 10.1006/mpat.1993.1083. [DOI] [PubMed] [Google Scholar]
  17. Kehoe M. A., Kapur V., Whatmore A. M., Musser J. M. Horizontal gene transfer among group A streptococci: implications for pathogenesis and epidemiology. Trends Microbiol. 1996 Nov;4(11):436–443. doi: 10.1016/0966-842x(96)10058-5. [DOI] [PubMed] [Google Scholar]
  18. Li P., Allen H., Banerjee S., Franklin S., Herzog L., Johnston C., McDowell J., Paskind M., Rodman L., Salfeld J. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell. 1995 Feb 10;80(3):401–411. doi: 10.1016/0092-8674(95)90490-5. [DOI] [PubMed] [Google Scholar]
  19. Malke H., Mechold U., Gase K., Gerlach D. Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen. FEMS Microbiol Lett. 1994 Feb 1;116(1):107–112. doi: 10.1111/j.1574-6968.1994.tb06683.x. [DOI] [PubMed] [Google Scholar]
  20. McKerrow J. H., Sun E., Rosenthal P. J., Bouvier J. The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol. 1993;47:821–853. doi: 10.1146/annurev.mi.47.100193.004133. [DOI] [PubMed] [Google Scholar]
  21. Musser J. M., Hauser A. R., Kim M. H., Schlievert P. M., Nelson K., Selander R. K. Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2668–2672. doi: 10.1073/pnas.88.7.2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Musser J. M., Kapur V., Kanjilal S., Shah U., Musher D. M., Barg N. L., Johnston K. H., Schlievert P. M., Henrichsen J., Gerlach D. Geographic and temporal distribution and molecular characterization of two highly pathogenic clones of Streptococcus pyogenes expressing allelic variants of pyrogenic exotoxin A (Scarlet fever toxin). J Infect Dis. 1993 Feb;167(2):337–346. doi: 10.1093/infdis/167.2.337. [DOI] [PubMed] [Google Scholar]
  23. Musser J. M., Kapur V., Szeto J., Pan X., Swanson D. S., Martin D. R. Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype M1 protein: recent intercontinental spread of a subclone causing episodes of invasive disease. Infect Immun. 1995 Mar;63(3):994–1003. doi: 10.1128/iai.63.3.994-1003.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Musser J. M., Stockbauer K., Kapur V., Rudgers G. W. Substitution of cysteine 192 in a highly conserved Streptococcus pyogenes extracellular cysteine protease (interleukin 1beta convertase) alters proteolytic activity and ablates zymogen processing. Infect Immun. 1996 Jun;64(6):1913–1917. doi: 10.1128/iai.64.6.1913-1917.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nelson K., Schlievert P. M., Selander R. K., Musser J. M. Characterization and clonal distribution of four alleles of the speA gene encoding pyrogenic exotoxin A (scarlet fever toxin) in Streptococcus pyogenes. J Exp Med. 1991 Nov 1;174(5):1271–1274. doi: 10.1084/jem.174.5.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Norrby-Teglund A., Kaul R., Low D. E., McGeer A., Andersson J., Andersson U., Kotb M. Evidence for the presence of streptococcal-superantigen-neutralizing antibodies in normal polyspecific immunoglobulin G. Infect Immun. 1996 Dec;64(12):5395–5398. doi: 10.1128/iai.64.12.5395-5398.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. OGBURN C. A., HARRIS T. N., HARRIS S. Extracellular antigens in steady-state cultures of the hemolytic Streptococcus: production of proteinase at low pH. J Bacteriol. 1958 Aug;76(2):142–151. doi: 10.1128/jb.76.2.142-151.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. OGBURN C. A., HARRIS T. N., HARRIS S. The determination of streptococcal antiproteinase titers in sera of patients with rheumatic fever and streptococcal infection. J Immunol. 1958 Nov;81(5):396–403. [PubMed] [Google Scholar]
  29. Pinkney M., Kapur V., Smith J., Weller U., Palmer M., Glanville M., Messner M., Musser J. M., Bhakdi S., Kehoe M. A. Different forms of streptolysin O produced by Streptococcus pyogenes and by Escherichia coli expressing recombinant toxin: cleavage by streptococcal cysteine protease. Infect Immun. 1995 Jul;63(7):2776–2779. doi: 10.1128/iai.63.7.2776-2779.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Podbielski A., Flosdorff A., Weber-Heynemann J. The group A streptococcal virR49 gene controls expression of four structural vir regulon genes. Infect Immun. 1995 Jan;63(1):9–20. doi: 10.1128/iai.63.1.9-20.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Podbielski A., Pohl B., Woischnik M., Körner C., Schmidt K. H., Rozdzinski E., Leonard B. A. Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production. Mol Microbiol. 1996 Sep;21(5):1087–1099. doi: 10.1046/j.1365-2958.1996.661421.x. [DOI] [PubMed] [Google Scholar]
  32. Podbielski A., Woischnik M., Pohl B., Schmidt K. H. What is the size of the group A streptococcal vir regulon? The Mga regulator affects expression of secreted and surface virulence factors. Med Microbiol Immunol. 1996 Nov;185(3):171–181. doi: 10.1007/s004300050028. [DOI] [PubMed] [Google Scholar]
  33. Potempa J., Pavloff N., Travis J. Porphyromonas gingivalis: a proteinase/gene accounting audit. Trends Microbiol. 1995 Nov;3(11):430–434. doi: 10.1016/s0966-842x(00)88996-9. [DOI] [PubMed] [Google Scholar]
  34. Schwartz B., Elliott J. A., Butler J. C., Simon P. A., Jameson B. L., Welch G. E., Facklam R. R. Clusters of invasive group A streptococcal infections in family, hospital, and nursing home settings. Clin Infect Dis. 1992 Aug;15(2):277–284. doi: 10.1093/clinids/15.2.277. [DOI] [PubMed] [Google Scholar]
  35. Schwartz R. H. Models of T cell anergy: is there a common molecular mechanism? J Exp Med. 1996 Jul 1;184(1):1–8. doi: 10.1084/jem.184.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shanley T. P., Schrier D., Kapur V., Kehoe M., Musser J. M., Ward P. A. Streptococcal cysteine protease augments lung injury induced by products of group A streptococci. Infect Immun. 1996 Mar;64(3):870–877. doi: 10.1128/iai.64.3.870-877.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Talkington D. F., Schwartz B., Black C. M., Todd J. K., Elliott J., Breiman R. F., Facklam R. R. Association of phenotypic and genotypic characteristics of invasive Streptococcus pyogenes isolates with clinical components of streptococcal toxic shock syndrome. Infect Immun. 1993 Aug;61(8):3369–3374. doi: 10.1128/iai.61.8.3369-3374.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tao L., LeBlanc D. J., Ferretti J. J. Novel streptococcal-integration shuttle vectors for gene cloning and inactivation. Gene. 1992 Oct 12;120(1):105–110. doi: 10.1016/0378-1119(92)90016-i. [DOI] [PubMed] [Google Scholar]
  39. Tokuda M., Duncan M., Cho M. I., Kuramitsu H. K. Role of Porphyromonas gingivalis protease activity in colonization of oral surfaces. Infect Immun. 1996 Oct;64(10):4067–4073. doi: 10.1128/iai.64.10.4067-4073.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Twining S. S. Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal Biochem. 1984 Nov 15;143(1):30–34. doi: 10.1016/0003-2697(84)90553-0. [DOI] [PubMed] [Google Scholar]
  41. Wessels M. R., Moses A. E., Goldberg J. B., DiCesare T. J. Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8317–8321. doi: 10.1073/pnas.88.19.8317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Whatmore A. M., Kapur V., Sullivan D. J., Musser J. M., Kehoe M. A. Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci. Mol Microbiol. 1994 Nov;14(4):619–631. doi: 10.1111/j.1365-2958.1994.tb01301.x. [DOI] [PubMed] [Google Scholar]
  43. Wolf B. B., Gibson C. A., Kapur V., Hussaini I. M., Musser J. M., Gonias S. L. Proteolytically active streptococcal pyrogenic exotoxin B cleaves monocytic cell urokinase receptor and releases an active fragment of the receptor from the cell surface. J Biol Chem. 1994 Dec 2;269(48):30682–30687. [PubMed] [Google Scholar]
  44. Yu C. E., Ferretti J. J. Frequency of the erythrogenic toxin B and C genes (speB and speC) among clinical isolates of group A streptococci. Infect Immun. 1991 Jan;59(1):211–215. doi: 10.1128/iai.59.1.211-215.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zheng H., Fletcher D., Kozak W., Jiang M., Hofmann K. J., Conn C. A., Soszynski D., Grabiec C., Trumbauer M. E., Shaw A. Resistance to fever induction and impaired acute-phase response in interleukin-1 beta-deficient mice. Immunity. 1995 Jul;3(1):9–19. doi: 10.1016/1074-7613(95)90154-x. [DOI] [PubMed] [Google Scholar]
  46. van de Rijn I., Kessler R. E. Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun. 1980 Feb;27(2):444–448. doi: 10.1128/iai.27.2.444-448.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES