Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome

Nature Biotechnology volume 24, pages 191–197 (2006)Cite this article

Abstract

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The M1_SF370 surface proteome.
Figure 2: Topological analysis of the 37 surface-exposed proteins belonging to the membrane protein family.

Similar content being viewed by others

References

  1. Lindahl, G., Stalhammar-Carlemalm, M. & Areschoug, T. Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens. Clin. Microbiol. Rev. 18, 102–127 (2005).

    Article  CAS  Google Scholar 

  2. Courtney, H.S., Dale, J.B. & Hasty, D.L. in Handbook of Bacterial Adhesion: Principles, Methods and Applications (eds. An, Y.N. & Friedman, R.J.) 553–579 (Human Press, Totowa, NJ, 2002).

    Google Scholar 

  3. Lin, J., Huang, S. & Zhang, Q. Outer membrane proteins: key players for bacterial adaptation in host niches. Microbes Infect. 4, 325–331 (2002).

    Article  CAS  Google Scholar 

  4. Niemann, H.H., Schubert, W.D. & Heinz, D.W. Adhesins and invasins of pathogenic bacteria: a structural view. Microbes Infect. 6, 101–112 (2004).

    Article  CAS  Google Scholar 

  5. Ton-That, H., Marraffini, L.A. & Schneewind, O. Protein sorting to the cell wall envelope of Gram-positive bacteria. Biochim. Biophys. Acta 1694, 269–278 (2004).

    Article  CAS  Google Scholar 

  6. Janulczyk, R. & Rasmussen, M. Improved pattern for genome-based screening identifies novel cell wall–attached proteins in gram-positive bacteria. Infect. Immun. 69, 4019–4026 (2001).

    Article  CAS  Google Scholar 

  7. Galperin, M.Y. & Koonin, E.V. Searching for drug targets in microbial genomes. Curr. Opin. Biotechnol. 10, 571–578 (1999).

    Article  CAS  Google Scholar 

  8. Pizza, M. et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287, 1816–1820 (2000).

    Article  CAS  Google Scholar 

  9. Maione, D. et al. Identification of a universal group B Streptococcus vaccine by multiple genome screen. Science 309, 148–150 (2005).

    Article  CAS  Google Scholar 

  10. Nakai, K. Protein sorting signals and prediction of subcellular localization. Adv. Protein Chem. 54, 277–344 (2000).

    Article  CAS  Google Scholar 

  11. Doytchinova, I.A., Taylor, P. & Flower, D.R. Proteomics in vaccinology and immunobiology: an informatics perspective of the immunone. J. Biomed. Biotechnol. 2003, 267–290 (2003).

    Article  Google Scholar 

  12. Molloy, M.P. et al. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267, 2871–2881 (2000).

    Article  CAS  Google Scholar 

  13. Phadke, N.D. et al. Analysis of the outer membrane proteome of Caulobacter crescentus by two-dimensional electrophoresis and mass spectrometry. Proteomics 1, 705–720 (2001).

    Article  CAS  Google Scholar 

  14. Molloy, M.P. et al. Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry. Proteomics 2, 899–910 (2002).

    Article  CAS  Google Scholar 

  15. Nouwens, A.S. et al. Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1. Electrophoresis 21, 3797–3809 (2000).

    Article  CAS  Google Scholar 

  16. Rhomberg, T.A. et al. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae. Proteomics 4, 3021–3033 (2004).

    Article  CAS  Google Scholar 

  17. Sabarth, N. et al. Identification of surface proteins of Helicobacter pylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis. J. Biol. Chem. 277, 27896–27902 (2002).

    Article  CAS  Google Scholar 

  18. Guina, T. et al. Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation. J. Am. Soc. Mass Spectrom. 14, 742–751 (2003).

    Article  CAS  Google Scholar 

  19. Ferretti, J.J. et al. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl Acad. Sci. USA 98, 4658–4663 (2001).

    Article  CAS  Google Scholar 

  20. Marques, M.A., Chitale, S., Brennan, P.J. & Pessolani, M.C. Mapping and identification of the major cell wall-associated components of Mycobacterium leprae. Infect. Immun. 66, 2625–2631 (1998).

    CAS  PubMed  Google Scholar 

  21. Dallo, S.F., Kannan, T.R., Blaylock, M.W. & Baseman, J.B. Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae. Mol. Microbiol. 46, 1041–1051 (2002).

    Article  CAS  Google Scholar 

  22. Spence, J.M. & Clark, V.L. Role of ribosomal protein L12 in gonococcal invasion of Hec1B cells. Infect. Immun. 68, 5002–5010 (2000).

    Article  CAS  Google Scholar 

  23. Kurar, E. & Splitter, G.A. Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response. Vaccine 15, 1851–1857 (1997).

    Article  CAS  Google Scholar 

  24. Tomoyasu, T. et al. Topology and subcellular localization of FtsH protein in Escherichia coli. J. Bacteriol. 175, 1352–1357 (1993).

    Article  CAS  Google Scholar 

  25. Akiyama, Y., Kihara, A., Mori, H., Ogura, T. & Ito, K. Roles of the periplasmic domain of Escherichia coli FtsH (HflB) in protein interactions and activity modulation. J. Biol. Chem. 273, 22326–22333 (1998).

    Article  CAS  Google Scholar 

  26. Amara, R.R., Shanti, S. & Satchidanandam, V. Characterization of novel immunodominant antigens of Mycobacterium tuberculosis. Microbiology 144, 1197–1203 (1998).

    Article  CAS  Google Scholar 

  27. Padmalayam, I., Anderson, B., Kron, M., Kelly, T. & Baumstark, B. The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ. J. Bacteriol. 179, 4545–4552 (1997).

    Article  CAS  Google Scholar 

  28. Paterson, G.K. & Mitchell, T.J. The biology of Gram-positive sortase enzymes. Trends Microbiol. 12, 89–95 (2004).

    Article  CAS  Google Scholar 

  29. Lancefield, R.C. Studies on the antigenic composition of group A hemolytic Streptococci. J. Exp. Med. 78, 465–476 (1943).

    Article  CAS  Google Scholar 

  30. Mora, M. et al. Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens. Proc. Natl Acad. Sci. USA 102, 15641–15646 (2005).

    Article  CAS  Google Scholar 

  31. Stalhammar-Carlemalm, M., Areschoug, T., Larsson, C. & Lindahl, G. The R28 protein of Streptococcus pyogenes is related to several group B streptococcal surface proteins, confers protective immunity and promotes binding to human epithelial cells. Mol. Microbiol. 33, 208–219 (1999).

    Article  CAS  Google Scholar 

  32. Reid, S.D. et al. Postgenomic analysis of four novel antigens of group A Streptococcus: growth phase-dependent gene transcription and human serologic response. J. Bacteriol. 184, 6316–6324 (2002).

    Article  CAS  Google Scholar 

  33. McMillan, D.J. et al. Identification and assessment of new vaccine candidates for group A streptococcal infections. Vaccine 22, 2783–2790 (2004).

    Article  CAS  Google Scholar 

  34. Ji, Y., Carlson, B., Kondagunta, A. & Cleary, P.P. Intranasal immunization with C5a peptidase prevents nasopharyngeal colonization of mice by the group A Streptococcus. Infect. Immun. 65, 2080–2087 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Massell, B.F., Michael, J.G., Amezcua, J. & Siner, M. Secondary and apparent primary antibody responses after group A streptococcal vaccination of 21 children. Appl. Microbiol. 16, 509–518 (1968).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Dale, J.B., Chiang, E.Y., Liu, S., Courtney, H.S. & Hasty, D.L. New protective antigen of group A Streptococci. J. Clin. Invest. 103, 1261–1268 (1999).

    Article  CAS  Google Scholar 

  37. Kawabata, S. et al. Systemic and mucosal immunizations with fibronectin-binding protein FBP54 induce protective immune responses against Streptococcus pyogenes challenge in mice. Infect. Immun. 69, 924–930 (2001).

    Article  CAS  Google Scholar 

  38. Guzman, C.A., Talay, S.R., Molinari, G., Medina, E. & Chhatwal, G.S. Protective immune response against Streptococcus pyogenes in mice after intranasal vaccination with the fibronectin-binding protein SfbI. J. Infect. Dis. 179, 901–906 (1999).

    Article  CAS  Google Scholar 

  39. Kuo, C.F. et al. Role of streptococcal pyrogenic exotoxin B in the mouse model of group A streptococcal infection. Infect. Immun. 66, 3931–3935 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Roggiani, M. et al. Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models of streptococcal toxic shock syndrome. Infect. Immun. 68, 5011–5017 (2000).

    Article  CAS  Google Scholar 

  41. Lei, B., Liu, M., Chesney, G.L. & Musser, J.M. Identification of new candidate vaccine antigens made by Streptococcus pyogenes: purification and characterization of 16 putative extracellular lipoproteins. J. Infect. Dis. 189, 79–89 (2004).

    Article  CAS  Google Scholar 

  42. Cunningham, M.W. Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13, 470–511 (2000).

    Article  CAS  Google Scholar 

  43. Cole, J.N. et al. Surface analyses and immune reactivities of major cell wall–associated proteins of group A Streptococcus. Infect. Immun. 73, 3137–3146 (2005).

    Article  CAS  Google Scholar 

  44. Lancefield, R.C. Persistence of type-specific antibodies in man following infection with group A streptococci. J. Exp. Med. 110, 271–292 (1959).

    Article  CAS  Google Scholar 

  45. Lancefield, R.C. Current knowledge of type-specific M antigens of group A Streptococci. J. Immunol. 89, 307–313 (1962).

    CAS  PubMed  Google Scholar 

  46. Hu, M.C. et al. Immunogenicity of a 26-valent group A streptococcal vaccine. Infect. Immun. 70, 2171–2177 (2002).

    Article  CAS  Google Scholar 

  47. Fernandez-Espla, M.D., Garault, P., Monnet, V. & Rul, F. Streptococcus thermophilus cell wall–anchored proteinase: release, purification, and biochemical and genetic characterization. Appl. Environ. Microbiol. 66, 4772–4778 (2000).

    Article  CAS  Google Scholar 

  48. Harris, T.O., Shelver, D.W., Bohnsack, J.F. & Rubens, C.E. A novel streptococcal surface protease promotes virulence, resistance to opsonophagocytosis, and cleavage of human fibrinogen. J. Clin. Invest. 111, 61–70 (2003).

    Article  CAS  Google Scholar 

  49. Lei, B., Mackie, S., Lukomski, S. & Musser, J.M. Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect. Immun. 68, 6807–6818 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.M. Musser and G. Orefici for providing some of the strains used in this work and Marco Tortoli for his expert assistance in animal studies. We are also grateful to Giorgio Corsi for the artwork and Antonietta Maiorino for her expert secretarial assistance. Manuel J. Rodríguez-Ortega had an Intra-European Marie Curie postdoctoral fellowship from the EU. This work was in part supported by National Institutes of Health/ National Institute of Allergy and Infectious Diseases grant number 5U1060595-02, and in part by the Italian Ministry of Education, University and Research grant no. 10811.

Author information

Authors and Affiliations

  1. Chiron Vaccines, Via Fiorentina, Siena, 1 53100, Italy

    Manuel J Rodríguez-Ortega, Nathalie Norais, Giuliano Bensi, Sabrina Liberatori, Sabrina Capo, Marirosa Mora, Maria Scarselli, Francesco Doro, Germano Ferrari, Ignazio Garaguso, Tiziana Maggi, Anita Neumann, Alessia Covre, John L Telford & Guido Grandi

Authors
  1. Manuel J Rodríguez-Ortega
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nathalie Norais
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Giuliano Bensi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sabrina Liberatori
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sabrina Capo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marirosa Mora
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maria Scarselli
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Francesco Doro
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Germano Ferrari
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ignazio Garaguso
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Tiziana Maggi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Anita Neumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Alessia Covre
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. John L Telford
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Guido Grandi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Guido Grandi.

Ethics declarations

Competing interests

All authors work in a private company as either full-time employees or contract scientists and, in particular, G.G. and J.L.T. are holders of company stock options.

Supplementary information

Supplementary Table 1

Surfome of Group A Streptococcus M1_SF370 strain. (PDF 45 kb)

Supplementary Table 2

List of M1_SF370 surfome proteins grouped in degree-of-confidence categories. (PDF 14 kb)

Supplementary Table 3

Surfome of Group A Streptococcus M3_CDCSS90 strain and comparison with the M1_SF370 surfome. (PDF 13 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rodríguez-Ortega, M., Norais, N., Bensi, G. et al. Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome. Nat Biotechnol 24, 191–197 (2006). https://doi.org/10.1038/nbt1179

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1179

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing