Summary
Streptococcus pyogenes secretes various proteins to the extracellular environment. During infection, these proteins interact with human macromolecules and contribute to pathogenesis. We describe a proteomic approach routinely used in our laboratory to characterize culture supernatant proteins using small-format two-dimensional gel electrophoresis. Proteins are collected after overnight growth of the bacteria in broth media. Compounds that inhibit isoelectric focusing, such as salts, are removed by enzymatic treatment and precipitation with trichloroacetic acid and acetone. Following resuspension in denaturing solution, the proteins are separated by isoelectric focusing using a 7-cm immobilized strip with a pH gradient of 4–7. Subsequently, proteins are further separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained with SYPRO Ruby. The small-gel format requires less time, reagents, and smaller culture volumes compared with large-format approaches, while still resolving and detecting a large proportion of the exoprotein fraction.
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References
Cunningham, M. W. (2000) Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13, 470–511.
Sumby, P., Barbian, K. D., Gardner, D. J., Whitney, A. R., Welty, D. M., Long, R. D., Bailey, J. R., Parnell, M. J., Hoe, N. P., Adams, G. G., DeLeo, F. R., and Musser, J. M. (2005) Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc. Natl. Acad. Sci. U.S.A. 102, 1679–1684.
Pancholi, V. and Fischetti, V. A. (1992) A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. J. Exp. Med. 176, 415–426.
Pancholi, V. and Fischetti, V. A. (1993) Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptoc occi is also an ADP-ribosylating enzyme. Proc. Natl. Acad. Sci. U.S.A. 90, 8154–8158.
Pancholi, V. and Fischetti, V. A. (1998) Alpha-enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J. Biol. Chem. 273, 14503–14515.
Rabilloud, T. (2000) Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2, 3–10.
Lei, B., Mackie, S., Lukomski, S., and Musser, J. M. (2000) Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect. Immun. 68, 6807–6818.
Rabilloud, T. (1998) Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis 19,758–760.
Gorg, A., Weiss, W., and Dunn, M. W. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.
Sanchez, J. C., Rouge, V., Pisteur, M., Ravier, F., Tonella, L., Moosmayer, M., Wilkins, M. R., and Hochstrasser, D. F. (1997) Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18, 324–327.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Chaussee, M.A., McDowell, E.J., Chaussee, M.S. (2008). Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes . In: DeLeo, F.R., Otto, M. (eds) Bacterial Pathogenesis. Methods in Molecular Biology™, vol 431. Humana Press. https://doi.org/10.1007/978-1-60327-032-8_2
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DOI: https://doi.org/10.1007/978-1-60327-032-8_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-740-2
Online ISBN: 978-1-60327-032-8
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