This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Torruellas Garcia, J.
Right arrow Articles by Plano, G. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Torruellas Garcia, J.
Right arrow Articles by Plano, G. V.

 Previous Article  |  Next Article 

Infection and Immunity, October 2006, p. 5645-5657, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00690-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag

Julie Torruellas Garcia,1,{dagger} Franco Ferracci,1,{dagger} Michael W. Jackson,1 Sabrina S. Joseph,1 Isabelle Pattis,3 Lisa R. W. Plano,2 Wolfgang Fischer,3 and Gregory V. Plano1*

Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida,1 Department of Pediatrics, University of Miami Miller School of Medicine, Miami, Florida,2 Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, Munich, Germany3

Received 28 April 2006/ Returned for modification 14 June 2006/ Accepted 24 July 2006

Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3ß kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3ß antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Miami Miller School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101. Phone: (305) 243-6310. Fax: (305) 243-4623. E-mail: gplano{at}med.miami.edu.

Editor: J. B. Bliska

{dagger} Julie Torruellas Garcia and Franco Ferracci contributed equally to this work.

Infection and Immunity, October 2006, p. 5645-5657, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00690-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Riordan, K. E., Sorg, J. A., Berube, B. J., Schneewind, O. (2008). Impassable YscP Substrates and Their Impact on the Yersinia enterocolitica Type III Secretion Pathway. J. Bacteriol. 190: 6204-6216 [Abstract] [Full Text]  
  • Lilo, S., Zheng, Y., Bliska, J. B. (2008). Caspase-1 Activation in Macrophages Infected with Yersinia pestis KIM Requires the Type III Secretion System Effector YopJ. Infect. Immun. 76: 3911-3923 [Abstract] [Full Text]  
  • Pattis, I., Weiss, E., Laugks, R., Haas, R., Fischer, W. (2007). The Helicobacter pylori CagF protein is a type IV secretion chaperone-like molecule that binds close to the C-terminal secretion signal of the CagA effector protein. Microbiology 153: 2896-2909 [Abstract] [Full Text]  
  • Urbanowski, M. L., Brutinel, E. D., Yahr, T. L. (2007). Translocation of ExsE into Chinese Hamster Ovary Cells Is Required for Transcriptional Induction of the Pseudomonas aeruginosa Type III Secretion System. Infect. Immun. 75: 4432-4439 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»