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, Franco Ferracci, Michael W. Jackson, Sabrina S. Joseph, Isabelle Pattis, Lisa R W Plano, Wolfgang Fischer, Gregory V Plano

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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-3β 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-3β 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.

Original languageEnglish
Pages (from-to)5645-5657
Number of pages13
JournalInfection and Immunity
Volume74
Issue number10
DOIs
StatePublished - Oct 1 2006

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Glycogen Synthase Kinases
Injections
Proteins
Glycogen Synthase Kinase 3
Eukaryotic Cells
Phosphorylation
Yersinia pestis
Peptides
Type IV Secretion Systems
Type III Secretion Systems
Protein Transport
HeLa Cells
Helicobacter pylori
Protein Kinases
Virulence
Plasmids
Phosphotransferases
Antibodies

ASJC Scopus subject areas

  • Immunology

Cite this

Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag. / Garcia, Julie Torruellas; Ferracci, Franco; Jackson, Michael W.; Joseph, Sabrina S.; Pattis, Isabelle; Plano, Lisa R W; Fischer, Wolfgang; Plano, Gregory V.

In: Infection and Immunity, Vol. 74, No. 10, 01.10.2006, p. 5645-5657.

Research output: Contribution to journalArticle

Garcia, Julie Torruellas ; Ferracci, Franco ; Jackson, Michael W. ; Joseph, Sabrina S. ; Pattis, Isabelle ; Plano, Lisa R W ; Fischer, Wolfgang ; Plano, Gregory V. / Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag. In: Infection and Immunity. 2006 ; Vol. 74, No. 10. pp. 5645-5657.
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