title: Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag.
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-3beta 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-3beta 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.
authors.affiliation: Department of Microbiology and Immunology, University of Miami Miller School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101, USA.
authors.forename: Julie Torruellas
authors.initials: JT
authors.lastname: Garcia
authors.forename: Franco
authors.initials: F
authors.lastname: Ferracci
authors.forename: Michael W
authors.initials: MW
authors.lastname: Jackson
authors.forename: Sabrina S
authors.initials: SS
authors.lastname: Joseph
authors.forename: Isabelle
authors.initials: I
authors.lastname: Pattis
authors.forename: Lisa R W
authors.initials: LR
authors.lastname: Plano
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Fischer
authors.forename: Gregory V
authors.initials: GV
authors.lastname: Plano
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D038341:Glycogen Synthase Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D015010:Yersinia pestis / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D038341:Glycogen Synthase Kinases