Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspF-mediated actin assembly and harbours N-terminal sequences that influence pedestal length

Cell Microbiol. 2006 Sep;8(9):1488-503. doi: 10.1111/j.1462-5822.2006.00728.x.


Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) both utilize type III secretion systems that translocate the effector protein Tir into the plasma membrane of mammalian cells in order to stimulate localized actin assembly into 'pedestals'. The Tir molecule that EPEC delivers is phosphorylated within its C-terminus on tyrosine-474, and a clustered 12-residue phosphopeptide encompassing this residue initiates an efficient signalling cascade that triggers actin polymerization. In addition to Y474, tyrosine-454 of EPEC Tir is phosphorylated, although inefficiently, and promotes actin polymerization at low levels. In contrast to EPEC Tir, EHEC Tir lacks Y474 and triggers pedestal formation in a phosphotyrosine-independent manner by interacting with an additional effector protein, EspF(U). To identify EHEC Tir sequences that regulate localized actin assembly, we circumvented the strict requirements for type III translocation and directly expressed Tir derivatives in mammalian cells by transfection. Infection of Tir-expressing cells with a Tir-deficient EHEC strain demonstrated that ectopically expressed Tir localizes to the plasma membrane, is modified by mammalian serine-threonine kinases and is fully functional for actin pedestal formation. Removal of portions of the cytoplasmic N-terminus of Tir resulted in the generation of abnormally long pedestals, indicating that this region of EHEC Tir influences pedestal length. In the presence of the entire N-terminal domain, a 12-residue peptide from the C-terminus of EHEC Tir is both necessary and sufficient to recruit EspF(U) and initiate actin pedestal formation. This peptide encompasses the portion of EHEC Tir analogous to the EPEC Tir-Y454 region and is present within the Tir molecules of all pedestal-forming bacteria, suggesting that this sequence harbours a conserved signalling function.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Actin Cytoskeleton / metabolism
  • Actins / metabolism*
  • Amino Acid Sequence
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Membrane / metabolism
  • Escherichia coli O157 / genetics*
  • Escherichia coli O157 / growth & development
  • Escherichia coli O157 / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Genetic Complementation Test
  • HeLa Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Microscopy, Fluorescence / methods
  • Models, Genetic
  • Mutation / genetics
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / physiology
  • Phosphorylation
  • Receptors, Cell Surface / chemistry
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Sequence Homology, Amino Acid


  • Actins
  • Carrier Proteins
  • Escherichia coli Proteins
  • EspFU protein, E coli
  • Intracellular Signaling Peptides and Proteins
  • Peptide Fragments
  • Receptors, Cell Surface
  • Tir protein, E coli