Characterization of TccP-mediated N-WASP activation during enterohaemorrhagic Escherichia coli infection.
Subversion of the host cell cytoskeleton is the hallmark of enterohaemorrhagic Escherichia coli (EHEC) infection. EHEC translocates the trans-membrane receptor protein Tir (translocated intimin receptor), which links the extracellular bacterium to the eukaryotic cell actin cytoskeleton, triggering formation of actin-rich pedestals beneath adherent bacteria. Tir-mediated actin accretion by EHEC requires TccP (Tir cytoskeleton coupling protein), a recently discovered type III secretion system effector protein which, following translocation, binds and activates Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the actin-related protein 2/3 complex leading to localized polymerization of actin. In this study, truncated N-WASP and TccP derivatives were generated and tested in in vitro actin polymerization and epithelial cell infection assays. The C-terminal amino acids 253-276 of the GTPase binding domain (GBD) of N-WASP were identified as essential, although not sufficient, for TccP:N-WASP protein:protein interaction, TccP-mediated N-WASP activation and induction of actin polymerization. TccP from EHEC O157:H7 strain EDL933 consists of a unique N-terminal domain and six proline-rich repeats. Progressive deletions within the N-terminus of TccP revealed that residues 1-21 are necessary and sufficient for its translocation, while amino acids 1-181, encompassing the N-terminal translocation signal and two proline-rich repeats, are sufficient for triggering actin polymerization in EHEC-infected epithelial cells and in in vitro actin polymerization assays. This study defines the modular domain structure of TccP and the molecular basis of TccP-mediated N-WASP activation and EHEC-induced remodelling of the host actin cytoskeleton.
Garmendia
Junkal
J
Division of Cellular and Molecular Biology, Imperial College, London, UK.
Carlier
Marie-France
MF
Egile
Coumaran
C
Didry
Dominique
D
Frankel
Gad
G
D000199:Actins / Q000378:metabolism*; D000465:Algorithms; D015153:Blotting, Western; D002460:Cell Line; D019281:Dimerization; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D051316:Wiskott-Aldrich Syndrome Protein; C505370:tccP protein, E coli