Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly
Figure 7
The GTPase-binding domain (GBD) of N-WASP is a dominant negative inhibitor of EHEC pedestal formation and binds with high efficiency to a single EspFU repeat in yeast two-hybrid assays.
(A) The modular structure of N-WASP is depicted, featuring WASP homology-1 (WH1), GTPase-binding (GBD), proline-rich (PRD), and WH2/verprolin-connector-acidic (VCA) domains. Several N-WASP-binding partners are shown above their interacting domains. (B) HeLa cells transfected with plasmids encoding Flag-N-WASP constructs were infected with EHECΔdam (a mutant that binds to mammalian cells and generates pedestals with considerably higher efficiency than wild type EHEC [44]), fixed, and treated with DAPI to identify bacteria, a Flag antibody to visualize tagged N-WASP (top panels), and phalloidin to detect F-actin (bottom panels). Flag-N-WASP recruitment was only evaluated in cells expressing low levels of these tagged proteins (top panels), while effects on actin pedestal formation were only assessed in cells expressing high levels of Flag-N-WASP (bottom panels). Pedestal formation indices were determined by calculating the percentage of mock-transfected or Flag-N-WASP overexpressing cells harboring five or more actin pedestals. Data represent the mean+/−SD of three experiments. (C) HeLa cells expressing GFP alone, a GFP-tagged GBD, or a GFP-tagged GBD H208D point mutant were infected with EHECΔdam or EPEC and treated with DAPI to identify bacteria and phalloidin to detect F-actin. Pedestal formation indices were determined as in (B). (D) Plasmids encoding the N-WASP GBD fused to the LexA DNA-binding domain and EspFU fragments fused to the Gal4 transcriptional activation domain were co-transformed into a yeast two-hybrid reporter strain. Data represent the mean+/−SD of β-galactosidase activity for three co-transformants for each pairwise combination.