Enteropathogenic Escherichia coli and vaccinia virus do not require the family of WASP-interacting proteins for pathogen-induced actin assembly

Abstract

The human pathogens enteropathogenic Escherichia coli (EPEC) and vaccinia virus trigger actin assembly in host cells by activating the host adaptor Nck and the actin nucleation promoter neural Wiskott-Aldrich syndrome protein (N-WASP). EPEC translocates effector molecules into host cells via type III secretion, and the interaction between the translocated intimin receptor (Tir) and the bacterial membrane protein intimin stimulates Nck and N-WASP recruitment, leading to the formation of actin pedestals beneath adherent bacteria. Vaccinia virus also recruits Nck and N-WASP to generate actin tails that promote cell-to-cell spread of the virus. In addition to Nck and N-WASP, WASP-interacting protein (WIP) localizes to vaccinia virus tails, and inhibition of actin tail formation upon ectopic expression of WIP mutants led to the suggestion that WIP is required for this process. Similar studies of WIP mutants, however, did not affect the ability of EPEC to form actin pedestals, arguing against an essential role for WIP in EPEC-induced actin assembly. In this study, we demonstrate that Nck and N-WASP are normally recruited by vaccinia virus and EPEC in the absence of WIP, and neither WIP nor the WIP family members CR16 and WIRE/WICH are essential for pathogen induced actin assembly. In addition, although Nck binds EPEC Tir directly, N-WASP is required for its localization during pedestal formation. Overall, these data highlight similar pathogenic strategies shared by EPEC and vaccinia virus by demonstrating a requirement for both Nck and N-WASP, but not WIP or WIP family members in pathogen-induced actin assembly.

Fig 1

Actin pedestal formation by EPEC and EHEC does not require Cdc42. Pedestal formation by EPEC and EHEC is independent of Cdc42. EPEC and EHEC infection result in pedestal formation (arrows) in both WT (left panel) and Cdc42^−/− FLCs (right panel) stained with phalloidin (F-actin, red) and DAPI (DNA, blue). Scale bar, 5 μm.

Fig 2

WIP localizes to EPEC- and EHEC-induced pedestals but is not required for actin assembly by EPEC, EHEC, Shigella, or vaccinia virus. WT (top row) and WIP^−/− (bottom row) fibroblasts were infected with EPEC, EHEC, Shigella, and vaccinia virus. Bacterial and viral DNA was identified by DAPI staining, and the sites of actin assembly were determined by staining with rhodamine-phalloidin. (A) WIP (arrows) localizes to actin pedestals induced by both EPEC and EHEC (upper panels); both EPEC and EHEC form actin-pedestals on WIP^−/− cells (lower panels). (B) Shigella and vaccinia virus form actin tails efficiently on WT and WIP^−/− FLC. Scale bar, 5 μm.

Fig 3

WIP is not essential for the recruitment of Nck or N-WASP during infection with vaccinia virus, EPEC or EHEC. F-actin (phalloidin staining, red) and DNA (DAPI, blue) were visualized in WT and WIP^−/− FLC infected with vaccinia virus (top row), EPEC (middle row), or EHEC (lower row). Actin tails and pedestals are formed efficiently in WIP^−/− FLC, and N-WASP (green) (arrows, left column) or Nck (green) (arrows, right column) are recruited to EPEC and vaccinia virus in the absence of WIP. *, Lack of Nck localization to EHEC. Scale bar, 5 μm.

Fig 4

Neither N-WASP nor WIP is recruited to EPEC in the absence of Nck. FLC transfected with a dominant-negative inhibitor of Nck (green) were infected with either EPEC (upper row) or EHEC (lower row) and were analyzed by DAPI staining of DNA and immunostained for N-WASP (left panel, red) or WIP (right panel, red). N-WASP localizes to EHEC (closed arrowhead), but not EPEC in DN-Nck transfected cells (open arrowhead). WIP localizes to EHEC (closed arrowhead), but not EPEC (open arrowhead) in DN-Nck transfected cells. Green anti-myc staining is omitted from the insets to improve phalloidin and DAPI visualization. Scale bar, 10 μm; inset scale bar, 2.5 μm.

Fig 5

N-WASP is required for the recruitment of both Nck and WIP. N-WASP^−/− FLC were infected with either EPEC (upper row) or EHEC (middle row) or vaccinia virus (lower row) and were analyzed by DAPI staining of DNA (blue), phalloidin staining for actin (red), and immunostaining with Nck (left panels, green) or transfection with WIP-GFP (right panels, green). Neither Nck nor WIP localized to EPEC (arrows), EHEC (arrows) or vaccinia virus (arrows) in the absence of N-WASP. Scale bar, 5 μm.

Fig 6

The WIP family member WIRE/WICH does not substitute for WIP in WIP^−/− cells during infection with EPEC or vaccinia virus. EPEC (upper row) and vaccinia virus (lower row) were equally able to induce actin pedestals and actin tails, respectively, in WIP WT (left panels) or KO cells (right panels) depleted of the WIRE/WICH by siRNA. Red is actin staining with Alexa Fluor 594-conjugated phalloidin; insets show DAPI staining of nuclei (omitted from the figure for clarity). Scale bar, 5 μm.

Fig 7

Model for strategies of Nck/N-WASP recruitment and activation shared by EPEC and vaccinia virus and contrasted with EHEC and Shigella.