Zettl, Markus Franz Johann; (2004) Signalling of pathogens to the actin cytoskeleton: Characterisation of the N-WASP/WIP complex in the actin based motility of EPEC, Shigella and vaccinia virus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Surprisingly organisms like viruses and bacteria developed common mechanisms of exploiting the host machinery for actin polymerisation mostly to spread their infection. A complex of N-WASP and WASP interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and is as well recruited to the surface of the gram-negative bacterium Shigella flexneri. Both processes are dependent on actin polymerisation, which is nucleated by the Arp2/3 complex. In contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex in vitro. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP information is lacking concerning the nature of its binding site, which could help to understand the role of WIP in vivo. This work reports the identification of the N-WASP WH1 binding motif in WIP, which turns out to be conserved in WIP homologues. To confirm our biochemical results in vivo we examined the effects of expressing WIP mutants deficient in N-WASP binding on actin based motility of Shigella and vaccinia. Expressions of these mutants led to a loss of recruitment of WIP to both pathogens and abrogated the inhibitory effects of the WASP binding domain (WBD) on vaccinia actin tail formation. Enteropathogenic E.coli (EPEC) like vaccinia remodels the host actin cytoskeleton and uses WASP and the Arp2/3 complex to form actin rich pedestal depending on tyrosine phosphorylation of pathogen surface membrane proteins. WIP was found to localise to the tip of actin pedestals and to be functionally involved in EPEC induced pedestal formation. Like vaccinia virus EPEC recruits WIP through its proline rich and WASP binding domain. The proline rich domain of WIP binds to the SH2/SH3 adaptor protein Nek, which is essential for EPEC actin pedestals and vaccinia actin tail formation. Deletion of the proline rich region of WIP but not N-WASP was essential for WIP recruitment to EPEC pedestals. Furthermore in absence of N-WASP neither WIP nor Nek were recruited to EPEC. Taken together data in this thesis suggest that EPEC like vaccinia recruit a complex of Nck, WIP and N-WASP. A comparison of vaccinia and EPEC however shows that the complex is recruited in differently and that in contrast to vaccinia actin tail, EPEC pedestal formation is independent of Src kinases.

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