Title: Activating mutations of N-WASP reveal novel roles for N-WASP during Shigella infection
Description: Shigella flexneri is an intercellular bacterium that infects human intestinal mucosa. Actin-based motility, which is essential for Shigella pathogenesis, occurs when the bacterial encoded IcsA directly binds and usurps control of the host protein N-WASP, a critical regulator of the actin cytoskeleton. Putative host control mechanisms that regulate the activity of N-WASP to polymerize actin include alterations in the conformation and the phosphorylation status of N-WASP. This dissertation examines Shigella -mediated actin-based motility in the presence of N-WASP molecules that contain regulatory point mutations that disrupt both its phospho-regulation and the structural conformation, and describes potential novel roles for N-WASP during Shigella infection. Point mutations that were predicted to alter the phospho-regulation and structural conformation of N-WASP were generated to assess Shigella 's ability to modulate host regulation of N-WASP. To determine the function of the mutant N-WASP proteins, a mammalian-based system to purify recombinant full length N-WASP was developed. Two in vitro biochemical assays were employed to demonstrate that N-WASP mutations predicted to lead to an open conformation were constitutively active.

This is the first work to describe point mutations in N-WASP that have constitutive actin polymerization activity that are believed to result from a constitutively open conformation. Using N-WASP deficient MEFs, stable cell lines expressing each of the N-WASP mutant constructs were created. These cell lines showed no signs of aberrant actin polymerization despite the expression of constitutively active N-WASP mutants suggesting that there are other levels of N-WASP regulation in addition to structural conformation and tyrosine phosphorylation. All cell lines were capable of supporting Shigella -mediated actin-based motility, indicating that regulation of N-WASP through tyrosine phosphorylation and structural conformation is not essential for Shigella -mediated actin polymerization. Reductions in the lengths of actin tails formed by Shigella , which is proportional to the rate of actin polymerization, demonstrate the importance of regulation of N-WASP conformation for efficient Shigella -mediated actin-based motility and pathogenesis. We demonstrate that phospho-regulation alone does not alter tail length, but that constitutively active N-WASP mutants lead to a reduction in actin tail lengths. The shorter tail lengths associated with this constitutively active construct can be rescued to wild type levels upon introduction of an additional phospho-mimicking mutation to the protein.

In contrast, the addition of a phospho-disruptive mutation has no effect on the shorter tail lengths. Despite producing smaller tails in cells expressing constitutively active form of N-WASP, Shigella do not have a reduced rate of intracellular motility. Shigella are able to form a greater number of plaques in N-WASP deficient cells expressing constitutively active N-WASP compared to those expressing wild type N-WASP. The increase in plaque numbers cannot be attributed to an increase in invasion rate or an increase in intercellular spreading, implying that increased survival of Shigella after invasion from an extracellular environment leads the elevated number of plaques formed. These data suggest a novel model for the role of N-WASP during the process of Shigella -mediated actin-based motility and infection.