Activating mutations of N-WASP alter Shigella pathogenesis

Abstract

The pathogenesis of Shigella requires binding to the host protein N-WASP. To examine the roles of structural conformation and phospho-regulation of N-WASP during Shigella pathogenesis, mutant N-WASP constructs predicted to result in a constitutively open conformation (L229P and L232P) or either a phospho-mimicking (Y253E) or phospho-disruptive (Y253F) structure were constructed. Pyrene actin assays demonstrated that the N-WASP L229P and L232P constructs are constitutively active. Despite the increase in actin polymerization seen in vitro, cell lines expressing N-WASP L229P and L232P supported shorter actin tails when infected with Shigella. Shigella actin tails were unchanged in cells expressing N-WASP phospho-regulation mutant proteins. Shigella invasion, intracellular, and intercellular motility were not altered in cells expressing N-WASP L229P or L232P. However, plaque numbers were increased in cells expressing N-WASP L229P and L232P. These data demonstrate that N-WASP structural conformation is an important regulator of Shigella pathogenesis in distinct segments of its lifecycle.

Fig. 1. NWASP mutations result in constitutive activation

(A) Protein sequence alignment of a portion of the GBD of murine WASP and rat NWASP. Predicted tertiary structure of the sequence denoting α-helixes is shown [4]. The residues selected for mutations are L229 in purple, L232 in red, and Y253 in green. Table represents the predicted structural phenotypes for each of the NWASP mutants. (B) Pyrene actin assay measuring the actin polymerization activity of NWASP mutations predicted to have an open conformation in the presence/absence of Cdc42-GTPγS. (C) Pyrene actin assay with phospho-regulatory NWASP in the presence/absence of Cdc42-GTPγS. (D). Histogram demonstrating that NWASP-deficient fibroblasts expressing NWASP phospho-regulatory (Y253E and Y253F) or constitutively active (L229P & L232P) constructs result in increased migration in a scratch assay (* p< 0.05).

Fig. 2. NWASP regulatory mutants localize appropriately and support Shigella-mediated actin based motility

(A) Stable cell lines expressing each of the NWASP constructs were infected with Shigella. Cells were stained for DNA (blue), NWASP (green), and actin (red). Insets show polar NWASP localization (arrow heads) and actin tail formation (arrows). (B) Tails formed in cells expressing NWASP Y253E and Y253F are similar in length to those expressed in cells expressing WT NWASP. (C) Tails formed in cells expressing NWASP L229P and L232P are shorter in length than those expressed in cells expressing WT NWASP. * P<.05 (Mann-Whitney test). (D) Time lapse microscopy was used to quantify the velocity of Shigella undergoing actin-based motility. Average speed of Shigella in cells expressing WT NWASP, L229P, or L232P is shown.

Fig. 3. Markedly increased plaque numbers, but not invasion rate or intercellular spread, in cells expressing constitutively active NWASP

(A) Increased number of plaques formed in cell lines expressing constitutively active NWASP L229 and L2332. Representative monolayers imaged 48h after infection with GFP expressing Shigella. Green signal represents Shigella plaques (inset indicates number of plaques in monolayer). Histogram on right is quantification of three independent experiments. (B) Shigella invasion rates are compared in stable cell lines expressing WT, L229 or L2332 NWASP constructs. Results shown are average of three independent experiments (* p < 0.05). (C) The average size of plaques formed in each cell line expressing mutant NWASP constructs was not significantly different from cell lines expressing WT NWASP. Combined results of three individual experiments are shown.