IpaA Targets β1 Integrins and Rho to Promote Actin Cytoskeleton Rearrangements Necessary for Shigella Entry

Kris A Demali,April L Jue,Keith Burridge

doi:10.1074/jbc.m605939200

Kris A Demali, April L Jue + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m605939200

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Abstract

Shigella invasion into the colonic epithelium involves many steps including the formation of large membrane protrusions by the epithelial cells that facilitate bacterial engulfment. IpaA, a Shigella protein secreted into target cells upon cell contact induces a loss of actin stress fibers in cells and promotes the reorganization of actin at the site of entry. The mechanism for this is not known but is thought to involve recruitment of the focal adhesion protein vinculin to IpaA. Here we have examined the mechanism for the effects of IpaA on the actin cytoskeleton. We show that IpaA-induced loss of actin stress fibers and cell rounding do not require vinculin expression or an intact vinculin binding site on IpaA. Rather, we find that cells expressing IpaA exhibited elevated Rho activity and increased myosin light chain phosphorylation. In addition, IpaA decreases integrin affinity for extracellular matrix ligands by interfering with talin recruitment to the integrin cytoplasmic tail. The combination of these two effects, namely weakened adhesion and increased contractility, account for the loss of actin stress fibers and cell rounding observed in cells exposed to IpaA.

Highlights

To facilitate invasion, the Shigella type III secretion system is stimulated upon contact with host cells and delivers bacterial “effector” molecules into the surrounding bacterial space and/or host cell membrane [1]
We provide evidence that there is a loss of stress fibers in vinculin-null mouse embryo fibroblasts (MEFs)2 expressing IpaA or in fibroblasts expressing a mutant of IpaA unable to bind vinculin
We find that cells expressing IpaA have elevated Rho activity and increased phosphorylation of myosin light chain resulting in increases in contractility that can be blocked by inhibitors of Rho kinase

Summary

EXPERIMENTAL PROCEDURES

Reagents and Cell Lines—The vinculin-null mouse embryo fibroblasts (VinϪ/Ϫ; Ref. 23) were a generous gift of E. VinϪ/Ϫ, HeLa, and REF52s were maintained in Dulbecco’s modified Eagle’s medium ϩ 10% fetal bovine serum as previously described [24]. Rho Activity—Rho activity assays were performed as previously described [27] using the RhoA-binding domain of Rhotekin expressed as a GST fusion protein. Nickel-nitrilotriacetic acid beads containing 50 ␮g of purified ␤1a integrin cytoplasmic domain were incubated with 300 ␮l of talin-enriched platelet extracts in the presence of 0.2 or 2 ␮g of GST or GST-IpaA. FACS Analysis—Subconfluent cultures of HeLa cells expressing Myc or Myc-IpaA were lifted from tissue culture dishes by incubation in phosphate-buffered saline ϩ 0.6 mM EDTA for 15 min at 37 °C, washed, and stained with TS2/16 (American Type Tissue Culture Collection) or 12G10 (Chemicon) for 1 h at 4 °C. For each wash step the cells were resuspended and pelleted in Dulbecco’s modified Eagle’s medium ϩ 0.2% fetal bovine serum

RESULTS

DISCUSSION

Methods