Valencia-Gallardo, C. | Search bioRxiv

bioRxiv2023

Polar interactions determine head domain-mediated vinculin oligomerization induced by the Shigella IpaA effector

Vinculin is a cytoskeletal linker strengthening cell adhesion. The Shigella IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a "closed" and "open" D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced Shigella invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell matrix adhesion.

cell biology↗

bioRxiv2019

Vinculin targeting by Shigella IpaA promotes stable cell adhesion independent of mechanotransduction

The Shigella effector IpaA co-opts the focal adhesion protein vinculin to promote bacterial invasion. Here, we show that IpaA triggers an unreported mode of vinculin activation through the cooperative binding of its three vinculin-binding sites (VBSs) leading to vinculin oligomerization via its D1 and D2 head subdomains and highly stable adhesions resisting actin relaxing drugs. Using cross-linking mass spectrometry, we found that while IpaA VBSs1-2 bound to D1, IpaA VBS3 interacted with D2, a subdomain masked to other known VBSs. Structural modeling indicated that as opposed to canonical activation linked to interaction with D1, these combined VBSs interactions triggered major allosteric changes leading to D1D2 oligomerization. A cysteine-clamp preventing these changes and D1D2 oligomerization impaired growth of vinculin microclusters and cell adhesion. We propose that D1D2-mediated vinculin oligomerization occurs during the maturation of adhesion structures to enable the scaffolding of high-order vinculin complexes, and is triggered by Shigella IpaA to promote bacterial invasion in the absence of mechanotransduction. SummaryThe Shigella IpaA effector binds to cryptic vinculin sites leading to oligomerization via its head domain. This vinculin oligomerization mode appears required for the maturation and strengthening of cell adhesion but is co-opted by invasive bacteria independent of actomyosin contractility.

microbiology↗