Caractérisation du rôle de la protéine IpaA sur l’ancrage cytosquelettique de Shigella flexneri

Shigella invades cells of the intestinal epithelium by injecting effector proteins through a Type III Secretion System (T3SS). The type III effector IpaA binds to talin and vinculin through three Vinculin Binding Sites (VBSs), promoting cytoskeletal anchorage to promote bacterial internalization. As opposed to other invasive bacteria, Shigella does not show constitutive cell binding activity but triggers transient adhesion through T3SS activation. IpaA VBS3 was found to be structurally analogous to talin H5 helix, which, together with H1-H4, forms the R1 talin bundle. Functional analysis identified IpaA VBS3 is necessary for talin recruitment to Shigella entry foci and formation of nascent adhesions and filopodia. IpaA VBS3 also stimulates filopodial capture of bacteria and stabilizes cell adhesions in invaded cells. On the other hand, structural models of IpaA VBSs/Vinculin interactions indicate a novel mode of vinculin activation, involving conformational changes in vinculin head domain that leads to trimerization and stabilization of focal adhesions. IpaA was also observed to be injected by "kissing-and-running" bacteria, facilitating additional bacterial binding and invasion events. This IpaA-dependent priming was dependent on IpaA VBS3 but not on IpaA VBS1-2. Injected IpaA was observed to form clusters apposed to the cell apical side and to induce integrin clustering. Overall, the results indicate that IpaA triggers general changes in cell adhesive properties and promotes a cooperative mechanism of Shigella invasion.Shigella envahit les cellules de l'épithélium intestinal en injectant des protéines effectrices via un système de sécrétion de type III (T3SS). L’effecteur de type III IpaA se lie à la taline et à la vinculine par l’intermédiaire de trois sites de liaison à la vinculine (VBS), favorisant ainsi l’ancrage cytosquelettique ainsi que l’internalisation des bactéries. Contrairement à d'autres bactéries invasives, Shigella ne montre pas d'activité de liaison cellulaire constitutive mais déclenche une adhésion transitoire par l'activation de T3SS. IpaA VBS3 s'est avéré être structurellement analogue à l'hélice de la taline H5, qui, avec H1-H4, forme le faisceau de taline R1. L'analyse fonctionnelle identifiée IpaA VBS3 est nécessaire au recrutement de la taline dans les foyers d'entrée de Shigella et à la formation d'adhérences naissantes et de filopodes. IpaA VBS3 stimule également la capture filopodiale des bactéries et stabilise les adhérences cellulaires dans les cellules envahies. D'autre part, les modèles structurels des interactions IpaA VBS ⁄ Vinculin indiquent un nouveau mode d'activation de la vinculine, impliquant des changements conformationnels dans le domaine de la tête de la vinculine, conduisant à la trimérisation et à la stabilisation des adhérences focales. IpaA a également été observé comme étant injecté par une bactérie "s'embrassant et courant", facilitant des événements supplémentaires de liaison bactérienne et d'invasion. Cet amorçage dépendant d'IpaA dépendait d'IpaA VBS3 mais pas d'IpaA VBS1-2. Une fois injecté, IpaA, forme des grappes apposées sur le côté apical de la cellule pour induire une grappe d'intégrines. Globalement, les résultats indiquent que l’IpaA provoque des modifications générales des propriétés adhésives des cellules et favorise un mécanisme coopératif d’invasion de Shigella

ipaA triggers vinculin oligomerization to strengthen cell adhesion during Shigella invasion

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

Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint

Summary: Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion