4 research outputs found
CaractĂ©risation du rĂŽle de la protĂ©ine IpaA sur lâancrage 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'Ă©pithĂ©lium intestinal en injectant des protĂ©ines effectrices via un systĂšme de sĂ©crĂ©tion de type III (T3SS). Lâeffecteur de type III IpaA se lie Ă la taline et Ă la vinculine par lâintermĂ©diaire de trois sites de liaison Ă la vinculine (VBS), favorisant ainsi lâancrage cytosquelettique ainsi que lâinternalisation des bactĂ©ries. Contrairement Ă d'autres bactĂ©ries invasives, Shigella ne montre pas d'activitĂ© de liaison cellulaire constitutive mais dĂ©clenche une adhĂ©sion transitoire par l'activation de T3SS. IpaA VBS3 s'est avĂ©rĂ© ĂȘtre structurellement analogue Ă l'hĂ©lice de la taline H5, qui, avec H1-H4, forme le faisceau de taline R1. L'analyse fonctionnelle identifiĂ©e IpaA VBS3 est nĂ©cessaire au recrutement de la taline dans les foyers d'entrĂ©e de Shigella et Ă la formation d'adhĂ©rences naissantes et de filopodes. IpaA VBS3 stimule Ă©galement la capture filopodiale des bactĂ©ries et stabilise les adhĂ©rences cellulaires dans les cellules envahies. D'autre part, les modĂšles structurels des interactions IpaA VBS â Vinculin indiquent un nouveau mode d'activation de la vinculine, impliquant des changements conformationnels dans le domaine de la tĂȘte de la vinculine, conduisant Ă la trimĂ©risation et Ă la stabilisation des adhĂ©rences focales. IpaA a Ă©galement Ă©tĂ© observĂ© comme Ă©tant injectĂ© par une bactĂ©rie "s'embrassant et courant", facilitant des Ă©vĂ©nements supplĂ©mentaires de liaison bactĂ©rienne et d'invasion. Cet amorçage dĂ©pendant d'IpaA dĂ©pendait d'IpaA VBS3 mais pas d'IpaA VBS1-2. Une fois injectĂ©, IpaA, forme des grappes apposĂ©es sur le cĂŽtĂ© apical de la cellule pour induire une grappe d'intĂ©grines. Globalement, les rĂ©sultats indiquent que lâIpaA provoque des modifications gĂ©nĂ©rales des propriĂ©tĂ©s adhĂ©sives des cellules et favorise un mĂ©canisme coopĂ©ratif dâinvasion 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