Pré-Publication, Document De Travail Année : 2022

Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system

Clara Lettl
  • Fonction : Auteur
Jérémy Guérin
Anaïs Vieille
  • Fonction : Auteur
Sven Furler
  • Fonction : Auteur
Sylvie Briand-Schumacher
  • Fonction : Auteur
Birgit Dreier
Célia Bergé
  • Fonction : Auteur
Andreas Plückthun
Sandrine Vadon-Le Goff
Rémi Fronzes
Wolfgang Fischer
  • Fonction : Auteur
Laurent Terradot

Résumé

Abstract The bacterial human pathogen Helicobacter pylori produces a type IV secretion system ( cag T4SS) to inject the oncoprotein CagA into gastric cells. The cag T4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shaped N-terminal domains (CagI N ) and prolonged by globular C-terminal domains (CagI C ). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagI C with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagI C were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagI C plays a key role in cag T4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cag T4SS, a crucial risk factor for gastric cancer development. Author summary Helicobacter pylori is a bacterial pathogen that colonises the human stomach in half of the world’s population. The most virulent strains use the cag- type IV secretion system ( cag T4SS), a molecular nanomachine capable of injecting the oncoprotein CagA into gastric cells. How CagA is delivered is unknown, but the cag T4SS produces an external appendage referred to as pilus, which interacts with host cell receptors, mediating CagA translocation from the cytoplasm of the bacteria to the inner membrane of the host cell. In this study we have investigated the structural and functional properties of CagI, a protein long-thought to be associated with the cag T4SS pilus but with yet unknown function. We found that CagI displays a unique dimeric structure and that its C-terminal domain is involved in interaction with the host cell. Designed Ankyrin Repeat Proteins were selected against CagI and found to interact with its C-terminal moiety with high affinity. DARPin binding was able to prevent CagI interaction with the host cell and inhibited CagA translocation by H. pylori . Our study reveals the role of CagI in cag T4SS interaction with gastric cells and provides a first example of a small protein binder inhibiting the cag T4SS activity.

Dates et versions

hal-03850702 , version 1 (14-11-2022)

Identifiants

Citer

Marine Blanc, Clara Lettl, Jérémy Guérin, Anaïs Vieille, Sven Furler, et al.. Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system. 2022. ⟨hal-03850702⟩
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