CE11 - Caractérisation des structures et relations structure-fonctions des macromolécules biologiques

Microtubule subversion by VgrG2b, a Pseudomonas aeruginosa Type 6 secretion effector – Microb6

Microtubules and bacterial infection

To invade eukaryotic host cells, pathogenic bacteria usually use the actin cytoskeleton. Our project will study the original recruitment of microtubules by the bacterium Pseudomonas aeruginosa for this.

Understand how the pathogenic bacterium Pseudomonas aeruginosa manipulates microtubles to invade host cells.

Main issues raised & general objectives : (number of characters : 0/3000)<br /> P. aeruginosa can invade non-phagocytic cells. We have shown that the VgrG2b effector is delivered by the type VI secretion machinery (T6SS) into the host where it targets the gamma-tubulin complex responsible for microtubule (MT) nucleation. This interaction precedes MT-dependent internalization of P. aeruginosa. We want to understand (i) how P. aeruginosa transforms the MT network to invade epithelial cells by studying MT remodeling & the recruitment of its motors (ii) the host targets & the function of the VgrG2b effector domain on them (iii) the ultrastructure of VgrG2b which constitutes the puncturing tip of T6SS in addition to being an effector.

We will use a combination of molecular microbiology (cloning, mutant constructs), cellular microbiology (infection of cell lines by P. aeruginosa), biochemistry (protein-protein interactions, enzyme activity assay, inhibition) & microscopy (fluorescence and electron microscopy). The project relies on the expertise of 2 teams on bacterial secretion systems & their effectors (partner 1), and Ton MT organization (partner 2), and on a collaborator for cryo-electron microscopy.

In progress

Understanding the basis of pathogenesis is key to developing new antibacterials.

Review published in July 2022: Reig, Le Gouellec, Bleves (2022) « What Is New in the Anti- Pseudomonas aeruginosa Clinical Development Pipeline Since the 2017 WHO Alert ? « in Front Cell Infect Microbiol DOI : 10.3389/fcimb.2022.909731

P. aeruginosa (Pa) can invade non-phagocytic cells. We showed that the VgrG2b effector is delivered by the T6SS machinery into the host cells where it targets the gamma-TuRC responsible for microtubule (MT) nucleation and formation. This interaction precedes a MT-dependent internalization of Pa. We propose to investigate (i) how Pa subverts the MT network to invade epithelial cells by studying MT remodeling and MT-motor recruitment upon infection (ii) interactants and function of the VgrG2b effector domain on its host targets (iii) the ultrastructure of VgrG2b which belongs to the T6SS puncturing device in plus of being an effector. We will use a combination of molecular and cellular (micro)biology, biochemical and microscopic approaches thanks to the expertise of the 2 teams on bacterial secretion systems & effectors (Partner 1), and on MT organization (Partner 2), and a collaboration for cryo-electron microscopy. Understanding pathogenic mechanisms is key for developing new antibacterial compounds.

Project coordination

Sophie BLEVES (Laboratoire d'ingénierie des systèmes macromoléculaires)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LISM Laboratoire d'ingénierie des systèmes macromoléculaires
MCD Unité de biologie moléculaire, cellulaire et du développement

Help of the ANR 367,088 euros
Beginning and duration of the scientific project: December 2021 - 36 Months

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