Eric DURAND

LISM (Marseille)

Eric DURAND

LISM (Marseille)

Résumé

Architecture and assembly of an anti-bacterial weapon: the Type VI secretion system

 

The Type VI secretion system (T6SS) is a multi-protein secretory machine that has been recently shown to be implicated in inter-bacterial competition through the delivery of anti-bacterial toxins with peptidoglycan, lipid or DNA hydrolysis activities directly into the target cell [1]. This machine is composed of 13 different subunits, categorized in two complexes: a cytoplasmic tubular structure related to bacteriophage contractile tails (TTC), built on an assembly platform – or baseplate (BPC), and anchored to the cell envelope by a membrane complex (MC). The tail is composed of puncturing device wrapped by a contractile sheath. In our laboratory, we have demonstrated that the model bacterium, Enteroaggregative E. coli (EAEC), utilizes the T6SS machinery to kill other Gram-negative bacteria [2]. This mechanism involves cell-cell and prolonged contacts between predator and prey bacterial cells. Prey lysis occurs rapidly, in a few tens of seconds after the contraction of the T6SS tail sheath. We recently succeeded to purify the membrane complex (MC), a 1.7 MDa structure that is composed of 10 copies of three proteins: TssJ, -L, and –M. Using negative stain electron microscopy, we collected tens of thousands of images of the complex in different orientation and reconstructed the membrane complex with a resolution of 11.6 A [3]. We were then able to connect this MC to the rest of the machinery by isolating a complex between TssJLM and the first protein to interact with the MC, namely TssA [4]. This study shed light on the function of TssA and demonstrated for the first time a direct association between the cytoplasmic protein TssA and the membrane embedded TssJLM MC complex. Our research project is dedicated to understand the structure, assembly and functioning of the T6SS in EAEC, as a model organism. The project is based on a multi-scale and integrated study of a macromolecular nanomachine, integrating several approaches:

  • Cellular scale: fluorescence microscopy, sub-cellular localisation of protein complexes (100-1000 Å), cryo-electron tomography (ECT: 20-60 Å)
  • From mid to near-atomic resolution: (Cryo)-electron microscopy and SAXS (3.5-25 Å).
  • Atomic resolution: X-ray crystallography (< 3.5 Å).
  • “Amino acid” resolution: CX-MS and site-directed mutagenesis.

The presentation will focus on our latest studies that all aim at understanding and deciphering cellular-scale behaviours – namely bacterial competition, pathogenesis/virulence, toxins secretion – at the molecular scale.

 

Bibliographique references

[1] Durand E, Cambillau C, Cascales E, Journet L. 2014. VgrG, Tae, Tle, and beyond: the versatile arsenal of Type VI secretion effectors. Trends Microbiol. Sep;22(9):498-507
[2] Brunet YR, Espinosa L, Harchouni S, Mignot T, Cascales E. 2013. Imaging type VI secretion-mediated bacterial killing. Cell Rep. 3:36-41
[3] Durand E, Nguyen VS, Zoued A, Logger L, Péhau-Arnaudet G, Aschtgen M-S, Spinelli S, Desmyter A, Bardiaux B, Dujeancourt A, Roussel A, Cambillau C, Cascales E and Fronzes R. 2015. Biogenesis and structure of a bacterial Type VI secretion membrane core complex. Nature. Jul 30;523(7562):555-60.
[4] Zoued A, Durand E, Brunet YR, Spinelli S, Douzi B, Guzzo M, Flaugnatti N, Legrand P, Journet L, Fronzes R, Mignot T, Cambillau C, Cascales E. Priming and polymerization of a bacterial contractile tail structure. Nature. 2016 Mar 3;531(7592):59-63.

Dans la session

MET et cryo-MET

10:45 - 12:30