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

Deciphering the mechanism of action of the type IX secretion system (T9SS) – T9-Mechanism

T9-Mechanism

Deciphering the mechanism of action of the type IX secretion system (T9SS)

Structure-function studies of the T9SS

The objective of this proposal aims at deciphering the mechanism of action of the recently discovered type IX secretion system (T9SS). The T9SS is responsible for Porphyromonas gingivalis pathogenesis through secretion of virulence factors, and for Flavobacterium johnsoniae motility through secretion of adhesins. Among the 18 identified proteins that are involved in T9SS function, the K-L-M-N proteins are supposed to form the core complex. We propose that the outer membrane ring-shaped K-N complex serves as an interaction platform to recruit the inner membrane L-M complex, and that the L-M complex is a novel rotary motor driven by proton motive force. Our goals are to solve the structures of the isolated soluble domains of L and N proteins by X-ray crystallography; to produce and to solve the structures of the L-M and K-L-M-N membrane complexes from the two bacterial models by cryo-EM; and to address the function of the L-M complex, and notably its role as a rotary motor, by in vivo approaches.

We use nanobodies directed against the soluble domains of GldL and GldM in order to purify the endogenous membrane complex directly from Flavobacterium johnsoniae membranes. After membrane solubilization, the complex is purified by nickel affinity chromatography (thanks to the His-tag fused to the nanobodies) and by size exclusion chromatography, and the resulting eluted fractions are analyzed by negative-stained EM. Concerning the purification of the endogenous GldKN complex, no nanobody directed against these proteins is available. Therefore, we use a F. johnsoniae mutant strain expressing GldK fused to the ALFA-tag (Götzke et al., 2019), and we will purify the complex using a nanobody directed against this tag.

- We solved the crystal structure of PorN and analyzed the function of its different parts.
- We deciphered the mechanism of action of the GldLM motor, revealing the role of the proton-motive force, of critical acidic residues in the transmembrane helices, and energy-dependent conformational changes.
- We revealed PorW as the protein link between the PorKN ring and the Sov translocon.

Native PAGE analysis of different fractions of the purified GldLM complex revealed the presence of the Alternative Complex III. Indeed, this membrane complex was purified from Flavobacterium johnsoniae membranes taking advantage of its opportunistic interaction with nickel (Sun et al., 2018). Therefore, we are now producing nanobodies fused to a Strep-tag instead of a His-tag.

1. Fuchsbauer O, Lunar Silva I, Cascales E, Roussel A, Leone P. (2022) Structural and functional analyses of the Porphyromonas gingivalis type IX secretion system PorN protein. J Biol Chem. 298:101618.
2. Vincent MS, Comas Hervada C, Sebban-Kreuzer C, Le Guenno H, Chabalier M, Kosta A, Guerlesquin F, Mignot T, McBride MJ, Cascales E, Doan T. (2022) Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium. PLoS Biol. 20:e3001443.
3. Gorasia DG, Lunar Silva I, Butler CA, Chabalier M, Doan T, Cascales E, Veith PD, Reynolds EC. (2022) Protein interactome analysis of the type IX secretion system identifies PorW as the missing link between the PorK/N ring complex and the Sov translocon. Microbiol Spectr. 10:e0160221.
4. Song L, Perpich JD, Wu C, Doan T, Nowakowska Z, Potempa J, Christie PJ, Cascales E, Lamont RJ, Hu B. (2022) A unique bacterial secretion machinery with multiple secretion centers. Proc Natl Acad Sci U S A. 119:e2119907119.

The objective of this proposal aims at deciphering the mechanism of action of the recently discovered type IX secretion system (T9SS). The T9SS is responsible for Porphyromonas gingivalis pathogenesis through secretion of virulence factors, and for Flavobacterium johnsoniae motility through secretion of adhesins. Among the 18 identified proteins that are involved in T9SS function, the K-L-M-N proteins are supposed to form the core complex. We propose that the outer membrane ring-shaped K-N complex serves as an interaction platform to recruit the inner membrane L-M complex, and that the L-M complex is a novel rotary motor driven by proton motive force. In this project, we will solve the structures of the isolated soluble domains of K, L and N proteins, and of the K-N, L-M and K-L-M-N membrane complexes from the two bacterial models, by X-ray crystallography and cryo-EM. The function of the L-M complex, and notably its role as a rotary motor, will be addressed by in vivo approaches.

Project coordination

PHILIPPE LEONE (Architecture et fonction des macromolécules biologiques)

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

AFMB Architecture et fonction des macromolécules biologiques
CNRS DR12 - LISM Laboratoire d'ingénierie des systèmes macromoléculaires
CBMN INSTITUT DE CHIMIE ET DE BIOLOGIE DES MEMBRANES ET DES NANOOBJETS

Help of the ANR 465,004 euros
Beginning and duration of the scientific project: December 2020 - 42 Months

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