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biochemical and structural study of actin-activated bacterial toxins

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General information

Reference : UMR9198-LOUREN-001
Workplace : GIF SUR YVETTE
Date of publication : Thursday, January 10, 2019
Scientific Responsible name : Louis RENAULT
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 April 2019
Proportion of work : Full time
Remuneration : 1 768,55 € gross monthly

Description of the thesis topic

Biochemical and structural study of the functional specificities of a new family of bacterial nucleotidyl cyclase toxins activated by actin in host cells.

Abstract:

Many pathogens manipulate inside infected eukaryotic cells the intracellular signaling of the secondary messenger cAMP to promote their survival and proliferation in hosts. The doctoral project aims to characterize at the molecular level a new atypical subfamily of nucleotidyl cyclase (NC) toxins called ExoY-like NC toxins. Nucleotidyl cyclases are enzymes synthetizing cyclic nucleotide monophosphates.

Exoenzyme Y (ExoY) was first identified as a toxin secreted via a type 3 secretion system (T3SS) by Pseudomonas aeruginosa, a major opportunistic and nosocomial human pathogen. P. aeruginosa is also responsible of progressive and severe chronic lung infections in patients with cystic fibrosis. ExoY is present in 90-98% of clinical isolates of P. aeruginosa, which suggests an important role in bacterial pathogenicity. Poorly characterized ExoY-related enzymatic modules or effector proteins (with 35 to 95% protein sequence similarity) have also been found recently in several toxins produced by various Gram-negative proteobacteria representing emerging human or animal pathogens [1, 2]. Some of these enzymes were shown to be essential for bacterial virulence [1].

We have recently shown that ExoY-like NC toxins are potently activated within the host target cells by using an atypical eukaryotic cofactor that is actin [2]. Yet, our new preliminary data suggest that they may differ significantly, for example in their substrate selectivity or interaction and activation mechanism with actin [3, 4]. In addition, since the actin cytoskeleton is hijacked by many pathogen effectors, it is also essential to analyze the potential impact of ExoY-like NC toxins on actin cytoskeleton dynamics during their interaction and activation with actin.

We thus aim here to characterize at the molecular and structural level and, in collaboration in cellular infection models, the functional specificities, precise role and virulence mechanisms of this novel class of actin-activated NC toxins in bacterial infections by P. aeruginosa and various other pathogenic proteobacteria. To decipher their structure-function relationships we will analyze several representative members of this subfamily of actin-activated NC toxins. Our main goals are to characterize in vitro and in cells their functional specificities and impact on actin self-assembly dynamics. We will study the structural bases for their activation mechanisms and enzymatic specificities, and search for inhibitors in high-throughput screening. On a longer term, we will finally consider the possible interplays between ExoY/ExoY-like virulence factors and other bacterial virulence factors that directly target and manipulate the actin cytoskeleton dynamics.

REFERENCES :
[1] Ziolo, K. J., et al. (2014) Vibrio vulnificus biotype 3 multifunctional autoprocessing RTX toxin is an adenylate cyclase toxin essential for virulence in mice. Infect Immun 82, 2148-57. doi: 10.1128/IAI.00017-14.
[2] Belyy A, et al. (2016) Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins. Nature Communications 2016 Dec 5; 7:13582. doi: 10.1038/ncomms13582. (Team publication on the research project)
[3] Raoux-Barbot D, et al. (2018) Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin. PLoS One. 13(11):e0206133. doi: 10.1371/journal.pone.0206133. (Team publication on the research project)
[4] Belyy A, et al. (2018) The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin. J. Biol Chem. 293(51):19785-19796. doi: 10.1074/jbc.RA118.003784. (Team publication on the research project)

Work Context

The work will be carried out at the Institut de Biologie Intgrative de la Cellule (I2BC), UMR9198, CEA, CNRS, University Paris-Sud, Univ. Paris-Saclay, in Gif-sur-Yvette, France. The laboratory is 45 minutes away from Paris (southwest) by the local train RER B.
The team is currently composed of 4 researchers (CNRS, INSERM, CEA) and 2 doctoral students.
team website:
https://www.i2bc.paris-saclay.fr/spip.php?article958&lang=en

Constraints and risks

none

Additional Information

To apply via employ.cnrs.fr.

Application requirements:
Master's degree in biochemistry, structural biology, biophysics, biology, or microbiology with excellent academic records. You should have as well excellent collaborative skills and strong interests for the studies of structure-function relationships of proteins.

Any additional research experience in areas relevant to the research project will be an advantage. This may be practical experience in bioinformatics analysis, biochemical, biophysical, molecular, structural or cellular biology methods... Such additional experience should be demonstrated and included in your application.

Names and contact details of at least two scientists that have offered to act as references for you must also be included, with a clear indication of their address and relationship to you.

Main technical approaches:
- Bioinformatic and structural analysis
- Expression and purification of recombinant proteins, protein labeling for biophysical studies
- Functionnal biochemical and biophysical analyses (kinetics of actin polymerization, functional assays in actin self-assembly, affinity measurements, hydrodynamic characterizations and modeling, single actin filament dynamics observed by epifluorescence TIRF microscopy, ...)
- Protein crystallization, structural determination and analysis by X-ray crystallography and maybe small angle X-ray scattering (SAXS) in solution
- Possible participation in cell biology studies and the search for inhibitors of ExoY-like toxins in high-thoughput screenings through collaborations


Keywords:
Life science, biochemistry, structural biology, host-pathogen interactions, bacterial toxins, actin cytoskeleton, enzymatic mechanisms, structure-function relationship

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