Molecular transport across the bacterial cell enveloppe

Introduction
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Our team aims to understand the assembly and function of conserved macrocomplexes present in the envelope of Gram-negative bacteria: the Tol and Ton systems. These protein assemblies, involved in cell adaptation and survival, are also the gateway for bacterial toxins and filamentous phages.

Both Tol and Ton systems harness the energy produced from the proton motive force generated at the inner membrane to transduce it at the outer membrane. These complexes are involved in functions that are essential for bacterial survival.

The Ton system mediates the active uptake across the outer membrane of metal, cyanocobalamin and carbohydrates. The Tol system has a more complex function, as it is involved in outer membrane integrity, in the correct localization of some polar factors and chemoreceptors, in outer-membrane phospholipid homeostasis, and in the constriction of the outer membrane during cell division.

Both Tol and Ton systems can be parasitized to open a gate for bacteriotoxins (called colicins), which result in bacterial cell death. These systems are also the target of filamentous phages during the infection process, such as the M13 phage targeting E. coli, and the CTX phage which is specific of the human pathogen Vibrio cholerae.

To obtain an integrate view of the inner membrane Tol and Ton complexes, we developpe an interdisciplinary study in collaboration with different laboratories, mixing genetic, biochemical and microscopy techniques.

1 – Structural and functional analysis of the Tol and Ton complexes.

Structural and functional studies on the Tol and Ton complexes isolated from various Gram negative bacteria are investigated in collaboration with international teams. More specifically, we are interested in the interactions occuring between the complex subunits in vivo, the structural study of soluble domain by NMR, the structural and dynamic studies of the complexes by EPR and the high-resolution structure of the membrane complexes.

The first structure/function results of such motor have been obtained with the E. coli Ton membrane complex. We have characterized the channels formed by the ExbB, ExbB/ExbD complexes and obtained the X-ray and cryo-EM high resolution structures of the ExbB/ExbD subcomplex.

2 – Dynamic study of the Tol-Pal proteins

We have started to develop the in vivo imaging techniques of the Tol-Pal proteins in different background strains using fluorescence microscopy. In collaboration, we use high resolution fluorescent microscopy and microfluidic device to follow the dynamic of the Tol-Pal proteins. The analyses are performed in relation with cell division, environmental conditions and using specific mutations.

3 – Filamentous phage transport across the envelope and role in host pathogenicity

The Tol system is the entrance gate for a family of bacterial viruses named filamentous phages. For some bacterial species, such as Pseudomonas aeruginosa or Vibrio cholerae, mutualistic association with these phages results in increased pathogenicity and virulence.

Our goal is to identify the determinants of phage-host-specific recognition and to characterize the sequence of molecular events involved during phage transport across the periplasm. Our models are the fd phage infecting E. coli, as well as the CTX and Pf phages, associated with the virulence of their hosts, V. cholerae and P. aeruginosa, respectively.

The expected results will shed new light on the mechanism of phage-dependent pathogenic conversion in bacteria and open new perspectives in the engineering of hybrid phages designed to deliver molecules of interest to pathogens.

Members

Denis Duché

CRCN, CNRS

Group Leader

Laetitia Houot

MCU, AMU

Group Leader

Christophe Bernard

MCU, AMU

Callypso Pellegri

PhD student

Lara Pelzer

PhD Student

Romane Guarino

PhD student

Roland Lloubès

DR, CNRS Emeritat

Group Leader

Pauline Baccelli

ITA, ANR

Group Leader

Past Members / Alumni

Researchers

Roland Lloubès – Former team leader (DR, CNRS Emeritat)

Technical Staff

Pauline BACCELLI (ITA, 2019-2021)
Bastien SERRANNO (Engineer, 2015-2017)
Hervé CELIA (Engineer CNRS, 2008-2018), now at NIDDK, NIH (USA)
Marthe GAVIOLI (TCE, retired in 2010)

Ph.D students

Mélissa PETITI (2015-2018) – Assemblage et dynamique in vivo du système Tol-Pal
Xiang ZHANG (2007-2010) – Organisation fonctionnelle d’un moteur moléculaire Tol et d’une protéine active contre une toxine bactérienne
Nadir SEDDIKI (codirection Dr Susan Buchanan, NIH, Bethesda) (2007-2010) – Import of macromolecules: Structural studies of the pesticin toxin and of an engineered variant
Aurélie Barnéoud-Arnoulet (2007-2010)- Mécanismes d’importation des collicines: détournement des fonctions physiologiques des protéines FtsH et FkpA
Emilie GOEMAERE (2004-2008) – Le complexe membranaire Tol d’E.coli: un modèle pour l’étude de l’organisation fonctionnelle d’un moteur moléculaire bacterien
Stephanie POMMIER (2002-2005) – Le système Tol-Pal d’E. coli

Post-Doctorants

Laetitia HOUOT (2010-2012)
Xiang ZHANG (2011)

Master 2

Callypso PELLEGRI (2021) – Analyse fonctionnelle du système Tol-Pal au cours de l’infection par les phages filamenteux.
Ambre MOREAU (2020) – Mécanisme moléculaire d’infection par les phages filamenteux: réseau d’interactions protéiques mis en jeu.
Melvin RENAULT (2019) – Etude de la localisation des moteurs Tol et Ton et de l’interchangeabilité des moteurs chez E. coli.
Poutoum SAMIRE (2018) – Importance du moteur énergisé TolQRA et de la flexibilité de TolA dans l’import des phages filamenteux.
Nicolas GOMEZ (2016) – Analyse fonctionnelle du système Tol-Pal en fonction de l’état redox de TolA
Laura FRADALE (2015) – Analyse transcriptionnelle et fonctionnelle du système Tol-Pal chez Vibrio cholera
Melissa PETITI (2014) – Etude du système Tol-Pal par mutagénèse dirigée et double hybride bactérien
Perinne VAN OVERTVELT (2011)
Xiang ZHANG (2007)
Aurélie BARNEOUD-ARNOULET (2007)

Master 1

Lara PELZER (2022)
Rachida SMIMIH (2022)
Megan CHARLES-ALFRED (2019)
Emeline LEMARIE (2019)
Melvin RENAULT (2018)
Poutoum SAMIRE (2017)
Raphaël RACHEDI (2017)
Pauline BACCELLI (2014)
Marie DESCAMPS (2014)
Alexandra LIPSKI (2006)

Undergraduates

Alice OLIVIER, L2 (2022)
Elsa PONCET, L3 Lyon (2022)
Léonie DELEUIL, BUT Toulon (2022)
Maxime RACCHINI, L2 (2022)
Maximilien ROUZAUD, L3 (2022)
Lola JUDOR, L2 (2022)
Garance CHABANNES (UE Labo in vivo, L2, 2012)
Chaima BOUDI, BTS (2022)
Jennifer GONCALVES DA SILVA (UE Labo in vivo, L2, 2019)
Myriam BOUSSOUFA (UE Labo in vivo, L2, 2019)
Defne AKSOY (UE Labo in vivo, L2, 2019)
Celia JONAS (UE Labo in vivo, L2, 2019)
Victoria SGOLUPPI, L3 (2018)
Faniry RABOANARIVOLA, L3 (2017)
Jad MOUALLEM, UE Labo in vivo L1 (2017)
Megan CHARLES-ALFRED, L3 (2017)
Ahmed NASRAOUI, UE Labo in vivo L1 (2016)
Clara FERNANDEZ, L2 (2016)
Laura BASSET, ESIL-Polytech (2015)
Marie-Sarah ROUVIERE, UE Labo in vivo L1 (2015)
Axel DEJARDIN, BTS (2014, 2015)
Lia FRIEIER, ERASMUS L3 (2013)
Audrey FAVARO, ESIL-Polytech (2012)
Clement NOEL, ESIL-Polytech (2012)
Stéphanie ROLLERO, ESIL-Polytech (2010)
Sarah DOCHE, ESIL-Polytech (2009)
Nathalie MIARD, INSA Toulouse (2007)
Patrick ROCKENFELLER, ERASMUS (2005)

Publications

2023

Direct interaction between fd phage pilot protein pIII and the TolQ–TolR proton-dependent motor provides new insights into the import of filamentous phages

Callypso Pellegri, Ambre Moreau, Denis Duché, Laetitia Houot

Journal of Biological Chemistry 299:105048 (2023)10.1016/j.jbc.2023.105048

2022

Timing of TolA and TolQ recruitment at the septum depends on the functionality of the Tol-Pal system

Pauline Baccelli, Raphaël Rachedi, Bastien Serrano, Mélissa Petiti, Christophe Bernard, Laetitia Houot, Denis Duché, Denis Duche #

Journal of Molecular Biology 434:167519 (2022)10.1016/j.jmb.2022.167519

2022

The Tol-Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate

Amine Ali Chaouche, Laetitia Houot, Denis Duché, Chantal Iobbi-Nivol, Marie-Thérèse Giudici-Orticoni, Michel Fons, Vincent Méjean

Research in Microbiology 173:103967 (2022)10.1016/j.resmic.2022.103967

2020

Fur -Dam regulatory interplay at an internal promoter of the enteroaggregative Escherichia coli Type VI secretion sci1 gene cluster

Yannick R Brunet, Christophe S Bernard, E. Cascales

Journal of Bacteriology (2020)

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2019

Connected partner-switches control the life style of Pseudomonas aeruginosa through RpoS regulation

Sophie Bouillet, Moly Ba, Laetitia Houot, Chantal Iobbi-Nivol, Christophe Bordi

Scientific Reports 9 (2019)10.1038/s41598-019-42653-5

2019

Similarities and Differences between Colicin and Filamentous Phage Uptake by Bacterial Cells

Denis Duché, Laetitia Houot

EcoSal Plus 8 (2019)10.1128/ecosalplus.ESP-0030-2018

2019

Tol Energy-Driven Localization of Pal and Anchoring to the Peptidoglycan Promote Outer-Membrane Constriction

Melissa Petiti, Bastien Serrano, Laura Faure, Roland Lloubes, Tam Mignot, Denis Duché

Journal of Molecular Biology 431:3275-3288 (2019)10.1016/j.jmb.2019.05.039

2019

Decoupling filamentous phage uptake and energy of the TolQRA motor in Escherichia coli .

Poutoum Samire, Bastien Serrano, Denis Duché, Emeline Lemarie, Roland Lloubes, Laetitia Houot

Journal of Bacteriology (2019)10.1128/JB.00428-19

2017

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

Martina Bielaszewska, Christian Rüter, Andreas Bauwens, Lilo Greune, Kevin-André Jarosch, Daniel Steil, Wenlan Zhang, Xiaohua He, Roland Lloubès, Angelika Fruth, Kwang Sik K. S. Kim, Alexander Schmidt, Ulrich Dobrindt, Alexander Mellmann, Helge Karch

PLoS Pathogens 13 (2017)10.1371/journal.ppat.1006159

2017

Electrostatic interactions between the CTX phage minor coat protein and the bacterial host receptor TolA drive the pathogenic conversion of Vibrio cholerae

Laetitia Houot, Romain Navarro, Matthieu Nouailler, Denis Duché, Francoise Guerlesquin, Roland Lloubès

Journal of Biological Chemistry 292:13584-13598 (2017)10.1074/jbc.M117.786061

2016

Structural insight into the role of the Ton complex in energy transduction

Hervé H. Celia, Nicholas Noinaj, Stanislav D Zakharov, Enrica Bordignon, Istvan Botos, Mónica Santamaria, Travis J Barnard, William A Cramer, Roland Lloubès, Susan K Buchanan

Nature 538:60-65 (2016)10.1038/nature19757

2016

(1)H, (15)N and (13)C resonance assignments of the C-terminal domain of Vibrio cholerae TolA protein.

Romain Navarro, Olivier Bornet, Laetitia Houot, Roland Lloubes, Francoise Guerlesquin, Matthieu Nouailler

Biomol NMR Assign (2016)10.1007/s12104-016-9690-y

2015

Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain

Lisa Kunsmann, Christian Rüter, Andreas Bauwens, Lilo Greune, Malte Glüder, Björn Kemper, Angelika Fruth, Sun Nyunt Wai, Xiaohua He, Roland Lloubes, M. Alexander Schmidt, Ulrich Dobrindt, Alexander Mellmann, Helge Karch, Martina Bielaszewska

Scientific Reports 1-18 (2015)10.1038/srep13252

2014

Fluorescence resonance energy transfer based on interaction of PII and PipX proteins provides a robust and specific biosensor for 2-oxoglutarate, a central metabolite and a signalling molecule.

Hai-Lin Chen, Christophe S Bernard, Pierre Hubert, Laetitia My, Cheng-Cai Zhang

FEBS Journal 281:1241-55 (2014)10.1111/febs.12702

2013

Non classical secretion systems.

Roland Lloubes, Alain Bernadac, Laetitia Houot, Stéphanie Pommier

Res. Microbiol. 164:655-63 (2013)10.1016/j.resmic.2013.03.015

2013

Mannitol and the mannitol-specific enzyme IIB subunit activate Vibrio cholerae biofilm formation.

Patrick Ymele-Leki, Laetitia Houot, Paula I Watnick

Applied and Environmental Microbiology 79:4675-83 (2013)10.1128/AEM.01184-13

2012

Characterization of colicin M and its orthologs targeting bacterial cell wall peptidoglycan biosynthesis.

Hélène Barreteau, Meriem El Ghachi, Aurélie Barnéoud-Arnoulet, Emmanuelle Sacco, Thierry Touzé, Denis Duché, Fabien Gérard, Mark Brooks, Delphine Patin, Ahmed Bouhss, Didier Blanot, Herman Van Tilbeurgh, Michel Arthur, Roland Lloubes, Dominique Mengin-Lecreulx

Microb. Drug Resist. 18:222-9 (2012)10.1089/mdr.2011.0230

2012

A bacterial two-hybrid genome fragment library for deciphering regulatory networks of the opportunistic pathogen Pseudomonas aeruginosa.

Laetitia Houot, Adeline Fanni, Sophie De Bentzmann, Christophe Bordi

Microbiology (Reading, Engl.) 158:1964-71 (2012)10.1099/mic.0.057059-0

2012

Energetics of colicin import revealed by genetic cross-complementation between the Tol and Ton systems.

Roland Lloubes, Emilie Goemaere, Xiang Y. Zhang, E. Cascales, Denis Duché

Biochem. Soc. Trans. 40:1480-5 (2012)10.1042/BST20120181

2012

Energetics of colicin import revealed by genetic cross-complementation between the Tol and Ton systems

Roland Lloubès, Emilie Goemaere, Xiang Zhang, E. Cascales, Denis Duché

Biochemical Society Transactions 40:1480-1485 (2012)10.1042/BST20120181

2012

Colicin M, a peptidoglycan lipid-II-degrading enzyme: potential use for antibacterial means?

Thierry Touzé, Hélène Barreteau, Meriem El Ghachi, Ahmed Bouhss, Aurélie Barnéoud-Arnoulet, Delphine Patin, Emmanuelle Sacco, Didier Blanot, Michel Arthur, Denis Duché, Roland Lloubes, Dominique Mengin-Lecreulx

Biochem. Soc. Trans. 40:1522-7 (2012)10.1042/BST20120189

2012

The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC.

Abdelrahim Zoued, Roland Lloubes, Laure Journet, E. Cascales, Marie-Stéphanie Aschtgen

MicrobiologyOpen 1:71-82 (2012)10.1002/mbo3.9

2011

Regulation of type VI secretion gene clusters by sigma54 and cognate enhancer binding proteins.

Christophe S Bernard, Yannick R Brunet, Marthe Gavioli, Roland Lloubes, E. Cascales

Journal of Bacteriology 193:2158-67 (2011)10.1128/JB.00029-11

2011

An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster

Yannick R Brunet, Christophe S Bernard, Marthe Gavioli, Roland Lloubes, E. Cascales

PLoS Genetics 7:e1002205 (2011)10.1371/journal.pgen.1002205

2011

The anaerobe-specific orange protein complex of Desulfovibrio vulgaris hildenborough is encoded by two divergent operons coregulated by ?54 and a cognate transcriptional regulator.

Anouchka Fiévet, Laetitia My, E. Cascales, Mireille Ansaldi, Sofia R Pauleta, Isabel Moura, Zorah Dermoun, Christophe S Bernard, Alain Dolla, Corinne Aubert

Journal of Bacteriology 193:3207-19 (2011)10.1128/JB.00044-11

2011

Mapping the interactions between Escherichia coli TolQ transmembrane segments

Xiang Y-Z Zhang, Emilie L Goemaere, Nadir Seddiki, Hervé H. Celia, Marthe Gavioli, E. Cascales, Roland Lloubes

Journal of Biological Chemistry 286:11756-64 (2011)10.1074/jbc.M110.192773

2010

Toxicity of the Colicin M Catalytic Domain Exported to the Periplasm Is FkpA Independent

Aurélie Barnéoud-Arnoulet, Hélène Barreteau, Thierry Touze, Dominique Mengin-Lecreulx, Roland Lloubes, Denis Duché

Journal of Bacteriology 192:5212-5219 (2010)10.1128/JB.00431-10

2010

Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

Hélène Barreteau, Ahmed Bouhss, Fabien Gérard, Denis Duché, Boubekeur Boussaid, Didier Blanot, Roland Lloubes, Dominique Mengin-Lecreulx, Thierry Touze

Journal of Biological Chemistry 285:12378-12389 (2010)10.1074/jbc.M109.093583

2010

Determinants of the Proton Selectivity of the Colicin A Channel

Stephen Slatin, Daniel Baty, Denis Duché

Biochemistry 49:4786-4793 (2010)10.1021/bi100122g

2010

Channel Domain of Colicin A Modifies the Dimeric Organization of Its Immunity Protein

Xiang Y-Z Zhang, Roland Lloubes, Denis Duché

Journal of Biological Chemistry 285:38053-38061 (2010)10.1074/jbc.M110.144071

2008

Immunity Protein Protects Colicin E2 from OmpT Protease

Denis Duché, Mohamed Issouf, Roland Lloubes

Journal of Biochemistry 145:95-101 (2008)10.1093/jb/mvn149

2007

Colicin biology.

E. Cascales, Susan Buchanan, Denis Duché, Colin Kleanthous, Roland Lloubes, Kathleen Postle, Margaret Riley, Stephen Slatin, Danièle Cavard

Microbiology and Molecular Biology Reviews 71:158-229 (2007)10.1128/MMBR.00036-06

2007

Colicin E2 Is Still in Contact with Its Receptor and Import Machinery When Its Nuclease Domain Enters the Cytoplasm

Denis Duché

Journal of Bacteriology 189:4217-4222 (2007)10.1128/JB.00092-07

2006

Release of Immunity Protein Requires Functional Endonuclease Colicin Import Machinery

Denis Duché, Aurélie Frenkian, Valérie Prima, Roland Lloubes

Journal of Bacteriology 188:8593-8600 (2006)10.1128/JB.00941-06

2005

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action

K. Möbius, A. Savitsky, C. Wegener, M. Plato, M. Fuchs, A. Schnegg, A. Dubinskii, Y. Grishin, I. Grigor'Ev, M. Kühn, Denis Duché, H. Zimmermann, H.-J. Steinhoff

Magnetic Resonance in Chemistry 43:S4-S19 (2005)10.1002/mrc.1690

2004