Cytoskeleton dynamics and motility

Functional and structural studies of bacterial virulence factors activated by actin,
or of multimodular proteins with intrinsically disordered actin-binding domains

Overview

Our projects address the biochemical and structural mechanisms by which cytoskeletal and signaling proteins coordinate in concert the actin cytoskeleton dynamics in physiological processes such as cell shape and morphogenesis, cell motility, or intracellular transport. We study how intrinsically disordered actin-binding domains in multimodular proteins regulate versatile functions with globular monomeric (G-actin) and/or polymerized filamentous (F-actin) actin and other cytoskeletal or signaling proteins. We are also interested in elucidating the molecular mechanisms by which pathogens subvert actin and its associated machineries via specific virulence factors.

Highlights

Bacterial ExoY-like virulence factors forms an emerging and atypical subfamily of nucleotidyl cyclase toxins, which are secreted by various pathogenic gram-negative bacteria and translocated or directly injected into eukaryotic cells. How they contribute to the virulence of various pathogens remains largely unknown. As a first step to characterize the molecular basis of their cytotoxicity and virulence mechanisms in host cells, we have identified how they are specifically activated within eukaryotic cells: by interacting with either F-actin or G-actin depending on ExoY-Like homologs existing in pathogens, and started to characterize their enzymatic specificities in overproducing canonical and non-canonical cyclic nucleotide monophosphate secondary messengers the actin cytoskeleton dynamics (Belyy et al., 2016;  Raoux-Barbot et al., 2018).

Main research topics

Role and functional specificities of actin-activated ExoY-like nucleotidyl cyclase virulence factors in gram-negative bacterial infections

(Magda Teixeira-Nunes, PhD student; Louis Renault, PI)

cAMP and CGMP act as universal secondary messengers to stimulate multiple and complex intracellular signalling cascades in prokaryotic or eukaryotic cells (Figure 1A). Many pathogens manipulate the cAMP intracellular signaling of host cells to promote their survival and proliferation in hosts. Bacterial ExoY-like virulence factors represent a new atypical subfamily of nucleotidyl cyclase (NC) toxins (Figure 1B). Nucleotidyl cyclase are ubiquitous enzymes, which bind nucleotide triphosphates to synthetize 3′,5′-cyclic monophosphate nucleotides (cNMPs). For example, Adenylate or Guanlylate Cyclase bind specifically ATP or GTP to synthetize cAMP or cGMP, respectively.

 

Exoenzyme Y (ExoY) is a toxin secreted, via a type 3 secretion system, by Pseudomonas aeruginosa, a major opportunistic and nosocomial pathogen. Bacterial P. aeruginosa (P. a.) strains cause severe infections in immunocompromised patients and exhibit remarkable capacity to resist antibiotics, which has led the World Health Organization (WHO) to rank P. a. among the top priority pathogens. ExoY-related enzymatic modules are also present in several toxins produced by various gram-negative bacterial strains from Vibrio, Providenciae, …. genus, some of which represent emerging human or animal pathogens (Ziolo et al. (2014) Infect Immun. 82(5):2148-57; Belyy et al., 2016).

 

ExoY-Like NC toxins are structurally related to the well-known canonical Adenylate Cyclase (AC) toxins edema factor (EF) of Bacillus anthracis and CyaA AC of Bordetella pertussis (Drum CL et al. (2002), Nature 415: 396–402; Guo et al. (2005) EMBO J. 24(18): 3190–3201). All these secreted NC toxins are inactive in bacteria. Upon their entry in host cells, their NC activity is potently stimulated by interacting with a cofactor that represents typically a specific and highly conserved protein/marker of their hosts (Figure 1B).

ExoY-Topic-Fig.1 : (A) Basic signal transduction pathways dependent on cAMP secondary messenger in human cells. (B) Bacterial ExoY-Like Nucleotidyl Cyclase (NC) toxins represent atypical secreted Adenylate Cyclases (ACs) of class II. Class II AC are structurally related and include well-known edema factor (EF) of Bacillus anthracis and CyaA AC of Bordetella pertussis in addition to ExoY-Like toxins. In contrast to Calmodulin-activated EF and CyaA toxins, ExoY-Like NC toxins uses actin as cofactor to be activated in host cells and the ExoY of Pseudomonas aeruginosa exhibits not only adenylate cyclase (AC) activity like EF and CyaA toxins but also intriguing guanylate- and uridylyl cyclase activities. The enzymatic properties of ExoY-Like homologs from different bacterial pathogens (structural mechanism of activation by G- or F-actin, substrate specificity, catalytic rates, …) and their functional and cytotoxic specificities in host cells remain to be determined.

We have shown that these NC toxins are potently activated by actin within the host target cells (Belyy et al., 2016; 2017). Yet, our preliminary data suggest that they differ significantly in their substrate selectivity, their interaction with actin and activation mechanisms, subcellular localizations, etc and could thus display a greater variability of cytotoxic effects in infected cells than initially anticipated (Raoux-Barbot D. et al. 2018; Belyy et al., 2018; unpublished data) (Figure 2).

ExoY-Topic-Fig.2 : Bacterial Nucleotidyl Cyclase (NC) toxins structurally related to ExoY-Like virulence factors. A. The different domain organization of EF from Bacillus anthracis, CyaA from Bordetella pertussis, ExoY from Pseudomonas aeruginosa, ExoY-Like module from Vibrio nigripulchritudo, and some of their different enzymatic properties (substrate specificity and which cofactor they use for activation in host cells). B-C Crystal structures of inactive EF alone (B) and Calmodulin-activated EF (C) explain how its AC domain is activated by the cofactor calmodulin by stabilizing the ATP binding site (Drum CL et al. 2002, Nature 415: 396–402). The important regions that stabilize allosterically the catalytic site and/or contribute to direct binding of the substrate are depicted in blue, cyan, and violet switch A, B, and C, respectively. The activation mechanisms of ExoY-Like toxins by either G- or F-actin remain to be determined.

Objectives of our research

We aim to characterize at the molecular and structural level and in cellular infection models the precise role and functional specificities of this novel class of actin-activated NC toxins in bacterial infections by P. aeruginosa and various pathogenic gram-negative organisms. This project currently funding by ANR (coordinator: LR) is a collaborative project with the teams of U. Mechold – D. Ladant and L. Touqui at Institut Pasteur, Paris, F. and P. Retailleau – J. Bignon (ICSN, CNRS, Gif-Sur-Yvette, F.).

We study ExoY-like homologs from different pathogens (between 30 to 70 % amino-acid sequence similarity) to decipher precisely the structure-function relationship of these atypical NC toxins. The main questions we address in our team are how ExoY-like toxins are specifically activated by filamentous (F-actin) or monomeric (G-actin) actin, is it possible to inhibit their detrimental enzymatic activation, what are the functional specificities and cytotoxicity of ExoY-like toxins that are associated with their use of F-/G-actin (while other structurally-related AC toxins such as EF and CyaA use the same cofactor calmodulin), their impact on the dynamic remodeling of actin cytoskeleton, which support numerous important physiological functions, and how do these toxins interplay with other bacterial toxins that are delivered simultaneously within host cells.

Structural basis of the versatility and multifunctionality of intrinsically disordrered actin-binding proteins/domains in actin self-assembly dynamics 

(Louis Renault, PI)

In eukaryotic cells, a tight control of actin assembly dynamics by actin binding proteins (ABPs) is central to many cytoskeletal processes such as muscle contraction, cell adhesion and motility and intracellular trafficking. More recently, actin appears to be directly involved in nuclear processes such as chromatin remodeling and gene transcription. ABPs often display complex multi-modular protein architectures that support their multiple interactions and functions with many different partners. Here we study how multi-domain ABPs use multifunctional intrinsically disordered regions (IDRs) to regulate the dynamics of actin filament assembly and disassembly [Herrada I et al. (2015) ACS Chem Biol. 10(12):2733-42; Renault (2016) Vitamines and Hormones 102:25-54, chapter 2 in the book “Thymosins”; Deville et al. (2016) FEBS. L. 590(20):3690-3699].

Intrinsically disordered proteins/domains define a new class of functional proteins/domains whose structural, interfacial and regulatory properties remain to be determined in many cellular processes. They are more abundant in eukaryotic proteins but remain however difficult to identify and characterize in vitro [Tompa P. (2012) Trends Biochem Sci 37(12):509-16]. Indeed, they do not adopt a unique and stable tertiary structure in solution, but display versatile adaptability in binding, often weak but specific binding, and frequent regulation by post-translational modifications or alternative splicings. The interest to understand acutely their molecular mechanisms at protein-protein interfaces is further accentuated by the involvement of numerous IDPs in many human diseases, such as amyloidoses and neurodegenerative diseases, but also cancer, cardiovascular disease, and diabetes [Uversky VN (2010) Expert Rev. Proteomics 7(4):543-64 ; Tompa P. (2012) Trends Biochem Sci 37(12):509-16].

We aim at understanding in detail the structure-function relationship and molecular mechanisms of regulation specific to multi-modular organizations that exploit intrinsically disordered domains, such as small, ubiquitous actin-binding WH2 domains, to interact with both actin monomers (called G-actin) and / or filament actin (F-actin) subunits, and other signalling or cytoskeletal proteins. Our general objective is to understand at the atomic scale the coordinated interactions of IDR-containing ABPs, to reconstitute appropriate integrated structural models and systems in vitro that approach the cellular context and reveal the cross-talks, synergies or antagonisms in actin assembly dynamics between different domains of ABPs and/or key cytoskeletal or nucleoskeletal proteins

Approaches

To build molecular models reliable at multiple scales, we combine functional analyses from complementary in-vitro approaches in biochemistry, molecular biology, biophysics, microscopy, and structural biology as shown in figure 3.

ExoY-Topic-Fig. 3: In-vitro approaches that we combine on our projects to decrypt proteins interactions, enzymatic reactions, protein assembly into dynamic, polarized large-scale structures and their regulations from the atomic to macroscopic scales.

Master internships / Open positions

We have open positions for M1, M2 and PhD students for candidates with strong interests in exploring the structure-function relationships of complex protein architectures, their dynamics and inhibition in pathological contexts by combining biochemical, biophysical and structural approaches. Candidates for a postdoc are welcome to contact us to discuss possible projects and fundings.

team

Louis RENAULT

Group Leader

Research Director

Magda TEIXEIRA NUNES

PhD student

Selected publications

* Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins.

Belyy A, Raoux-Barbot D, Saveanu C, Namane A, Ogryzko V, Worpenberg L, David V, Henriot V, Fellous S, Merrifield C, Assayag E, Ladant D, Renault L#, Mechold U#.

Nat Commun. 2016 Dec 5;7:13582. doi: 10.1038/ncomms13582. (# : corresponding authors)

 

* ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview.

Belyy A, Mechold U, Renault L, Ladant D.

Toxicon. 2018 Jul;149:65-71. doi: 10.1016/j.toxicon.2017.12.046.

 

* The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin.

Belyy A, Santecchia I, Renault L, Bourigault B, Ladant D, Mechold U.

J Biol Chem. 2018 Dec 21;293(51):19785-19796. doi: 10.1074/jbc.RA118.003784.

 

* Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin.

Raoux-Barbot D, Belyy A, Worpenberg L, Montluc S, Deville C, Henriot V, Velours C, Ladant D, Renault L, Mechold U.

PLoS One 2018 Nov 12;13(11):e0206133. doi: 10.1371/journal.pone.0206133.

 

* Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

Renault L.# (2016) in Vitamines and Hormones 102:25-54, chapter 2 in the book “Thymosins” (#: corresponding author).

 

Major Publications before 2015

Multifunctionality of small intrinsically disordered actin-binding WH2 repeats

  • Louis Renault#, Célia Deville, Carine van Heijenoort. (2013) Structural features and interfacial properties of WH2, β-thymosin domains and other intrinsically disordered domains in the regulation of actin cytoskeleton dynamics Cytoskeleton (Hoboken) 70(11):686-705. (#: corresponding author).
  • D. Didry, F.X. Cantrelle, C. Husson, P. Roblin, A. M. Eswara Moorthy, J. Perez, C. Le Clainche, M. Hertzog, E. Guittet, M.F. Carlier, C. van Heijenoort# and L. Renault#. (2012) How a Single Residue in Individual ß-Thymosin/WH2 Domains Controls their Functions in Actin Assembly.
    EMBO J. 31(4):1000-13 (# : corresponding authors)
  • C. Husson, L. Renault, D. Didry, D. Pantaloni, M.F. Carlier#. (2011) Cordon-Bleu uses WH2 domains as multifunctional dynamizers of actin filament assembly.
    Molecular Cell 43, 464-77.
  • Bosch M, Le KH, Bugyi B, Correia JJ, Renault L#, Carlier MF#. (2007) Analysis of the Function of Spire in Actin Assembly and Its Synergy with Formin and Profilin.
    Molecular Cell 28(4), 555-568. (# : corresponding authors).

Interferon-gamma inducible GTP-binding proteins involved in innate immunity regulation, or proteins / virulence factors of pathogens

  • Ghosh A., Praefcke G. J. K., Renault L.#, Wittinghofer A.#, Herrmann C. (2006) How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP.
    Nature 440, 101-4. (# : corresponding authors).
  • Hible G., Christova P., Renault L., Seclaman E., Thompson A., Girard E., Munier-Lehmann H., and Cherfils J. (2006) Unique GMP-binding site in Mycobacterium tuberculosis guanosine monophosphate kinase.
    Proteins 62, 489-500.
  • Würtele M., Renault L., Barbieri J.T., Wittinghofer A. & Wolf E. (2001) Structure of the ExoS GTPase activating domain.
    FEBS L. 491, 26-29.
  • Prakash, B., Praefcke, G.J.K., Renault, L., Wittinghofer, A. & Hermann, C. (2000) Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins.
    Nature 403, 567-71.

Eukaryotic factors activating GTP-binding proteins involved in intracellular trafficking and their inhibition mechanisms by mutations or a small fungal metabolite

  • Renault L., Guibert B., Cherfils J. (2003) Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor.
    Nature 426, 525-530.
  • Renault L., Kuhlmann J., Henkel A. & Wittinghofer A. (2001) Structural basis for guanine nucleotide exchange on Ran by the Regulator of Chromosome Condensation (RCC1).
    Cell 105, 245-255.
  • Renault, L., Nassar, N., Vetter, I., Becker, J., Klebe, C., Roth, M. & Wittinghofer, A. (1998) The 1.7 Å crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller.
    Nature 392, 97-101.

For all the publications of the Team click on the button below.

External funding

ANR activExoY – 18-CE44-0004-01 (2019-2023)

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