rVAP: Vaccins recombinants Anti Parasitaires

Vaccines against parasitic diseases remain a challenge in terms of target identification, antigen production and formulation to obtain efficient vaccine. Vaccines based on recombinant antigenic proteins constitute an interesting alternative strategy to inactivated or live vaccines but their development is often more difficult.. Aside such recombinant vaccines, companion products are needed to make diagnosis and assess the risk of disease, manage the vaccination scheme in human or animal populations, and evaluate the protection status of individuals. Such management tools are particularly needed for veterinary products and sanitary procedures. . For instance, a vaccine that allows the Differentiating Infected from Vaccinated Animals (DIVA vaccine) could be mandatory for animal exportation. Moreover, the need of Correlate of Protection (CoP) assays for human populations is increasing, as the immunological status of individuals is one of the most important parameter in public health management in case of epidemic diseases.

The identification and development of vaccines and diagnostic tools take advantage of deep basic research on host-parasite interfaces. Our model system, Babesia divergens, an intraerythrocytic Apicomplexa parasite transmitted to bovines by ticks, illustrates such potential: an established vaccine system based on a recombinant protein (BD37), a vaccine antigen switching between protective or non-protective induced responses, and investigation levels ranging from molecular structures to experiments in live animals. To address questions about mechanisms of host cell invasion and host immune system escape, Babesia genus offers original and powerful models. Our previous studies highlighted the role of glycosylphosphatidylinositol (GPI)-anchored proteins in "invasion and escape" mechanisms. GPI-anchored proteins are produced as membrane-bound antigens by the parasites, and shed as soluble proteins in the extracellular space (e.g. host blood circulation) Our hypothesis is that the antigen physical state (soluble or membrane-bound) is modulating molecular interactions with the host, and that these interactions drive the immune system toward a protective or non-protective response.

Our current research aims at establishing this protein-embedded immune system escape mechanisms by omparative analysis of GPI-anchored proteins in various Babesia-host models (human, bovine, canine and rodent). This could foster the identification of general principles and methods to decipher parasite invasion and escape mechanisms as well as the influence of GPI-anchors on the recognition of antigenic proteins. Together with an increased knowledge of host-parasite interactions at molecular level, outcomes of these researches provide solutions for animal and human babesiosis studies and management (vaccines, diagnostic tools...). Rational vaccine design against other pathogens (Apicomplexa or not) could also benefit from such knowledge.

rvap-gpi-ap

Glycosylphosphatidyl-anchored proteins (GPI-AP) mediate host-parasite interactions according to their physical state:
1: membrane-bound GPI-AP at the surface of Babesia merozoite (extracellular)
2: host cell surface interaction before invasion of red blood cell
3: soluble GPI-AP interaction with host cell surface
4: immune system produces non-protective antibodies against soluble form of GPI-AP
5: protective immune response shifted against membrane-bound form of GPI-AP
6: soluble form of GPI-AP

Wieser et al., Int J Parasitol. 2019; 49(2):175-181: DOI: 10.1016/j.ijpara.2018.12.002

 

 

RMN haute pression

C. Roumestand, N. Declerck, P. Barthe, C. Dubois

The team has done pioneering works in the field of High-Pressure (HP) NMR. Through a tight collaboration with Catherine A. Royer, an expert in HP Biophysics and former director of the CBS, the group of C. Roumestand was the first French NMR group to develop High-Pressure NMR methodology applied to protein folding analysis (for reviews see Roche et al., Prog. Nucl. Magn. Reson. Spectrosc. 2017; Roche et al., Meth. Enzymol. 2019). Protein folding constitutes still a very active research field for the team, with several on-going projects. Besides, the group is involved in ambitious projects concerning Microbial adaptation to high Pressure.


High-Pressure NMR and Protein Unfolding

C. Roumestand, P. Barthe, C. Dubois

High hydrostatic pressure (HHP) is a very useful reversible perturbation method for exploring the thermodynamics of the folding/unfolding equilibrium of biomolecules. When combined to NMR spectroscopy, HHP offers the possibility to monitor at a single residue level the structural transitions occurring upon protein denaturation.

 hp1
NMR detected high pressure unfolding of Titin I27 single-module. Upper panels: examples of [1H-15N] HSQC NMR spectra at different pressures and residue-specific denaturation curves obtained for 3 residues exhibiting distinct unfolding profiles. Lower panels: ribbon representations of Titin I27 solution structure showing in thick red the contacts that are weakened at the indicated pressures. Adapted from Herrada et al., Biophys J, 2018.

 

 

We use high-pressure NMR to reach a detailed structural and energetic characterization of the folding process of proteins such as the leucine-reach repeat protein pp32 or immunoglobulin-like domains from the dengue virus envelope protein or the protein titin (single- or bi-domains) from sarcomeres. Special emphasis has been put on the study of folding cooperativity inside a single domain (pp32 leucine-reach repeat domain) or between two domains in modular protein (titin). Similar studies are on-going, trying to establish the relationships between protein sequence, protein topology, protein 3D structure and folding routes.

We also use HP-NMR to address fundamental issues regarding protein conformational landscapes or the links between specific thermodynamic parameters and biological properties, such as between thermal expansion and conserved pattern of interactions in proteins, or between protein-ligand binding volumes and affinity.

Collaborations: F. Rico (Marseille); C. Royer (RPI, Troy, USA); J. Roche (Iowa State Univ., USA); Y. Kuroda (Tokyo University, Japan)

References: Dellarole et al., J Am Chem Soc, 2015; Fossat et al., Biophys J, 2016; Toleikis et al., J Phys Chem B, 2016; Herrada et al., Biophys J, 2018; Roche et al., Prog Nucl Magn Reson Spectrosc., 2017; Meth Enzymol, 2019; Saotome et al., Biomolecules, 2019

 


Microbial Adaptation to High-Pressure

 

Adaptation to life under HHP

C. Roumestand

hp2 400x480

Hydrothermal vents in the Mariana trench: the cradle of life ?

  Adaptation to life under high hydrostatic pressure is a trait shared between the first microbial cells and today's piezophiles (HP-adapted). Understanding the basis of this adaptation in contemporary cells is essential to understand the origin of life. To identify these adaptive signatures, we propose to study the molecular evolution of key proteins in piezophilic and piezosensitive Archae, in collaboration with Microbiologists specialized in extremophiles (P. Oger, ENSA Lyon) and a Physicist specialized in HP-SANS measurements (J. Peter, ILL Grenoble) 

 

Pathogens Adaptation to High Pressure

N. Declerck, C. Roumestand

 

hp3
Fluorescence intensity images (13x13 µm) of E. coli cells expressing GFP-Mrr at atmospheric pressure (0.1 MPa), under high pressure (100 MPa) and after pressure is released (back 0.1 MPa).

 

Resistance to high pressure can be acquired by potential human pathogens exposed to barotromatic treatments such as those now routinely used in the food industry as non-thermal sterilization processes. In E. coli, the Mrr endonuclease is responsible for the activation of the SOS reponse after a HP shock and mrr mutations conferring resistance to pressure up to 2 GPa can be selected in only a few generation. We are currently investigating the oligomeric switch on which rely Mrr HP-induced activation, in collaboration with A. Aertsen at KU Leuven (Belgium) and C. Royer at RPI (Troy, USA) who developed a microscopy setup for fluorescence correlation measurements under high pressure.

 

Pathogens & Infectious Diseases

 

Mechanistic and structural bases of recognition between avirulence proteins (AVR) and resistance proteins (R).

André Padilla & Karine de Guillen

 

 

The molecular bases of plant resistance against pathogenic fungi aggressions are poorly understood. This immunity is based on two levels defence system. The first line involves the detection of microbial molecules, such as flagellin or components of the cell wall of the pathogenic microorganism, leading to resistance. To avoid this first level the fungus secretes, during infection, small proteins called avirulence (AVR), which are considered as key elements of fungal pathogenicity but whose functions are largely unknown. The recognition of these plants pathogenic effectors is triggered by resistance proteins (R), which constitute the second layer of defence. This recognition leads to a "hypersensitive" response, which is characterized by localized cell death, avoiding plant colonization by the pathogen.

The fungus Magnaporthe oryzae is the major pathogen of rice. The disease triggered by this fungus is called rice blast. It is responsible for large economic losses and is a major threat to food security. In recent years collaboration with the team of T. Kroj (INRA-Montpellier BGPI) was established. Our collaborators have demonstrated the interaction between avirulence protein of M.oryzae AVR-Pia and AVR-CO39 and resistance proteins RGA4-RGA5 (Cesari et al, Plant Cell, 2013, Ribot et al, The Plant Journal 2013). The production of recombinant avirulences proteins allowed us to determine their three-dimensional structures by NMR. These recent findings raise several questions:

- How are AVR proteins specifically recognized by "their" R protein?

- What are the key amino acids involved in the interaction?

- How is AVR recognition linked to defence activation in R proteins hetero pair?

Validation of structures (AVR and R-domains) by functional studies will help us to understand the mechanisms of recognition and co-evolutions between AVR and R

Interaction between fungus Avr proteins and plant R proteins. (adapted from Liu et al 2014)

 

External collaboration: T. Kroj INRA-BGPI – Montpellier – France

 

Structure and dynamics of RYMV viral encoded P1 protein

Hélène Déméné & Yinshan Yang

 

The P1 protein is a crucial protein of the RYMV (Rice Mottle Yellow) virus that infects the most productive rice plants in Africa. Using an integrative approach combining X-Ray and NMR spectroscopy at the CBS, we have characterized the structure and revealed the dynamics of the RYMV P1 protein that are linked to the mode of viral activation.

 

External collaboration: Florence Vignols and Christophe Brugidou (IRD)

 

Structure of Major Antigens from Apicomplexa.

Christian Roumestand & Yinshan Yang.

 

Babesiosis (formerly known as piroplasmosis) is a tick-borne disease caused by the intraerythrocytic development of protozoa parasites from the genus Babesia. Like Plasmodium falciparum, the agent of Malaria, or Toxoplasma gondii, responsible for human toxoplasmosis, Babesia belongs to the Apicomplexa family. Babesia canis is the agent of the canine babesiosis, while Babesia divergens infects essentially bovine. The identification and characterization of parasite surface proteins represent major goal, both to understand the molecular bases of Apicomplexa invasion process and for the potential as vaccines of such antigens. Indeed, it has been shown that the GPI membrane anchored protein Bd37, a 37 kDa antigenic adhesion protein from B. divergens, was able to induce complete protection against various parasite strains. Its orthologue in B. canis, Bc28.1, has been described as a 28 kDa membrane protein GPI anchored at the surface of the merozoite. We have defined the erythrocyte binding function of these two proteins and determined their high-resolution solution structure using NMR spectroscopy (Delbecq et al., 2008: Yang et al., 2012). Surprisingly, although these proteins are thought to play a similar role in the adhesion process, the structure of Bc28.1 (see Figure) appears unrelated to the structure of Bd37. Site-directed mutagenesis experiments also suggest that the mechanism of the interaction with erythrocyte membrane could be different for the two proteins. The resolution of the structure of these adhesion proteins represents a milestone for the characterization of the parasite erythrocyte binding and its interaction with the host immune system.

 

Solution structure of adhesion proteins from Babesia.

(Left) Bd37 from B. divergens; (Right) Bc28 from B. canis

 

External Collaborations:

· R. Cerdan – DIMNP – Montpellier – France

· S. Delbecq – University Montpellier I – Montpellier – France

Connexion