PhD

Regulation of autophagy by HIV-1 proteins

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The team has demonstrated that HIV-1 envelope (Env) modulates autophagy in its target cells. In bystander CD4 T cells, Env-mediated autophagy is massive and leads to apoptosis. Autophagy is thus involved, at least in part, in the immunodeficiency induced by the virus. In contrast, autophagy is totally inhibited during their productive infection, suggesting that one or several HIV-1 proteins could inhibit this process. The project is to decipher how autophagy is blocked in the productively infected cells. We are working on Vpr, a HIV-1 protein incorporated at high levels in the virions which has multiple functions in the cell, such as cell cycle arrest, apoptosis and nuclear import of the viral DNA. Using a yeast two-hybrid screen, we discovered that many autophagy proteins (ATGs) can bind to Vpr, especially BNIP3, a mitochondrial protein that can trigger apoptosis and autophagy, and ULK1, an ATG absolutely required for the initiation of the autophagy process. First results indicate that Vpr coming from the virus can decrease autophagy in the CD4 T cells. The main objective of the project will be to understand how Vpr can inhibit autophagy and the role of the interaction with BNIP3 and/or ULK1 in this process. This research project should help to better understand the interactions between HIV-1 and its host and to discover new routes for therapeutic intervention.

Klionsky DJ, …Biard-Piechaczyk, ... Espert L, … Zughaier SM. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016,12(1):1-222.
Sophie Sagnier, Coralie F. Daussy, Sophie Borel, Véronique Robert-Hebmann, Mathias Faure, Fabien P. Blanchet, Bruno Beaumelle, Martine Biard-Piechaczyk and Lucile Espert. Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4+ T lymphocytes. J Virol. 2015, 89(1):615-25. Article online in Global Medical Discovery (https://globalmedicaldiscovery.com/)
Sophie Borel, Véronique Robert-Hebmann, Jamal Alfaisal, Ashish Jain, Mathias Faure, Lucile Espert, Laurent Chaloin, Jean-Christophe Paillart, Terje Johansen, Martine Biard-Piechaczyk. HIV-1 Vif interacts with LC3 and inhibits autophagy. AIDS, 2015 29 :275-286.
Papin, CF. Daussy, J. Alfaisal, L. Espert, FP. Blanchet, J. Blanco, M. Biard-Piechaczyk. Autophagy and HIV infection. Encyclopedia of AIDS, 2015.
Alfaisal, C.F. Daussy, F.P. Blanchet, Lucile Espert, Sara Salinas, M. Biard-Piechaczyk. To eat or not to eat: the intricate relationship between autophagy and HIV-1. Current Topics in Virology. 2014, 12:23-51.

Structural and functional studies of proteins involved in synthesis and transport of mycolic acids in Mycobacterium tuberculosis

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According to the World Health Organization, tuberculosis (TB) has infected 9.6 millions and killed 1.5 million people in 2014. The mortality remains very high, especially due to co-infection with HIV, the difficulty in diagnosing latent and/or extrapulmonary forms of the infection and the emergence of multi- and ultra-drug resistant Mycobacterium tuberculosis strains. Thus, to stop the progression of the disease, it appears urgent to develop innovative therapeutic strategies based on the discovery and characterization of new pharmacological targets.

The atypical cell wall of M. tuberculosis is extremely hydrophobic due to the presence of very long fatty acids, designated mycolic acids, which are responsible for the natural resistance of the bacilli to most antibiotics. Paradoxically, because of its unique structure and composition, the mycobacterial cell wall represents a source of potential attractive chemotherapeutic targets. In this context, we are studying proteins involved in the synthesis, recycling and transport of these essential mycobacterial cell envelope components. This PhD project will mainly deal with the structural and functional aspects of newly identified proteins involved in the synthesis and/or transport of mycolic acids in M. tuberculosis. The proposed study will include a wide array of techniques ranging from molecular biology (PCR, cloning), microbiology (mycobacterial cultures, generation of defined mutant strains), biochemistry (production and purification of recombinant proteins, enzymology) to structural biology (X-ray crystallography and cryo-electron microscopy).

This project will unravel the contribution of new genes in mycobacterial cell wall metabolism and virulence and to propose new therapeutic targets against tuberculosis.


Role of the M.tuberculosis RbpA protein in gene regulation and antibiotic resistance

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Mycobacterium tuberculosis is the most successful human pathogens that has infected one third of the world population and continues to kill ~1.5 million people each year. The spreading of multi-drug resistant forms of tuberculosis become a serious threat for public health worldwide. Most of the drugresistance mechanisms are based on the regulation of activity of bacterial RNA polymerase (RNAP), the central enzyme of transcription and the target for the anti-tuberculosis drug rifamipicin (Rif). RbpA is a recently discovered protein implicated in stress response and tolerance to Rif in Mycobacterium. Because RbpA is indispensable for bacterial growth it can be considered as potential target for antituberculosis drug discovery. Recently we mapped the RbpA binding site and showed that RbpA acts as transcriptional activator stimulating RNAP containing initiation factors sigma-A and sigma-B. The molecular mechanism of this activation is unknown. The aims of this project are: (1) to explore the mechanism of RbpA action, (2) its role in regulation of gene expression and (3) sensitivity of RNAP to antibiotics using the in vitro transcription system of M.tuberculosis developed in our laboratory. The work will include genetic characterization of the RbpA-RNAP binding interface by site-directed mutagenesis of RNAP subunits and biochemical characterization of the mutations effects on transcription. Also the effect of RbpA on tolerance to the antibiotic Rif, its analogs (rifapentine and rifabutin) and lipiarmycin will be explored..

Morichaud Z, Chaloin L, Brodolin K. Regions 1.2 and 3.2 of the RNA polymerase sigma subunit promote DNA melting and attenuate action of the antibiotic Lipiarmycin. J Mol Biol. 428 (2 Pt B):463-76 (2016).
Hu Y, Morichaud Z, Sudalaiyadum Perumal A, Roquet-Baneres F, Brodolin K. Mycobacterium RbpA cooperates with the stress-response sigmaB subunit of RNA polymerase in promoter DNA unwinding. Nucleic Acids Res. 42(16):10399-408 (2014).
Hu Y, Morichaud Z, Chen S, Leonetti JP, Brodolin K. (2012) Mycobacterium tuberculosis RbpA protein isa new type of transcriptional activator that stabilizes the σA-containing RNA polymeraseholoenzyme. Nucleic Acids Res. 40, 6547-57.
Tupin A, Gualtieri M, Leonetti JP, Brodolin K. (2010) The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site. EMBO J. 29, 2527-37.
Zenkin, N., Kulbachinsky, A., Yuzenkova, Yu., Mustaev, A., Bass,I., Severinov K., Brodolin K. (2007) Region 1.2 of the RNA polymerase sigma subunit controls recognition of the -10 promoter element. EMBO J. 26, 955-64.

Role of selective autophagy during HIV-1 infection

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Autophagy is a ubiquitous degradation pathway involved in innate immunity. This degradation can be highly selective, thanks to the intervention of autophagy receptors, like p62 or NDP52, involved in the specific targeting of substrates to autophagosomes after their interaction with the Atg8 family (mainly LC3). The role of selective autophagy during bacterial infection is a highly evolving field. However, its role in viral infections remains poorly investigated. Our team has demonstrated that the HIV-1 envelope proteins (Env) are responsible for autophagy triggering in CD4 T lymphocytes. If the target cells become productively infected, the autophagy process is controlled by the virus. On the contrary, when the target cells are not productively infected because the viral cycle is interrupted after the entry step, autophagy is not controlled and leads to apoptosis.
Several studies have shown the importance of the autophagic process during HIV infection in different cellular contexts. However, excepting the viral transactivator Tat (work from our group), the targets that are selectively degraded by autophagy during HIV-1 infection are not known. Consequently, we have started a proteomic screen that will allow us to identify the viral and cellular factors that specifically interact with the autophagic adaptors p62 and/or NDP52 during HIV-1 infection. After the selection of the more interesting candidates, we will analyse the role of this selective degradation during HIV-1 infection. The results will allow us to obtain new clues on the mechanisms leading to CD4 T cell apoptosis associated with this infection and to progress in the understanding of how the virus is able to counteract the cellular defences.

Klionsky DJ, …. Espert L, … Zughaier SM. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016 Jan 2;12(1):1-222.
S Sagnier, C Daussy, S Borel, V Robert-Hebmann, M Faure, FP Blanchet, B Beaumelle, M Biard-Piechaczyk and L Espert. Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4+ T lymphocytes. J Virol.  2015 Jan;89(1):615-25.
Daussy CF, Beaumelle B, Espert L. Autophagy restricts HIV-1 infection. Oncotarget. 2015 Aug 8;6(25):20752-3.
Espert L, Beaumelle B, Vergne I. Autophagy in Mycobacterium tuberculosis and HIV infections. Front Cell Infect Microbiol. 2015 Jun 2;5:49.eCollection 2015.
S Borel, V Robert-Hebmann, J Alfaisal, A Jain, M Faure, L Espert, L Chaloin, JC Paillart, T Johansen, M Biard-Piechaczyk. HIV-1 Vif interacts with LC3 and inhibits autophagy. AIDS, 2015 29 :275-286.

Characterization of the pathogenic potential and of the adaptive capacities of atypical Brucella species

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Brucellae are facultatively intracellular, highly infectious bacteria responsible of brucellosis, one of the major bacterial zoonoses, transmitted to humans by contact, ingestion and inhalation. The recent description of atypical Brucella species raises the question of the possible re-emergence of human brucellosis in countries declared as being exempt. They have been isolated from unusual hosts, with a yet unknown pathogenic potential and possible mode of transmission to humans. Among these species are Brucella microti, Brucella inopinata and Brucella sp. strains isolated from amphibians. Despite marked phenotypic differences, the sequences of the genomes of classical and atypical species are almost identical. We put forward the hypothesis that these phenotypic differences are due to differential gene expression rather than existence of specific genes. The subject of this thesis is the characterization of the strategies of atypical Brucella species in the adaptation to different environmental conditions and to the host cells.

This thesis project will be structured into two parts: (A) Identification of the genes and the molecular mechanisms of resistance of B. microti to acid stress by comparative transcriptome analysis, coupled to functional studies. These studies will be extended to other atypical species of Brucella, (B) In a second part, the atypical species of Brucella will be characterized regarding their pathogenic potential in in vitro models of infection. To this end, complementary approaches in molecular microbiology and cell biology will be developed. The whole of these studies will allow a better comprehension of the virulence mechanisms of these species, barely studied until now.

Jiménez de Bagüés MP, Ouahrani-Bettache S, Quintana JF, Mitjana O, Hanna N, Bessoles S, Sanchez S, Scholz HC, Lafont V, Köhler S, Occhialini A. 2010. The new species B. microti replicates in macrophages and causes death in murine models of infection. J. Infect. Dis. 202: 3-10.
Hanna N, Jimenez de Bagues MP, Ouahrani-Bettache S, El Yakhlifi Z, Köhler S, Occhialini A. 2011. The virB operon is essential for lethality of B. microti in the Balb/c murine model of infection. J. Infect. Dis. 203: 1129-35.
Occhialini A, Jiménez de Bagüés MP, Saadeh B, Bastianelli D, Hanna N, De Biase D, Köhler S. 2012. The glutamic acid decarboxylase system of the new species B. microti contributes to its acid resistance and to oral infection of mice. J. Infect. Dis. 206: 1424-32.
Hanna N, Ouahrani-Bettache S, Drake KL, Adams LG, Köhler S, Occhialini A. 2013. Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses. BMC Genomics. 14: 459.
Damiano MA, Bastianelli D, Al Dahouk S, Köhler S, Cloeckaert A, De Biase D,Occhialini A. 2015. Glutamate decarboxylase-dependent acid resistance in Brucella spp.: Distribution and contribution to fitness under extreme acid conditions. Appl. Environ. Microbiol. 81: 578-586.

Lipid transporters in pathogenic mycobacteria : from genes to pharmacological applications

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The envelope of pathogenic mycobacteria comprises a large panel of complex (glycol)lipids involved in virulence and host-pathogen interactions. The transport of these lipids across the cell wall remains largely. We focus on the MmpL proteins that belong to the superfamily of efflux pumps thought to transport complex lipids and to participate in physiopathology of the infection caused by Mycobacterium abscessus, an emerging pathogen responsible for severe pulmonary infections, especially in cystic fibrosis patients. In addition, MmpL proteins seem to play a role in the intrinsic resistance of M. abscessus to most antibiotics.

We propose to inactivate several MmpL genes in M. abscessus and to analyze the lipid composition of the mutants to identify and characterize the MmpL substrates. The project will also consist to dissect the infectious process of the MmpL mutants at a spatio-temporal level using the zebrafish (Danio rerio) model of infection, recently developed in our laboratory. In particular, studies in zebrafish embryos will to decipher the contribution the MmpL proteins in phagocytosis, intracellular survival, macrophage/neutrophil recruitment and granuloma formation. The mutants will also be assessed for their susceptibility/resistance to various antibiotics. Overall, this project combining a vast panoply of techniques (genetics, microbiology, biochemistry, microinjection, microscopy) should add new insights into the functional role of MmpL in i) biogenesis of the cell wall, ii) virulence mechanisms and iii) innate resistance of M. abscessus to classical antibiotherapy and may open the avenue for new pharmacological developments.


Role of actin cytoskeleton and membrane curvature co-factors in HIV-1 biogenesis

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HIV-1 is a deadly virus that infects mainly the CD4 T lymphocytes in human bodies. During HIV-1 particle formation, the retroviral Gag proteins are targeted and assembled at the inner leaflet of the cell plasma membrane. Gag interacts specifically with phospholipids that belong to the plasma membrane nanodomains. These domains can contain actors of membrane dynamics and curvature, also dependent on underneath cortical actin. Actin cytoskeleton dynamic can promote localized membrane reorganization and thus could play a role in Gag assembly and viral particle production. Among the actin regulators, one can found the small RhoTPases and their effectors. Our research team has shown the implication of Rac1 and some specific effectors on HIV-1 Gag membrane localization and viral particle release in Jurkat and primary T lymphocytes. Our results showed an activation of Rac1 and the involvement of the IRSp53-Wave2-Arp2/3 signalling pathway in HIV-1 biogenesis. This current work uncovers a new role for cortical actin and membrane dynamics in HIV-1 particle formation in CD4 T lymphocytes. We now want to characterize, at the cellular and molecular level, the recruitment of this complex at HIV-1 assembly site in CD4 T cells. For that purpose, we will perform virology and cell membrane trafficking biology in addition to biophotonic technics (FRET/FLIM and FCCS for protein-protein interaction) and high resolution microscopy (dual color PALM/STORM for localisation).

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.
Thomas A, Mariani-Floderer C, López-Huertas MR, Gros N, Hamard-Péron E, Favard C, Ohlmann T, Alcamí J, Muriaux D.
J Virol. 2015 Aug;89(16):8162-81.
Role of Gag and lipids during HIV-1 assembly in CD4(+) T cells and macrophages.
Mariani C, Desdouits M, Favard C, Benaroch P, Muriaux D.
Front Microbiol. 2014 Jun 25;5:312.
Intracellular expression of Tat alters mitochondrial functions in T cells: a potential mechanism to understand mitochondrial damage during HIV-1 replication.
Rodríguez-Mora S, Mateos E, Moran M, Martín MÁ, López JA, Calvo E, Terrón MC, Luque D, Muriaux D, Alcamí J, Coiras M, López-Huertas MR.
Retrovirology. 2015 Sep 16;12:78.
Biocompatible photoresistant far-red emitting, fluorescent polymer probes, with near-infrared two-photon absorption, for living cell and zebrafish embryo imaging.
Adjili S, Favier A, Fargier G, Thomas A, Massin J, Monier K, Favard C, Vanbelle C, Bruneau S, Peyriéras N, Andraud C, Muriaux D*, Charreyre MT*. *co-last authors.
Biomaterials. 2015 Apr;46:70-81.
RNA control of HIV-1 particle size polydispersity.
Faivre-Moskalenko C, Bernaud J, Thomas A, Tartour K, Beck Y, Iazykov M, Danial J, Lourdin M, Muriaux D*, Castelnovo M*.
PLoS One. 2014 Jan 24;9(1):e83874. *co-last authors.

 


     
       

   

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IRIM
Institut de Recherche en Infectiologie de Montpellier
UMR 9004 - CNRS / UM
1919 route de Mende - 34293 Montpellier cedex 5
FRANCE

 

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