PhD

Study of the factors and mechanisms of adaptation of Brucella Spp. to their intracellular environment

Contact: alessandra.occhialini[at]irim.cnrs.fr

Brucella is the causative agent of brucellosis, a world-wide zoonosis. These facultative intracellular coccobacilli are transmitted to humans by ingestion of dairy products based on raw milk, by contact and inhalation of aerosolized bacteria.

The 12 species that make up this genus are genetically very close but they have a strong host preference. In the recent past, new strains and species have been isolated from wild animals (vole, various rodents, wild boar, frogs…). Compared to classical Brucella species (Brucella abortus, Brucella melitensis, Brucella suis), that are pathogenic to humans and farm animals, these new strains show atypical characteristics: they have growth rates in vitro and in cellulo and resistance to extreme and intermediate acid conditions that are increased. This thesis project will study the factors and mechanisms of adaptation of Brucella to intermediate acid pH in relation to its survival capacity in the macrophagic vacuole. Genetic and physiological analyzes will be performed with candidate genes identified by RNA seq data analysis involving 2 Brucella species (B. suis and B. microti) and two pH conditions (4.5 and 7). Results of this work will provide a better understanding of the virulence and host specificity mechanisms of these species.

1.    Al Dahouk S, Köhler S, Occhialini A, Jiménez de Bagüés MP, Hammerl JA, Eisenberg T, Vergnaud J, Cloeckaert A, Zygmunt MS, Whatmore AM, Melzer F, Drees KP, Foster JT, Wattam AR, Scholz HC. 2017. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts. Scientific Reports (Sci Rep UK), 7: 44420.
2.    Freddi L, Damiano MA, Chaloin L, Pennacchietti E, Al Dahouk S, Köhler S, De Biase D, Occhialini A. 2017. The glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella. Front Microbiol, 8: 2236.
3.    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 extremely acidic conditions. Appl Environ Microbiol. 81(2):578-86.
4.    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.
5.     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(1):3-10.

 

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

Contact: konstantin.brodolin[at]irim.cnrs.fr

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 drug-resistance 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 rifampicin. RbpA is a recently discovered transcriptional activator increasing tolerance of Mycobacterium to rifampicin. Findings from our team indicated that activation of RbpA-controlled genes may play a pivotal role in tuberculosis pathogenesis and virulence. The molecular mechanism of this activation is unknown. Objectives of this project are: (1) to explore the molecular mechanism of RbpA action, (2) its role in regulation of gene expression and (3) sensitivity of RNAP to antibiotics. The work will include genome-wide mapping of the RbpA- dependent genes (RNA-seq and ChIP-seq) and structural analysis of RNAP from M. tuberculosis. Effect of RbpA on the RNAP tolerance to the rifampicin and its analogs will be explored.


Optimizing allosteric inhibitors against 5'-nucleotidases to restore the anticancerous immune response

Contact: laurent.chaloin[at]irim.cnrs.fr

The research proposal is dedicated to the optimization of allosteric inhibitors targeting two important enzymes involved in nucleotide metabolism, the 5’-nucleotidases cN-II and CD73. Scientific reports within the field of oncology have confirmed their role in cancer progression or in drug resistance. Indeed, cN-II affects the phosphorylation level and the pharmacological activity of cytotoxic nucleoside analogues (used as anticancer drugs). As for CD73, by dephosphorylating AMP into adenosine, its activity has been shown to favor the blockade of the antitumor immune response and protects cancer cells against immunosurveillance. These two enzymes are now proven targets in oncology. Indeed, the development of drug-resistant clones in acute lymphoblastic leukemia (ALL) has been attributed to high cN-II activity and the inhibition of cN-II activity restores the sensitivity to these drugs. Also, the promotion of tumor growth, cancer cell invasion and metastasis has been correlated to the over-expression of CD73 in many cancers. Our project consists in i) the improvement of inhibitors already identified for cN-II and ii) the development of new selective inhibitors targeting CD73 by using an original approach based on the development of allosteric inhibitors. Allosteric regulators will block the enzyme in an inactive conformation independently of the presence of natural ligand with a better selectivity, thereby limiting off-target effects of the future drug. These allosteric inhibitors will be evaluated using in vitro assays (recombinant enzymes and model cell lines for cN-II and CD73) to guide their chemical optimization. Best optimized compounds will be tested in tumor and immune cells.

 

Contribution of host cell RNA helicases in Chikungunya virus replication

Contact: laurence.briant[at]irim.cnrs.fr

RNA helicases are ubiquitous biological machines that separate a double helix made of DNA or RNA and allow cellular proteins to access, read or rearrange genetic information. Because RNA viruses synthesize their genomes in a template-dependent manner, they all require a helicase in addition to a RNA polymerase, to displace single stranded genomes after replication so they can be translated or packaged in new particles. Among the host-encoded helicases documented, RNA helicase A (RHA) has been described as a cofactor for RNA viruses acting directly on the RNA genome replication, or by enhancing the innate response to infection. Based on our preliminary results pointing the contribution of RHA in the replication of several human RNA viruses and in their pathogenesis, this program will explore the role played by RHA during replication of Chikungunya virus (CHIKV), a medically important arbovirus present in more than 40 countries, with high risk of emergence in temperate climate area (e.g. Montpellier limited outbreak in October 2014). We have recently evidenced RHA contribution in CHIKV RNA translation. Our aim will be to investigate the molecular underlying mechanisms, especially the contribution of RHA helicase activity. The molecular basis of RHA interaction with CHIKV RNA and nonstructural proteins will be determined and finally RHA will be evaluated as a potential antiviral target. This work will benefit from an access to infectious viral strains, access to BSL3 facilities and collaborations with experts in drug design.

1.    Fullam A, Schröder M. Biochim Biophys Acta. 2013 Aug;1829(8):854-65.
2.    Lee T, Pelletier J. Oncotarget. 2016 Jul 5;7(27):42716-42739.
3.    Fros JJ, Pijlman GP. Viruses. 2016 Jun 11;8(6).
4.    Manojlovic Z, Stefanovic B. RNA. 2012 Feb;18(2):321-34.
5.    Solignat M et al. Virology. 2009 Oct 25;393(2):183-97

 

Chikungunya virus at the host cell membrane interface: structural and functional characterization of replication organelles and associated cofactors

Contact: laurence.briant[at]irim.cnrs.fr

Chikungunya virus (CHIKV) is the causative agent of the acute febrile illness that often leads to painful and long lasting chronic arthritis. There is currently no preventive vaccine and an important barrier to producing an effective antiviral drug against CHIKV is the paucity of information about its replication. In this proposal we aim to focus on CHIKV replication at the host cell membrane interface. As many other RNA viruses from various family, CHIKV hijacks the host cell membranes to create replication organelles that host the viral replication complexes together with cellular cofactors and also prevent detection of viral proteins and nucleic acids by the host innate immune system. These membranous organelles de facto represent attractive targets for antiviral strategies. The aims of this proposal are (1) to define the organization of replication organelles and associated complexes by cryoelectron microscopy approaches; (2) to develop a genetic screen to identify the host machinery hijacked by CHIKV to reorganize the cell membranes; (3) to determine protein-protein and protein-nucleic acid interactions allowing cofactors recruitment to replication compartments. This research program will be developed in collaboration with P. Bron (CBS, Montpellier) and JL Battini (IGMM, Montpellier). By achieving these aims, we will provide an intellectual basis for future rational drug design against essential components of the CHIKV replication organelles with potential broad implications in RNA virology in general.

1.    Shulla A, Randall G. (+) RNA virus replication compartments: a safe home for (most) viral replication. Curr Opin Microbiol. 2016 Aug;32:82-8. doi: 10.1016/j.mib.2016.05.003. PMID: 27253151
2.    Harak C, Lohmann V. Ultrastructure of the replication sites of positive-strand RNA viruses. Virology. 2015 May;479-480:418-33. doi: 10.1016/j.virol.2015.02.029.PMID: 25746936
3.    Paul D, Bartenschlager R. Architecture and biogenesis of plus-strand RNA virus replication factories. World J Virol. 2013 May 12;2(2):32-48. doi: 10.5501/wjv.v2.i2.32. PMID: 24175228
4.    Burt FJ, Chen W, Miner JJ, Lenschow DJ, Merits A, Schnettler E, Kohl A, Rudd PA, Taylor A, Herrero LJ, Zaid A, Ng LF, Mahalingam S. Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen. Lancet Infect Dis. 2017 Jan 31. pii: S1473-3099(16)30385-1. doi: 10.1016/S1473-3099(16)30385-1. PMID: 28159534

 

How HIV-1 favors the multiplication of opportunistic pathogens

Contact: bruno.beaumelle[at]irim.cnrs.fr

HIV-1 Tat protein is actively secreted by infected cells [1], and Tat concentrations in the order of the nM were accordingly measured in the sera from infected patients. Circulating Tat enter several cell types using endocytosis [2] and Tat is able to cross the endosome membrane to reach the cytosol of target cells [2, 3]. Tat then binds to phosphatidylinositol 4,5 bisphosphate (PIP2) at the plasma membrane and becomes palmitoylated [4]. Tat can thereby interfere with the recruitment by this phosphoinositide of several proteins involved in different cellular activities such as endocytosis or phagocytosis [5]. We observed that Tat can favour the intracellular multiplication of pathogens such as Toxoplasma gondii and Mycobacterium tuberculosis (MTB) in macrophages. Tat is thus likely involved in the development of these infections in HIV-1 infected patients. Indeed, MTB infection is a major cause of morbidity and mortality associated with HIV-1 infection, and more than 25% of AIDS-related deaths result from MTB infection. Along the same lines, one quarter of HIV-1 infected patients suffers from cerebral toxoplasmosis. The mechanisms underlying these synergies between pathogens remain to be elucidated. The aim of the thesis is to elucidate the molecular bases of Tat facilitating effect on Toxoplasma and Mycobacterium multiplication in macrophages. Preliminary data indicate that autophagy is involved. We will use techniques such as production of recombinant proteins, cell culture and transfection, preparation and infection of human primary macrophages, cell imaging (photonic and electronic), microinjection of zebrafishes and other modern approaches of cell biology and biochemistry.

1.    Rayne, F., S. Debaisieux, H. Yezid, Y.L. Lin, C. Mettling, K. Konate, N. Chazal, S.T. Arold, M. Pugniere, F. Sanchez, A. Bonhoure, L. Briant, E. Loret, C. Roy, and B. Beaumelle, Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells. EMBO J, 2010. 29(8): p. 1348-62.
2.    Tryoen-Toth, P., S. Chasserot-Golaz, A. Tu, P. Gherib, M.F. Bader, B. Beaumelle, and N. Vitale, HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol (4,5) bisphosphate. J Cell Sci, 2013. 126(2): p. 454-463.
3.    Debaisieux, S., F. Rayne, H. Yezid, and B. Beaumelle, The Ins and Outs of HIV-1 Tat. Traffic, 2012. 13(3): p. 355-63.
4.    Chopard, C., P. Toth, M. Schatz, H. Yezid, S. Debaisieux, C. Mettling, A. Gross, M. Pugnière, A. Tu, J.M. Strub, J.M. Mesnard, N. Vitale, and B. Beaumelle, Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells. Nature Communications, 2018. under correction.
5.    Debaisieux, S., S. Lachambre, A. Gross, S. Mettling, S. Besteiro, H. Yezid, D. Henaff, C. Chopard , J. Mesnard, and B. Beaumelle, HIV-1 Tat inhibits phagocytosis by preventing the recruitment of Cdc42 to the phagocytic cup. Nature Communication, 2015. 6: p. 6211.

 

Study of the role of the Fra-2 protein in the development of ATL

Contact: jean-marie.peloponese[at]irim.cnrs.fr

HTLV-1 (Human T-cell leukemia type 1) is responsible for the development of adult T-cell leukemia (ATL), an aggressive and monoclonal proliferation of CD4 T lymphocytes. In the APIR team, we are interested in the roles of AP-1 transcription factors in the development of ATL. In order to better understand their role, we analyzed the expression of Jun and Fos family members in ATL cells freshly isolated from leukemic patients. We observed that the T lymphocytes of asymptomatic patients express a cJun/cFos heterodimer whereas the leukemic cells express a JunD/Fra-2 heterodimer. Regarding the role of AP-1 proteins in carcinogenesis, the commonly accepted model is a change in the expression profile of AP-1 factors plays a key during tumor progression. Numerous studies have shown that the deregulation of cFos or Fra-1 is important for cell transformation; on the other hand, little is known about the role of Fra-2. The objective of this thesis project is to study the role of the different isoformes of Fra-2 in the proliferation of HTLV-1 infected cells and in HTLV-1-mediated cell transformation.

 

In vivo study of the nuclear export of intron-containing mRNA: the undeciphered model of MLV

Contact: marylene.mougel[at]irim.cnrs.fr

Splicing and nuclear export of RNA are obligatory steps in gene expression by eukaryotic cells. General discard pathways eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Nevertheless, some microorganisms, as retroviruses produce a primary genomic RNA (gRNA) which avoids splicing and reaches the cytoplasm to be either translated into proteins or packaged in progeny virus as genome. Cytoplasmic expression of intron-containing mRNA is a key regulatory step for virus replication. Retroviruses (HIV, MPMV) have proven to be invaluable model systems to study nuclear export of intron-containing RNA and for the discovery of cellular export factors such as Crm1 and Tap. The murine leukemia virus (MLV) as a simple retrovirus, encodes no trans-acting regulators of gene expression. Thus, it must fully rely to the cellular machinery for the expression of its unspliced gene. Although MLV is among the first retroviruses discovered, the nuclear export pathway of its gRNA remains unknown. Preliminary results of our lab showed that the MLV gRNA nuclear export is Tap dependent. By using RNA imaging at the level of the unique molecule based on MS2 fluorescence system, we propose to further study the MLV gRNA nuclear export. We will characterize the export pathway(s) used by MLV for its replication in the host cells.

1.    Chamontin C, Yu B, Racine PJ, Darlix JL and Mougel M (2012). MoMuLV and HIV-1 nucleocapsid proteins have a common role in genomic RNA packaging but distinct in late reverse transcription.  Plos One 7(12):e51534
2.    Pessel-Vivares L, Ferrer M, Lainé S and Mougel M (2014) MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs. Retrovirology, 11:21.
3.    Pessel-Vivares L, Houzet H, Lainé S and Mougel M (2015) Insights into the nuclear export of murine leukemia virus intron-containing RNA. RNA Biol. 2015;12(9):942-9.
4.    Ferrer M, Clerté C, Chamontin C, Basyuk E, Lainé S, Hottin J, Bertrand E, Margeat E and Mougel M (2016) Imaging HIV-1 RNA dimerization in cells by multicolour super-resolution and fluctuation microscopies Nucleic Acids Res. 44(16): 7922–7934.
5.    Ferrer F, Henriet S, Chamontin C, Lainé S and Mougel M (2016) From cells to virus particles: quantitative methods to monitor RNA packaging Viruses, 8(8), 239.

     
       

   

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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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