Role of tetraspanins in the invasion and survival of mycobacteria in the host cell
Mycobacterium tuberculosis remains one of the most devastating human pathogens. In some industrialized countries, infections caused by atypical mycobacteria surpasse tuberculosis in terms of prevalence.
Among these, Mycobacterium abscessus is a fast-growing species, particularly resistant to conventional antibiotic therapy. M. abscessus is responsible for soft tissue infections but causes also severe pulmonary infections, especially in patients with cystic fibrosis. The success of these infections is largely due to the exceptional ability of pathogenic mycobacteria to invade macrophages and survive intracellularly through the establishment of mechanisms to escape the bactericidal activity of the macrophage. This includes the inhibition of the phagolysosomal fusion. In some cases, bacteria egress phagosomes to multiply in the cytoplasm freeing themselves from the macrophage’s grip in order to infect new cells and then perpetuate the infectious cycle. Thus, the identification of molecules capable of blocking the entry, and therefore the invasion, of the host cell may constitute a new concept to alter the early steps of the infectious process.
We have recently identified novel surface receptors belonging to the tetraspanin family, favoring the internalization of mycobacteria by macrophages and lung epithelial cells. Tetraspanins are an important family of 33-member membrane proteins with four transmembrane domains. All exhibit a large extracellular loop comprising 70 to 130 amino acids. Several amino acid residues located in the large extracellular loop allow the association of tetraspanins with each other which, in the presence of other membrane proteins, form membrane microdomains. Tetraspanins play a variety of roles in adhesion, migration and cell invasion. Our original work suggests that M. abscessus uses different tetraspanins interacting together as a surface platform to penetrate and invade the host cell during the early stages of colonization. It is also conceivable that mycobacteria can exploit tetraspanins within the phagosomes in which they reside to promote their own intracellular growth.
The proposed thesis project will consist of a multidisciplinary approach combining biochemical techniques (recombinant protein production, immunoprecipitation), molecular biology (PCR, cloning, mutants, CRISPR/Cas9, RNA interference), cell biology (culture of various cell lines, transfection, immunofluorescence, microscopy, video-microscopy) and microbiology to study, at the cellular and molecular levels, the contribution and importance of different tetraspanins in infection with pathogenic mycobacteria. From a fundamental perspective, the expected results should improve our current knowledge regarding the early events of the infectious process. They could also lead, in the future, to new innovative therapeutic approaches targeting these surface receptors to inhibit mycobacterial internalization, thereby altering the infectious process.
Study of the factors and mechanisms of adaptation of Brucella Spp. to their intracellular environment
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.
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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
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.
Contribution of host cell RNA helicases in Chikungunya virus replication
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.
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Chikungunya virus at the host cell membrane interface: structural and functional characterization of replication organelles and associated cofactors
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.
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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
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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
Study of the role of the Fra-2 protein in the development of ATL
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
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.