ERC FUNDED PROJECTS

Understanding the establishment and persistence of bacterial infections in the gut requires integrating an ensemble of factors including bacterial and host components and the presence of other microorganisms. We will capitalize on 25 years of studies on the bacterium Listeria monocytogenes used as a model, to focus on three objectives which will significantly increase our knowledge of the bacterium, of the cell biology of infection and of the epigenetic reprogramming upon infection. Our aims are: - at the bacterial level : to describe for the first time, the proteomic landscape of a bacterium during switch from saprophytism to virulence. We will use a proteogenomic approach together with ribosome profiling, to analyze the translation of the whole transcriptome after bacterial growth in several conditions, including in vivo, in order to barcode all the proteins which play a role in infection. This will open the way to assess the role of 1) small proteins; 2) internal translation initiation sites ; 3) the coupling of transcription and translation. - at the host cell level : To decipher the molecular mechanisms underlying the dynamics and role in infection of host intracellular organelles, starting with mitochondria. - At the host epigenetic level : To explore how the microbe reprograms host transcription and how tolerance to a commensal such as Akkermansia muciniphila differs from responsiveness to a pathogen insult, at the level of histones and mRNA modifications by studying 1) chromatin remodeling, in particular histones modifications during infection ; 2) modifications of the epitranscriptome during Listeria infection and colonization with Akkermansia ; 3) whether there is an epigenetic memory of infection and colonization. This ambitious multidisciplinary project will not only generate new concepts in infection biology but also will unravel fundamental mechanisms in microbiology, cell biology, and epigenetics opening new avenues for further research.

1 147 500 €

Start date: 2015-10-01, End date: 2019-09-30

The overall goal of this project is to understand the molecular mechanisms that lead to the generation of potent and broadly neutralizing antibodies against medically relevant pathogens, and to identify the factors that limit their production in response to infection or vaccination with current vaccines. We will use high-throughput cellular screens to isolate from immune donors clonally related antibodies to different sites of influenza hemagglutinin, which will be fully characterized and sequenced in order to reconstruct their developmental pathways. Using this approach, we will ask fundamental questions with regards to the role of somatic mutations in affinity maturation and intraclonal diversification, which in some cases may lead to the generation of autoantibodies. We will combine crystallography and long time-scale molecular dynamics simulation to understand how mutations can increase affinity and broaden antibody specificity. By mapping the B and T cell response to all sites and conformations of influenza hemagglutinin, we will uncover the factors, such as insufficient T cell help or the instability of the pre-fusion hemagglutinin, that may limit the generation of broadly neutralizing antibodies. We will also perform a broad analysis of the antibody response to erythrocytes infected by P. falciparum to identify conserved epitopes on the parasite and to unravel the role of an enigmatic V gene that appears to be involved in response to blood-stage parasites. The hypotheses tested are strongly supported by preliminary observations from our own laboratory. While these studies will contribute to our understanding of B cell biology, the results obtained will also have translational implications for the development of potent and broad-spectrum antibodies, for the definition of correlates of protection, and for improving vaccine design.

1 867 500 €

Start date: 2015-10-01, End date: 2020-09-30

This proposal aims to provide insight into how bacteria are killed by the complement system, an important part of the host immune response against bacterial infections. Complement is a large protein network in plasma that labels bacteria for phagocytosis and directly kills them via the formation of a pore-forming complex (Membrane Attack Complex (MAC)). Currently we do not understand how complement activation results in bacterial killing. This knowledge gap is mainly caused by the lack of tools to study the enzymes that trigger MAC formation: the C5 convertases. In my lab, we recently established a novel assay system for C5 convertases that allows us for the first time to study these enzymes under purified conditions. This model, combined with my expertise in microbiology, places my lab in a unique position to understand C5 convertase biology (Aim 1), determine the enzyme's role in MAC functioning (Aim 2) and elucidate how the MAC kills bacteria (Aim 3). Thus, I aim to provide insight into the molecular events necessary for bacterial killing by the complement system. I will use biochemical, structural and microbiological approaches to elucidate the precise molecular arrangement of C5 convertases in vitro and on bacterial cells. I will generate unique tools to study how C5 convertases regulate MAC insertion into bacterial membranes. Finally, I will engineer fluorescent bacteria and labeled complement proteins to perform advanced microscopy analyses of how MAC kills bacteria. These insights will lead to fundamental knowledge about the functioning of complement and will create new avenues for blocking the undesired complement activation during systemic infections and acute inflammatory processes. Furthermore this knowledge will improve desired complement activation by therapeutic antibodies and vaccination strategies in infectious diseases. Finally, this work opens up new possibilities to understand how both humans and bacteria regulate complement.

1 497 290 €

Start date: 2015-03-01, End date: 2020-02-29

T lymphocytes display potent pro- or anti-inflammatory properties, which typically associate with distinct effector (Teff) versus regulatory (Treg) cell subsets. Based on published and our preliminary data showing a major impact of microRNAs on T cell differentiation and (auto)immune pathology, my proposal aims to dissect the miRNA networks that control the balance between Teff and Treg subsets in vivo, in various experimental models of infection and autoimmunity. We will focus on three critical mediators of T cell functions: interferon-gamma (IFN-g) and interleukin-17A (IL-17), highly pro-inflammatory Teff cytokines; and Foxp3, the transcription factor that confers Treg suppressive properties. To track the activity of these key genes, we will generate a new Ifng/ Il17/ Foxp3 triple reporter mouse, from which we will isolate Teff and Treg subsets to determine their genome-wide miRNA profiles and specific signatures in vivo. We will investigate both natural (thymic-derived and present in naïve mice) and induced (in the periphery upon challenge) Teff and Treg subsets, as they make distinct contributions to the immune response. We will identify miRNAs selectively expressed in Teff (Ifng+ or Il17+) versus Treg (Foxp3+) subsets of various lineages (CD4+, CD8+, gamma-delta or NKT) in each in vivo model; assess whether they are induced during thymic development or upon peripheral activation; and determine the robustness of subset-specific miRNA profiles across various in vivo challenges. We will then use loss- and gain-of-function strategies to define the individual miRNAs that impact Teff or Treg differentiation and disease pathogenesis; dissect the external cues and intracellular mechanisms that regulate miRNA expression; and identify the mRNA networks controlled by key miRNAs in Teff and Treg differentiation. I expect this project to provide major conceptual and experimental advances towards manipulating miRNAs either to boost immunity or to treat autoimmunity.

2 000 000 €

Start date: 2015-07-01, End date: 2020-12-31