Thèse au Coeur du Noyau Cellulaire Analyse Fonctionnelle de l'Invasion Nucléaire par Listeria Monocytogenes H/F - 75

Établissement : Université Paris-Saclay GS Life Sciences and Health
École doctorale : Structure et Dynamique des Systèmes Vivants
Laboratoire de recherche : MICALIS- Microbiologie de l'Alimentation au service de la santé humaine
Direction de la thèse : Alessandro PAGLIUSO ORCID 0000000204215442
Début de la thèse : 2026-09-01
Date limite de candidature : 2026-04-01T23:59:59

Les agents pathogènes intracellulaires ont développé diverses stratégies pour survivre au sein des cellules hôtes, résidant généralement dans le cytosol ou dans des organites membranaires. Très peu sont connus pour envahir le noyau, faisant de ce compartiment l'un des environnements les moins explorés en biologie des infections. Listeria monocytogenes, un modèle bien établi d'agent pathogène intracellulaire, est considéré comme limité aux compartiments cytosoliques et vacuolaires. Nos données non publiées apportent des preuves solides que Listeria peut également envahir le noyau des cellules hôtes in vitro.

L'objectif de ce projet est de caractériser et de quantifier de manière exhaustive l'invasion nucléaire de Listeria dans une variété de modèles cellulaires, comprenant des lignées cellulaires humaines cancéreuses, des cellules primaires dérivées de souris, ainsi que des échantillons histologiques de tissus murins infectés par Listeria. En utilisant des techniques de microscopie de pointe, des approches de quantification à haut débit et la transcriptomique, ce projet visera à élucider la fréquence et la variabilité de l'invasion nucléaire par Listeria et à déterminer les conséquences de cette colonisation nucléaire sur la physiologie de la bactérie et de la cellule hôte.

Notre travail a le potentiel de modifier le paradigme associé à ce modèle bactérien majeur et de stimuler la recherche dans le domaine des bactéries intranucléaires.

Nuclear-localized bacteria represent a fascinating but underexplored frontier in the study of host-pathogen interactions. These bacteria, which have the remarkable ability to invade and survive within the nucleus of eukaryotic cells, challenge our understanding of intracellular parasitism. Despite the potential significance of nuclear invasion in bacterial pathogenesis and host reprogramming, research in this area has been hampered by a major obstacle: most of intranuclear bacteria are obligate intracellular microbes, non-culturable under laboratory conditions. Therefore, the molecular mechanisms enabling intranuclear bacteria to invade and replicate within their hosts remain unexplored. Furthermore, the impact of such nuclear invasion on host cell physiology has yet to be investigated.
During our examination of infected samples via electron microscopy, we serendipitously spot the presence of Listeria inside host cell nuclei. To our knowledge, the presence of Listeria within the nucleus appears as terra incognita. By leveraging Listeria's tractability, we can now dissect the genetic and molecular pathways involved in nuclear localization, allowing for precise exploration of how intracellular bacteria breach the nuclear envelope, interact with nuclear components, and impact host cell physiology. Once again Listeria appears to be a great model to push the boundaries of cellular microbiology and provide insights into previously inaccessible aspects of intracellular bacterial behavior.

This project is structured around two scientific aims.

AIM1. Quantification and characterization of Listeria nuclear invasion in vitro and in vivo.
To understand the relevance of nuclear invasion during Listeria infection, we will quantify Listeria nuclear invasion on different cancerous cell models that are widely used to study Listeria-host interaction (epithelial cells such as Jeg3, HepG2; and macrophages such as RAW264.7). Cells will be infected and the presence of Listeria in the nucleus will be quantified via confocal microscopy (in collaboration with Vlad Costache, Micalis). We will quantify the percentage of cells showing intranuclear Listeria as well as the number of nuclear bacteria per cell. We will then extend our analysis to primary mouse cells that are highly susceptible to Listeria infection to ensure that nuclear localization is not limited to transformed cells (in collaboration with Anna Oevermann, University of Bern). Finally, we will demonstrate the physiological relevance of Listeria nuclear localization analyzing liver sections from infected mice (in collaboration with Marc Lecuit, Pasteur Institute).
As a complementary approach to study the process of nuclear invasion, we will employ two imaging techniques: Focus Ion Beam Scanning Electron Microscopy (FIB-SEM, in collaboration with Claire Boulogne, I2BC) and live imaging analysis. FIB-SEM will enable visualization of bacteria within the nucleus and their interaction with nuclear sub-compartments in fully and accurately reconstructed 3D space. In addition this technique will provide an ultrastructural view at the nanometer scale of infected nuclei, allowing us to characterize rare and possibly transient events such as vacuolar bacteria inside the nucleus with unprecedented detail. Live imaging microscopy will allow us to i) understand bacteria and host behavior during infection (eg. does bacteria multiply inside the nucleus? At which rate compared to cytosolic bacteria? Do cells with nuclear bacteria undergo cell death?); ii) get insights into the process of nuclear invasion (eg. does nuclear invasion require bacterial actin-based motility?; do mitotic cells retain nuclear bacteria during cell division? Are bacteria trapped inside the nucleus during mitosis?).

AIM2. Mechanistic insights on nuclear invasion and impact on bacteria and host physiology
How do bacteria breach the nuclear barrier? Are there specific virulence factor involved? Is nuclear invasion a bacteria- or host-mediated process? What are the consequences of nuclear invasion on bacteria and host physiology?
Our extensive collection of bacterial mutants that we have obtained over the years through multiple collaborations with leading scientists in the field of Listeria will allow us to get mechanistic details about the process of nuclear invasion. We also plan to use a transposon mutant library (recently available in the lab) to identify- on a genome-wide scale- the contribution of Listeria genes involved in nuclear invasion and intranuclear proliferation. In a complementary approach, a genome-wide siRNA screening will help in defining the genetic features on the host side. The physiology of nuclear bacteria and parasitized eukaryotic cells will be studied by microscopy and live imaging approaches to characterize bacterial and host multiplication rate as well as bacterial and cell viability. Finally, via a combination of super-resolution microscopy and single cells RNA sequencing approaches (in collaboration with Claudia Bevilacqua, INRAE), we will define if intranuclear bacteria could affect chromatin compaction, gene expression, RNA stability or splicing.

The methods are extensively described in the Objectifs section