Le système immunitaire inné repose sur la détection de motifs microbiens et l'activation de réponses adaptées, parmi lesquelles les voies de signalisation dépendantes des facteurs NF-κB jouent un rôle primordial. Ces voies sont finement régulées afin d'éviter une réponse immunitaire excessive et soutenue dans le temps qui peut causer de nombreuses pathologies, comme les maladies auto-immunes et pro-inflammatoires. Au cours de ma thèse, j'ai élucidé certains mécanismes de régulation des voies de signalisation NF-κB, Toll et IMD, chez la drosophile, qui reposent sur l'ubiquitination de protéines et leur dégradation par la voie endocytaire ou le protéasome. L'ubiquitination réversible des protéines est une modification post-traductionnelle qui permet de réguler leur activité, leur stabilité et leur localisation subcellulaire. En particulier, l'ubiquitination des récepteurs membranaires peut servir de signal d'endocytose et de dégradation lysosomale. Chez la drosophile, le récepteur PGRP-LC reconnaît spécifiquement le peptidoglycane (PGN) bactérien de type acide diaminopimélique et induit la voie de signalisation IMD. J'ai montré que PGRP-LC est ubiquitiné, internalisé et dégradé par la voie endocytaire. Dans ce processus, j'ai identifié le rôle majeur de la déubiquitinase USP8 qui contrôle la dégradation de PGRP-LC ubiquitiné. J'ai aussi mis en évidence que la stimulation de la voie IMD par les PGN augmente l'internalisation et la dégradation de PGRP-LC, assurant l'élimination des récepteurs après que la voie IMD ait été activée. En outre, j'ai participé à des études visant à comprendre le rôle des déubiquitinases USP2, USP34 et USP36, préalablement sélectionnées par l'équipe comme des régulateurs négatifs des voies IMD et/ou Toll. Mes résultats ont notamment contribué à montrer que USP2 agit principalement au niveau de la protéine adaptatrice Imd, en permettant l'hydrolyse de ses chaînes d'ubiquitine K48 et sa dégradation par le protéasome. Finalement, j'ai observé que USP2 interagit également avec PGRP-LC et favorise l'hydrolyse des chaînes K48 associées à ce récepteur, bien que dans ce cas, la dégradation des formes poly-ubiquitinées K48 de PGRP-LC ne dépende pas du protéasome, mais des protéines de la voie endocytaire Hrs, Rab5 et de la déubiquitinase USP8. / The innate immune system relies on the recognition of “non-self” and on the activation of adapted responses, among which NF-κB signaling pathways play a crucial role. These pathways are tightly regulated, in order to prevent an excessive and sustained immune response, responsible for several pathologies, such as autoimmune and pro-inflammatory diseases. During my PhD thesis, I elucidated some Drosophila regulatory mechanisms of NF-κB pathways, Toll and IMD, which rely on protein ubiquitination and their subsequent degradation by the endocytic pathway or proteasome. Reversible ubiquitination of proteins is a post-translational modification, regulating their activity, their stability and the subcellular localization. In particular, ubiquitination of membrane receptors could trigger their internalization and their subsequent lysosomal degradation. In Drosophila, the PGRP-LC receptor specifically recognizes diaminopimelic acid containing peptidoglycan (PGN) and induces the IMD signaling pathway. I proved that PGRP-LC receptor is ubiquitinated, internalized and degraded by the endocytic pathway. In this process, I identified the major role of the USP8 deubiquitinating enzyme, which controls the degradation of ubiquitinated PGRP-LC. Besides, I showed that the IMD stimulation by PGN enhances the PGRP-LC internalization and its degradation, ensuring receptors elimination once the IMD pathway has been activated. Moreover, I took part to studies, aiming to understand the role of USP2, USP34 and USP36, previously selected by the team as negative regulators of the IMD and/or Toll pathways. In particular, my results showed that USP2 principally acts at the Imd level, allowing for the hydrolysis of its K48 poly-ubiquitin chains and its proteasomal degradation. Finally, I observed that USP2 also interacts with PGRP-LC and favors the hydrolysis of PGRP-LC associated K48 chains, whereas the degradation of K48 poly-ubiquitinated PGRP-LC is independent from the proteasome, but rather depends on the Hrs and Rab5 endocytic proteins and on the USP8 deubiquitinating enzyme.

In general the work behind this thesis has revolved around the interesting pattern recognition gene family PGRPs (peptidoglycan recognition proteins). In particular the transmembrane PGRP-LC and to investigate its multifaceted role in the immune response of the fruit fly. As a well characterized model organism living on, and surrounded by, a multitude of microorganisms, Drosophila melanogaster serves as a great tool to gain insights about innate immunity. The two pillars of Drosophila innate immunity are the humoral and the cellular defense. Together they are very potent and can vanquish many infections, but if one of these pillars is damaged, chances are that the defense will collapse and the organism will succumb to the infection. The initial step in any immune response is to become aware of the pathogen. To accomplish this, innate immunity relies on recognizing common molecular building blocks necessary each group of microorganisms. One such building block is the bacterial cell wall component peptidoglycan. PGRPs are a widely spread gene family, and proteins of this family can bind peptidoglycan. We describe that there are 13 PGRP genes in Drosophila, one these codes for PGRP-LC. As it sits in the cell membrane in any of its three different splice forms, PGRP-LC can bind peptidoglycan, dimerize, and subsequently activate the imd/relish signalling pathway, and thereby trigger a vast production of antimicrobial peptides. These short peptides are the firearms of the humoral response. We identified three new inducible antimicrobial peptide genes, Diptericin B, Attacin C and Attacin D. Analyses of their sequences shed light on the evolution and relationship of these antimicrobial peptides The antimicrobial peptides are potent weapons, but without a functional cellular response the animal is at loss. Animals lacking blood cells are gravely compromised. It is interesting to find that PGRP-LC is involved at this end of the immune response equation as well. We have found that PGRP-LC is able to activate blood cells and increase numbers of circulating cells, in a JNK (Jun N-terminal kinase) dependent manner. Intriguingly this activation is not dependent on Relish, the NF-kB transcription factor of the Imd/Relish pathway. PGRP-LC activation funnels into both Imd/Relish and the JNK pathways. When PGRP-LC is lost, it appears that some basal, or background, JNK activation is lost. These effects are very mild, however the animal appears to become more sensitive to additional perturbations in this signalling pathway. This was the starting point when we started to re-evaluate Dredd, the caspase responsible for cleaving and activating Relish. Dredd also contributes to the JNK signalling pathway.

grainy head (grh) genes encode a family of transcription factors conserved from fly to human. Drosophila grh is the founding member of this gene family and has multiple functions, including tracheal tube size control, epidermal barrier formation and reconstruction after wounding. To understand the underlying molecular mechanism of grh functions, we tried to isolate its direct targets and analyze their function. We identified ten grh targets by combining bioinformatics and genetics. Grh directly controls the expression of stitcher (stit), which encodes a Ret family receptor tyrosine kinase (RTK), during both development and wound healing. Stit promotes actin cable assembly and induces extracellular signal-regulated kinase (ERK) phosphorylation around the wound edges upon injury. Stit also activates barrier repair genes and its own expression at the wound sites in a Grh-dependent manner. This positive feedback loop ensures efficient epidermal wound repair. In addition, Grh regulates the expression of multiple genes involved in chitin biosynthesis or modification. Most of the genes are required for tracheal tube size control. Two of them, verm and serp, encode related putative luminal chitin deacetylases. The functional analysis of verm and serp identifies an important role of luminal chitin matrix modification in limiting tracheal tube elongation. Therefore, it is very likely that Grh controls tracheal tube size through regulating multiple targets involved in the assembly or modification of luminal chitin matrix. Grh also directly activates the epidermal expression of Peptidoglycan recognition protein LC (PGRP-LC) gene that is required for the induction of antimicrobial peptides (AMPs) upon infection. Furthermore, ectopically expressing Grh is sufficient to induce AMP Cecropin A lacZ reporter (CecA-LacZ) in the embryonic epidermis. These results suggest a new function of Grh in the local immune responses in Drosophila barrier epithelia. / At the time of the doctoral defense, the following papers was unpublished and had a status as follows: Paper 1: Manuscript.