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GREMI Award 2003 - Ph.D. Thesis Summary :


 François LEULIER


Centre de Génétique Moléculaire - CNRS UPR2167,

 Batiment 26, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France


The aim of my thesis in the laboratory of Bruno Lemaitre at the CNRS in Gif-sur-Yvette, France, was to study the molecular basis of bacterial infection and the corresponding host defence responses using Drosophila as a model system. Drosophila represent an ideal model system in which to study host-pathogen interactions since insects have a particularly effective immune system that appears to be evolutionarily conserved.


In response to microbial challenge, Drosophila induces the expression of a variety of small peptides with potent antifungal and antibacterial activities. These peptides provide efficient host protection because they directly kill invading microbes. Depending on the invading pathogen, Drosophila activates either the Imd- or Toll-signalling pathways, which in turn induce the expression of pathogen-specific sets of antimicrobial peptides. While fungal infection stimulates the Toll pathway, antibacterial defence is mediated through the Imd pathway. Activation of either Toll or Imd signalling results in the activation of distinct NF-kB-like transcription factors. Similarly in mammals, members of the NF-kB protein family play a central role in the regulation of inflammatory and innate immune responses. The conservation of the NF-kB regulatory mechanisms between organisms as diverse as insects and mammals indicate that the regulation of the innate immune response is evolutionarily conserved.


At the beginning of my Ph.D. little was known regarding the Imd signalling cascade. The identification of the immune deficiency (imd) mutation and its implication in activating the NF-kB family member Relish independent of the Toll pathway suggested the presence of a second Toll-independent NF-kB activating pathway. To identify new component of the Imd signalling cascades we used genetic screens and reverse genetic analysis. My research led to the identification of two new components of the Imd pathway, namely the caspase Dredd and the adaptor protein dFADD. Furthermore, I have also participated in the characterization of the MAPKKK dTAK1, a third component of the Imd pathway. Importantly, Dredd, dFADD and dTAK1 are the Drosophila orthologues of mammalian Caspase-8, FADD and TAK1 respectively. Thus, we find that the Imd pathway of Drosophila is highly similar to the mammalian TNF-R1 pathway (see figure). Consistently, TNF-R1 signalling in mammals also plays a central role in the regulation of immune and inflammatory responses highlighting the strong conservation between the TNF-R1 signalling pathway in mammals and the Imd pathway in insects.


Since different invading pathogens trigger either the Toll- or Imd-signal transduction pathway, we wished to characterise the specificity of the antimicrobial responses in Drosophila. Recent evidence suggested that Toll signalling was required to fend off fungal and Gram-positive bacterial infection. In contrast, the Imd pathway appeared to be required to protect Drosophila against Gram-negative bacterial infections. The observation that different invading pathogens trigger distinct signalling cascades led to the model whereby different classes of invading microbes may activate distinct members of the NF-kB protein family (see figure).


To identify the molecular components of invading microbes that selectively engage the Toll or Imd pathway we collaborated with members of the IBBMC lab in Orsay. We found that bacteria-specific peptidoglycans of the bacteria cell wall were responsible for activating the Toll or Imd pathway. Peptidoglycans are macromoleular structures that are present in bacteria-specific forms in the cell wall of Gram-positive and Gram-negative bacteria. While peptidoglycans of the Lys-type are found in Gram-positive bacteria, meso-DAP type peptidoglycans are predominantly present in Gram-negative bacteria. These results together with studies from mammalian intra-cellular Nod receptors have indicated that peptidoglycans are complex microbial components that selectively activate inflammatory and immune responses during bacterial infection.


Taken together, our results have corroborated the conservation of innate immune responses between species as diverse as insects and mammals. Furthermore, our studies exemplify the value of studying fundamental biological processes using model organisms such as Drosophila melanogaster where in vivo analysis is greatly helped by the availability of powerful genetic tools.


 Figure: In Drosophila, antimicrobial genes expression is controlled by the Toll and Imd pathways.


            The Toll signal transduction pathway is activated in response to fungal and Gram-positive bacterial infections. Toll activation results in the expression of a subset of genes encoding antimicrobial peptides. The Toll signal transduction cascade shares striking similarities to the TLR/NF-kB pathway of mammals. The following Drosophila mammalian proteins share strong homologies: Toll/TLR, dMyd88/Myd88, Pelle/IRAK, Cactus/I-kappaB and Dif,Dorsal/NF-kappaB. However, unlike in the mammalian TLR/NF-kB pathway, in Drosophila the Toll receptor is activated by the cytokine ligand Spaetzle that is cleaved by proteases activated by circulating “Pattern Recognition Receptors” (PGRP-SA and GNBP-1 for Gram positive bacteria).

            The Imd pathway is activated in response to Gram-negative bacterial infection. Imd activation causes the expression of antibacterial peptides. This pathway share similarities with the TNF-R1 pathway. The following Drosophila/mammalian proteins share homologies: Imd/RIP, dTAK1/MEKK3, dmIKKg/IKKg, dmIKKb/IKKb, Dredd/Caspase-8, and Relish/p105.

            Differential activation of the Toll or Imd pathway during bacterial infection is triggered though exposure to bacteria-specific peptidoglycans. The lysine-type peptidoglycan from Gram-positive bacteria is recognized by PGRP-SA and GNBP-1 and activates the Toll pathway while the meso-DAP peptidoglycan from Gram negative bacteria is recognized by PGRP-LC activating the Imd pathway.

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