Genetic analysis has delineated two major regulatory pathways, the Toll and Imd IMD immune deficiency pathways, that control the expression of AMP genes via NF-κB transcription factors [Ferrandon et al., 2007]. The Toll pathway is essential for fighting fungal and some Gram-positive bacterial infections [Lemaitre et al., 1996]. Toll, the founding member of the Toll-like receptor family, is not itself a PRR. Rather, it is activated by a ligand of the nerve growth factor family, the Spz cytokine. To bind to the Toll receptor, Pro-Spz needs to be proteolytically processed by a protease, the Spz-processing enzyme (SPE) [Jang et al., 2006], which is itself activated by upstream proteolytic cascades. One such cascade is activated in response to a Gram-positive bacterial challenge by a complex of PGRP-SA [Michel et al. 2001], PGRP-SD [Bischoff et al., 2004], and Gram-negative binding protein 1 (GNBP1) [Gobert et al., 2003] and to fungal challenge by GNBP3 [Gottar et al., 2006].
Since 2007, we are working on the molecular mechanisms of the immune response in Drosophila in tight collaboration with the laboratory RIDI (IBMC, Strasbourg, France). The first task of this project was to install the Drosophila S2 cell expression system to achieve recombinant proteins production. The idea was to use cells from Drosophila melanogaster to produce proteins from the same organism. During the 4 last years, more than 60 constructions were cloned into the pMT/V5-His vector (Invitrogen). Among them, about 40 were expressed in medium or large quantity either for crystallization or for functional test. Most of the proteins we are working on are glycosylated. Therefore the crystallization step remains highly challenging. Nevertheless we have obtained 8 crystal structures, of which 5 are directly involved in the Toll pathway. The combination of structural and genetic data within the partnership allowed us to confirm and/or refine the existing models and in some cases to propose new models as briefly shown in the following examples.
Bacterial recognition by PGRP-SD [Leone et al., 2008]
It is generally admitted that Gram-positive and Gram-negative bacteria activate Toll and Imd IMD immune deficiency pathways, respectively. One of the major differences in their peptidoglycans (PGN) is the sequence of the stem peptide. The third amino acid is mostly a lysine in Gram-positive bacteria PGN and a diaminopimelic acid (DAP) in Gram-negative bacteria PGN. PGRP-SD is involved in the Toll pathway activation. Indeed, a gene knockout of PGRP-SD is sufficient to reduce the ability of flies to resist infection by Staphylococcus pyogenes and S. aureus [Bischoff et al., 2004]. We have determined the crystal structure of PGRP-SD to a resolution of 1.5 Å . Comparison with available structures of PGRPs in complex with their ligand suggests a DAP-type specificity for PGRP-SD. This result is supported by pull-down assays with insoluble PGNs. This result was rather surprising and shed light on an unexpected complexity not described by the genetic approach. The precise role of a DAP binding protein in the activation of the Toll pathway will need further studies to be assessed. (FIGURE-3)
Detection of fungal infection [Mishima et al., 2009]
Flies mutant for GNBP3 fail to activate the Toll pathway in response to killed fungi and succumb rapidly to fungal but not bacterial infections [Gottar et al., 2006]. Members of the GNBP/GRP family are extracellular proteins composed of a small N-terminal domain of about 100 residues and a longer C-terminal domain of about 350 residues. We have shown that a recombinant protein encoding the N-terminal domain of GNBP3 binds to fungi and to long β-1,3-glucan chains but not to short laminarioligosaccharides. The determination of the crystal structure of GNBP3 N-terminal domain reveals an immunoglobulin fold in which the -glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long-chain structured polysaccharides. Thus, our results allow us to propose a novel mechanism for β-glucan recognition.
Signal transmission by proteolytic cascades [Kellenberger et al., 2011]
The detection of microbial motifs by PRRs triggers proteolytic cascades ending with the cleavage of Spz and the subsequent activation of the Toll pathway. At present, two clip-Serine Proteases (clip-SP), namely SPE [Jang et al., 2006] and Grass Grass [El Chamy et al., 2008], have been demonstrated to participate in these cascades. We have solved the crystal structure of Grass zymogen, which represents the first structure of an entire clip-SP from Drosophila. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the vicinity of the activation site. We have established a classification of the clip-SPs based on the 75-loop of the SP domain and on the conformation of the clip domain. According to this classification, Grass should be a terminal protease, a prediction not in accordance with genetic data. Therefore we proposed a novel model for Toll pathway activation, where the function of Grass, downstream of ModSP would be to cleave a regulatory plasma protein necessary for the activation of SPE. Very recently, we have solved the structure of Persephone, another clip-SP involved in the sensing of danger signal upstream of the Toll receptor.