Evolutionary novelty in the apoptotic pathway of aphids

Apoptosis, a conserved form of programmed cell death, shows interspecies differences that may reflect evolutionary diversification and adaptation, a notion that remains largely untested. Among insects, the most speciose animal group, the apoptotic pathway has only been fully characterized in Drosophila melanogaster, and apoptosis-related proteins have been studied in a few other dipteran and lepidopteran species. Here, we studied the apoptotic pathway in the aphid Acyrthosiphon pisum, an insect pest belonging to the Hemiptera, an earlier-diverging and distantly related order. We combined phylogenetic analyses and conserved domain identification to annotate the apoptotic pathway in A. pisum and found low caspase diversity and a large expansion of its inhibitory part, with 28 inhibitors of apoptosis (IAPs). We analyzed the spatiotemporal expression of a selected set of pea aphid IAPs and showed that they are differentially expressed in different life stages and tissues, suggesting functional diversification. Five IAPs are specifically induced in bacteriocytes, the specialized cells housing symbiotic bacteria, during their cell death. We demonstrated the antiapoptotic role of these five IAPs using heterologous expression in a tractable in vivo model, the Drosophila melanogaster developing eye. Interestingly, IAPs with the strongest antiapoptotic potential contain two BIR and two RING domains, a domain association that has not been observed in any other species. We finally analyzed all available aphid genomes and found that they all show large IAP expansion, with new combinations of protein domains, suggestive of evolutionarily novel aphidspecific functions

Caractérisation structurale et fonctionnelle de PGRP-LB : Modulation de la voie immunitaire IMD dans les interactions avec les bactéries pathogéniques et mutualistiques.

Au sein des animaux, les protéines de reconnaissance du peptidoglycane (PGRPs) sont ubiquitaires et jouent un rôle majeur dans l’immunité innée. Ces protéines ont été caractérisées à différents niveaux du processus immunitaire comme récepteurs, modulateurs et effecteurs. Leur considérable diversité taxonomique et fonctionnelle rend difficile leur classification et l’identification de leur rôle immunitaire. Le premier chapitre de ma thèse répond à cette question. Ainsi, j’ai utilisé la méthode basée sur les réseaux de similarité de séquences pour proposer une nouvelle approche dans l’analyse des PGRPs. Cette étude a permis l’identification de résidus clés et des propriétés structurales impliqués dans la diversification des PGRPs. Cette analyse a aussi montré la faible caractérisation des PGRPs modulatrices comparées aux PGRPs réceptrices. Afin de compléter la connaissance sur les PGRPs, je me suis concentré sur la protéine immunomodulatrice PGRP-LB, capable de cliver le peptidoglycane (PGN) en composés non-immunogéniques. Dans le second chapitre, j’ai étudié le mécanisme réactionnel des PGRPs amidasiques grâce à des analyses enzymatiques et de la cristallographie aux rayons X sur la PGRP-LB de drosophile. Enfin, dans le troisième chapitre, j’ai cherché à comprendre comment l’expression du gène pgrp-lb et son activité ont pu évoluer sous les contraintes endosymbiotiques au sein de l’association entre le charançon des céréales Sitophilus et la bactérie intracellulaire Sodalis pierantonius. J’ai montré que la séquence N-terminale propre à la PGRP-LBi de Sitophilus est impliquée dans la dégradation du PGN synthétisé par l’endosymbiote. Durant cette thèse, j'ai mis en évidence la diversification des PGRPs dans l'adaptation de l'immunité innée.Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play a pivotal role in innate immunity. These proteins have been characterized at various levels of the immune process as receptors, modulators, and effectors. Because of this substantial functional and taxonomic diversity, PGRP classification and immune role identification can be challenging. To address this question, in the first chapter, I used a sequence similarity network method to provide a new approach in the analysis of PGRPs. This study allowed the identification of key residues and the protein features involved in the PGRPs diversification. However, this analysis pointed to the low characterization of the PGRP modulators of the immunity compared to the PGRP receptors. To extend the knowledge on the PGRPs, I focused on the immunomodulator PGRP-LB, which is able to cleave peptidoglycan (PGN) into non-immunogenic compounds. In the second chapter, I deciphered the reaction mechanism of amidase PGRPs, using enzymatic assays and X-ray crystallography on the Drosophila PGRP-LB. Finally, in the third chapter, I analyzed how the pgrp-lb gene expression and activity have evolved under endosymbiotic constraints in the association between the weevil Sitophilus spp. and the intracellular bacterium Sodalis pierantonius. I showed that the specific N-terminal sequence of Sitophilus spp. PGRP-LBi is involved in the degradation of the PGN synthesized by the endosymbiont. During this PhD, I highlighted the large diversification of the PGRPs in the adaptation of the innate immunity

PGRP-LB: An Inside View into the Mechanism of the Amidase Reaction

International audiencePeptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+

Droplet microfluidics for time-resolved serial crystallography

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here we report the use of droplet miniaturisation for the controlled production of uniform crystals, providing an avenue for controlled diffusion and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3-µm lysozyme crystals and 2-µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times o

Anaerobic fixed-target serial crystallography

International audienceCryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryoconditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O-2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the 'sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics

iGEM REPORT: Gotta Detect ‘Em All: a multi-STI sensor based on aptamers

/International audienceNowadays, STIs constitute a major public health issue. Indeed, treatments are often started too late because of belated diagnosis resulting in health problems, such as sterility. If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection could help limit their deleterious effects. In this work, a new diagnosis approach based on aptamers is presented. Bound to paper, they allow the detection of HIV and Hepatitis B biomarkers from a blood sample. The associated device is composed of an anchor, the streptavidin protein, allowing the fixation of the aptamer to the paper via biotin (see graphical abstract). With this system, the HIV-1 Reverse Transcriptase (BBa_K1934060 and BBa_K1934061: protein subunits p51 and p66) and HBsAg (surface antigen of Hepatitis B) are specifically targeted. Then, the biomarker/aptamer complex is detected by two methods. The first one is based on fluorescence. As a proof of concept, a paired ATP/aptamer was used and enabled to successfully detect ATP up to 10 µmol.L-1. However, the signal was not detectable with naked eyes or with a cell phone equipped with blue and green filters either. Therefore, a lateral flow assay with nano-sized latex black beads was tested. This second technique showed that a protein biomarker, such as thrombin, could be complexed with latex beads coated with aptamers, in liquid. Finally, the ultimate step, migration of the latex beads inside paper, needs further optimization. Moreover, to easily handle several STI-tests on a single paper strip, an innovative bio-sourced PLA casing was designed and 3D printed to offer an additional intuitive user-interface

iGEM REPORT: Gotta Detect ‘Em All: a multi-STI sensor based on aptamers

/International audienceNowadays, STIs constitute a major public health issue. Indeed, treatments are often started too late because of belated diagnosis resulting in health problems, such as sterility. If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection could help limit their deleterious effects. In this work, a new diagnosis approach based on aptamers is presented. Bound to paper, they allow the detection of HIV and Hepatitis B biomarkers from a blood sample. The associated device is composed of an anchor, the streptavidin protein, allowing the fixation of the aptamer to the paper via biotin (see graphical abstract). With this system, the HIV-1 Reverse Transcriptase (BBa_K1934060 and BBa_K1934061: protein subunits p51 and p66) and HBsAg (surface antigen of Hepatitis B) are specifically targeted. Then, the biomarker/aptamer complex is detected by two methods. The first one is based on fluorescence. As a proof of concept, a paired ATP/aptamer was used and enabled to successfully detect ATP up to 10 µmol.L-1. However, the signal was not detectable with naked eyes or with a cell phone equipped with blue and green filters either. Therefore, a lateral flow assay with nano-sized latex black beads was tested. This second technique showed that a protein biomarker, such as thrombin, could be complexed with latex beads coated with aptamers, in liquid. Finally, the ultimate step, migration of the latex beads inside paper, needs further optimization. Moreover, to easily handle several STI-tests on a single paper strip, an innovative bio-sourced PLA casing was designed and 3D printed to offer an additional intuitive user-interface

iGEM REPORT: Gotta Detect ‘Em All: a multi-STI sensor based on aptamers

/International audienceNowadays, STIs constitute a major public health issue. Indeed, treatments are often started too late because of belated diagnosis resulting in health problems, such as sterility. If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection could help limit their deleterious effects. In this work, a new diagnosis approach based on aptamers is presented. Bound to paper, they allow the detection of HIV and Hepatitis B biomarkers from a blood sample. The associated device is composed of an anchor, the streptavidin protein, allowing the fixation of the aptamer to the paper via biotin (see graphical abstract). With this system, the HIV-1 Reverse Transcriptase (BBa_K1934060 and BBa_K1934061: protein subunits p51 and p66) and HBsAg (surface antigen of Hepatitis B) are specifically targeted. Then, the biomarker/aptamer complex is detected by two methods. The first one is based on fluorescence. As a proof of concept, a paired ATP/aptamer was used and enabled to successfully detect ATP up to 10 µmol.L-1. However, the signal was not detectable with naked eyes or with a cell phone equipped with blue and green filters either. Therefore, a lateral flow assay with nano-sized latex black beads was tested. This second technique showed that a protein biomarker, such as thrombin, could be complexed with latex beads coated with aptamers, in liquid. Finally, the ultimate step, migration of the latex beads inside paper, needs further optimization. Moreover, to easily handle several STI-tests on a single paper strip, an innovative bio-sourced PLA casing was designed and 3D printed to offer an additional intuitive user-interface

iGEM REPORT: Gotta Detect ‘Em All: a multi-STI sensor based on aptamers

/International audienceNowadays, STIs constitute a major public health issue. Indeed, treatments are often started too late because of belated diagnosis resulting in health problems, such as sterility. If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection could help limit their deleterious effects. In this work, a new diagnosis approach based on aptamers is presented. Bound to paper, they allow the detection of HIV and Hepatitis B biomarkers from a blood sample. The associated device is composed of an anchor, the streptavidin protein, allowing the fixation of the aptamer to the paper via biotin (see graphical abstract). With this system, the HIV-1 Reverse Transcriptase (BBa_K1934060 and BBa_K1934061: protein subunits p51 and p66) and HBsAg (surface antigen of Hepatitis B) are specifically targeted. Then, the biomarker/aptamer complex is detected by two methods. The first one is based on fluorescence. As a proof of concept, a paired ATP/aptamer was used and enabled to successfully detect ATP up to 10 µmol.L-1. However, the signal was not detectable with naked eyes or with a cell phone equipped with blue and green filters either. Therefore, a lateral flow assay with nano-sized latex black beads was tested. This second technique showed that a protein biomarker, such as thrombin, could be complexed with latex beads coated with aptamers, in liquid. Finally, the ultimate step, migration of the latex beads inside paper, needs further optimization. Moreover, to easily handle several STI-tests on a single paper strip, an innovative bio-sourced PLA casing was designed and 3D printed to offer an additional intuitive user-interface

iGEM REPORT: Gotta Detect ‘Em All: a multi-STI sensor based on aptamers

/International audienceNowadays, STIs constitute a major public health issue. Indeed, treatments are often started too late because of belated diagnosis resulting in health problems, such as sterility. If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection could help limit their deleterious effects. In this work, a new diagnosis approach based on aptamers is presented. Bound to paper, they allow the detection of HIV and Hepatitis B biomarkers from a blood sample. The associated device is composed of an anchor, the streptavidin protein, allowing the fixation of the aptamer to the paper via biotin (see graphical abstract). With this system, the HIV-1 Reverse Transcriptase (BBa_K1934060 and BBa_K1934061: protein subunits p51 and p66) and HBsAg (surface antigen of Hepatitis B) are specifically targeted. Then, the biomarker/aptamer complex is detected by two methods. The first one is based on fluorescence. As a proof of concept, a paired ATP/aptamer was used and enabled to successfully detect ATP up to 10 µmol.L-1. However, the signal was not detectable with naked eyes or with a cell phone equipped with blue and green filters either. Therefore, a lateral flow assay with nano-sized latex black beads was tested. This second technique showed that a protein biomarker, such as thrombin, could be complexed with latex beads coated with aptamers, in liquid. Finally, the ultimate step, migration of the latex beads inside paper, needs further optimization. Moreover, to easily handle several STI-tests on a single paper strip, an innovative bio-sourced PLA casing was designed and 3D printed to offer an additional intuitive user-interface