Rôle du système de sécrétion de type III de la bactérie entomopathogène Photorhabdus luminescens au cours du processus infectieux

Les entérobactéries des genres Xenorhabdus et Photorhabdus associées symbiotiquement à des nématodes des sols sont pathogènes par injection chez de nombreux insectes. Un locus codant pour un système de sécrétion de type III (ou TTSS) qui permet de délivrer des protéines bactériennes dites effectrices dans le cytosol de la cellule hôte, a été identifié chez Photorhabdus luminescens. Ce locus code pour la machinerie de sécrétion/translocation, des régulateurs de l'expression génique et une protéine effectrice LopT, homologue à la cytotoxine YopT de Yersinia. Un deuxième effecteur potentiel, homologue au premier et appelé LopT2 a été identifié dans une autre partie du génome à proximité d'un vestige de phage. L'objectif de ce travail a été de caractériser ce système de sécrétion et d'évaluer son rôle dans le processus infectieux chez l'insecte. Dans un premier temps, nous avons démontré en utilisant des souches de Xenorhabdus et Photorhabdus constitutivement marquées à la GFP (Green Fluorescent Protein) que ces deux bactéries avaient une localisation extracellulaire dans l'insecte. Le système de sécrétion de type III est présent chez toutes les espèces de Photorhabdus suggérant une acquisition avant la séparation des espèces mais pas chez Xenorhabdus. De plus, les composants de la machinerie de sécrétion sont hautement conservés mais les effecteurs prédits diffèrent entre P. luminescens et P. asymbiotica, deux espèces au spectre d'hôte différent (respectivement les Invertébrés et l'Homme). L'expression hétérologue chez Yersinia nous a permis de montrer que LopT ou LopT2 sont transloqués dans le cytoplasme de cellules de mammifères, comme le démontre l'apparition d'une forme électrophorétique différente de la GTPase RhoA. De plus, des études in vitro, ont mis en évidence que LopT est capable de libérer RhoA et Rac des membranes de cellules humaines et d'insectes. En outre, des essais d'infection réalisés in vivo chez le lépidoptère Spodoptera littoralis et chez l'orthoptère Locusta migratoria avec une souche de TT01 arborant une fusion traductionnelle des gènes lopT ou lopT2 avec le gène rapporteur GFP ont révélé que ces deux gènes sont induits uniquement dans des sites de défense tels que les nodules. Le mutant de la machinerie de sécrétion d'une part, n'induit plus la formation de nodules et d'autre part, est phagocyté par les macrophages d'insecte suggérant que LopT et LopT2, ou d'autres effecteurs, joueraient un rôle primordial dans l'inhibition de la phagocytose et indiquant un lien entre l'expression du TTSS et la nodulation chez l'insectLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Genes involved in the establishment of hepatic steatosis in Muscovy, Pekin and mule ducks

International audienceOur main objectives were to determine the genes involved in the establishment of hepatic steatosis in three genotypes of palmipeds. To respond to this question, we have compared Muscovy ducks, Pekin ducks and their crossbreed the mule duck fed ad libitum or overfed. We have shown a hepatic overexpression of fatty acid synthase (FAS) and di-acyl glycerol acyl transferase 2 (DGAT2) in overfed individuals, where DGAT2 seemed to be more regulated. This increase in lipogenesis genes is associated with a decrease of lipoprotein formation in Muscovy and mule ducks, especially apolipoprotein B (ApoB) and Microsomal Triglyceride Transfer Protein (MTTP), leading to lipid accumulation in liver. In Pekin ducks, MTTP expression is upregulated suggesting a better hepatic lipids exportation. Regarding lipids re-uptake, fatty acid-binding protein 4 and very-low-density-lipoprotein receptor are overexpressed in liver of mule ducks at the end of the overfeeding period. This phenomenon puts light on a mechanism unknown until today. In fact, mule can incorporate more lipids in liver than the two other genotypes leading to an intensified hepatic steatosis. To conclude, our results confirmed the genotype variability to overfeeding. Furthermore, similar observations are already described in non-alcoholic fatty liver disease in human, and ask if ducks could be an animal model to study hepatic triglyceride accumulation

Pre- and post-prandial expression of genes involved in lipid metabolism at the end of the overfeeding period of mule ducks

In palmipeds, overfeeding leads to hepatic steatosis, also called ‘‘foie gras’’ which is the result of many metabolic mechanisms. In order to understand these mechanisms, we decided to measure the expression of genes implicated in lipid metabolism during 12 hours (h) following the last meal of the overfeeding period. We have shown that there is a precocious expression (within 2 h) of fatty acid synthase and acyl CoA synthetase longchain 1 in liver and muscle of mule ducks in addition with a later peak. Furthermore, di-acyl glycerol acyl transferase presents the highest induction of expression in liver and it is overexpressed quite a long time, positioning this enzyme as a key factor in hepatic steatosis. These observations are quite similar in muscle. Lipoprotein secretion is upregulated later in postprandial period, with an upregulation of apolipoprotein and microsomal triglycerides transfer protein beginning at 5 h in liver or muscle. Regarding hepatic re-uptake of lipid, lesser variations are observed, suggesting that fatty acid binding protein and very low-density lipoprotein receptor (VLDLR) are already at their maximum expression specifically in these tissues. In muscle, VLDLR and LDLR upregulation is observed 5 h after the meal, associated with an overexpression in the adipose tissue of lipase maturation factor 1 involved in the maturation of lipoprotein lipase. These findings will allow us to better understand the kinetic treatment in lipid metabolism after a meal in overfed ducks. This first report on kinetic expression will allow researcher to better target their sampling time knowing the optimal point of expression of each gen

Kinetics of expression of genes involved in glucose metabolism after the last meal in overfed mule ducks

International audienceIn waterfowls, overfeeding leads to a hepatic steatosis, also called "foie gras." We decided to investigate the role of glucose metabolism in steatosis emergence. For this, we measured the expression of genes during the 12 h following the last meal of the overfeeding period. As expected, it showed that the expression of glucose transporter is more precocious in jejunal mucosa, especially for SGLT1, known to be the major transporter at the apical surface. In the liver, GLUT2 and HK1 are upregulated at the same time and seem to work together to import glucose. In peripherals tissues, such as muscle and subcutaneous adipose tissue (SAT), expression of genes of interest occurs later than the one in jejunum and liver. These results are in accordance with the evolution of glycemia. This study allows us to better understand the kinetic treatment of glucose after a meal in overfed ducks. It also will allow researchers to better target their sampling time knowing the optimal point of expression of each gene

Evolution of intestinal microbiota and body compartments during spontaneous hyperphagia in the Greylag goose

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L'engraissement spontané du foie chez les palmipèdes : Etat des lieux et perspectives de recherche

International audienceLa production de foie gras, telle que pratiquée aujourd'hui, est remise en question par les associations de protection animale, mais aussi par le Conseil de l'Europe. Le Comité Permanent de la Convention Européenne pour la protection des animaux dans les élevages a ainsi recommandé que des études portant sur des méthodes alternatives à la prise forcée d'aliment chez les palmipèdes soient mises en place dans les pays européens producteurs de foie gras. Cette synthèse a pour objectif de présenter l'état d'avancement des recherches sur ces méthodes alternatives. Les voies de synthèse et de stockage de lipides chez les oiseaux seront abordées ainsi que le comportement alimentaire et le métabolisme hépatique des oiseaux migrateurs, ayant permis de définir les bases biologiques des recherches menées sur la stéatose spontanée chez les palmipèdes domestiques. Les résultats des recherches conduites sur l'engraissement hépatique spontané des palmipèdes, les effecteurs de celui-ci et l'impact en terme de durabilité du système seront discutés. Enfin, plusieurs perspectives de recherche sur des leviers possibles tels que l'alimentation, la conduite d'élevage, la sélection génétique et l'étude du microbiote intestinal, seront proposées. Françai

Variation in the Effectors of the Type III Secretion System among Photorhabdus Species as Revealed by Genomic Analysis

Entomopathogenic bacteria of the genus Photorhabdus harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P. asymbiotica, two highly related species with different hosts

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects

Ontogeny of hepatic metabolism in mule ducks highlights different gene expression profiles between carbohydrate and lipid metabolic pathways

International audienceBackground: The production of foie gras involves different metabolic pathways in the liver of overfed ducks suchas lipid synthesis and carbohydrates catabolism, but the establishment of these pathways has not yet beendescribed with precision during embryogenesis. The early environment can have short- and long-term impacts onthe physiology of many animal species and can be used to influence physiological responses that is calledprogramming. This study proposes to describe the basal hepatic metabolism at the level of mRNA in mule duckembryos in order to reveal potential interesting programming windows in the context of foie gras production. To this end, a kinetic study was designed to determine the level of expression of selected genes involved in steatosis-related liver functions throughout embryogenesis.The livers of 20 mule duck embryos were collected every 4 days from the 12th day of embryogenesis (E12) until 4days after hatching (D4), and gene expression analysis was performed. The expression levels of 50 mRNAs werequantified for these 7 sampling points and classified into 4 major cellular pathways.Results: Interestingly, most mRNAs involved in lipid metabolism are overexpressed after hatching (FASN, SCD1,ACOX1), whereas genes implicated in carbohydrate metabolism (HK1, GAPDH, GLUT1) and development (HGF, IGF,FGFR2) are predominantly overexpressed from E12 to E20. Finally, regarding cellular stress, gene expression appears quite stable throughout development, contrasting with strong expression after hatching (CYP2E1, HSBP1, HSP90AA1).Conclusion: For the first time we described the kinetics of hepatic ontogenesis at mRNA level in mule ducks and highlighted different expression patterns depending on the cellular pathway. These results could be particularly usefulin the design of embryonic programming for the production of foie gras

Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization

International audiencePhotorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insect