A Model of Bacterial Intestinal Infections in Drosophila melanogaster

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis

Relative Roles of the Cellular and Humoral Responses in the Drosophila Host Defense against Three Gram-Positive Bacterial Infections

BACKGROUND: Two NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense. METHODOLOGY/PRINCIPAL FINDINGS: In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response. CONCLUSIONS/SIGNIFICANCE: Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen

Etudes des relations hôte-pathogène entre Drosophila melanogaster et une bactérie entomopathogène Serratia marcescens

J ai établi un système d infection par voie orale. L étude de ce système d'infection intestinale de la drosophile m a permis de démontrer l importance de deux mécanismes complémentaires et indépendants de la défense de l hôte: la réponse locale imd-dépendante au niveau de l épithélium intestinal et la phagocytose par les plasmatocytes. J ai participé à la caractérisation d un nouveau récepteur de phagocytose, Eater. Les mouches dépourvues de ce récepteur sont plus sensibles à l infection par S. marcescens.Nous avons décidé d utiliser cette propriété pour réaliser un crible au niveau du génome entier afin d identifier tous les gènes impliqués dans la défense contre les infections intestinales. Nous avons criblé de manière exhaustive, par interférence à l ARN double brin, une banque de 13760 lignées transgéniques1 permettant de cibler presque tous les gènes prédits du génome de la drosophile.Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In the fly, S. marcescensescapes from the gut and reaches the body cavity.We have demonstrated that two mechanisms act together against such food-borne infection: an immune-deficiency pathway-dependent antimicrobial response in the intestine and phagocytosis by blood cells in the hemolymph. In addition, a strong oxidative burst takes place in the gut lumen in response to the presence of microorganisms. The molecular dissection of the interaction between S. marcescens and Drosophila, thanks to the genetic tools available in both host and pathogen, will provide a useful paradigm to decipher intestinal pathogenesis. The insights gained from this approach may also help us better understand intestinal innate immunity invertebrates. We are using a third generation mutagenesis screen to identify all the genes involved in the host defense against S. marcescens intestinal infections, using the Vienna transgenic RNAi library.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Influence of Coronal Flaring on the Shaping Ability of Two Heat-Treated Nickel-Titanium Endodontic Files: A Micro-Computed Tomographic Study

Nickel-titanium (NiTi) usage is associated in endodontics with some complications including canal transportation. Centering ability of a NiTi file is the ability to stay centered in the root canal system during instrumentation. Any undesirable deviation from the natural canal path is indicated as canal transportation. A possible strategy to improve the centering ability of NiTi instruments is the pre-enlargement of the coronal third of the root canal to minimize coronal interferences. This procedure is known as coronal flaring. The aim of this study was to perform a micro-computed tomographic (micro-CT) evaluation of the effect of coronal flaring on canal transportation and centering ability of two heat treated nickel-titanium rotary instruments, 2Shape (Micro Mega, Besançon, France) and HyFlex CM (Coltène Whaledent, Altstätten, Switzerland). Thirty extracted mandibular molars with two independent mesial canals were selected and randomly instrumented (n = 15 canals) with One Flare (Micro Mega, Besançon, France) before HyFlex CM, HyFlex CM (without coronal flaring), One Flare before 2Shape and 2Shape (without coronal flaring). One Flare (Micro Mega, Besançon, France) was introduced 4 mm below the canal entrance for canals prepared with coronal flaring. HyFlex CM and 2Shape were used accordingly to manufacturers’ instructions. New files were used for each canal. During and after instrumentation, irrigation procedures were performed. Micro-CT images were obtained pre- and post-preparation to measure and record root canal transportation and centralization. They were reconstructed from root apex to canal orifices, generating approximately 1000 sections per specimen. The anatomical thirds were determined by dividing the number of cross-sectional slices by three. Root canal transportation and centralization were determined by Gambil method, and the mean values were analyzed by repeated measures analysis of variance followed by multiple comparisons of Bonferroni to compare the different instrumentations procedures and the root thirds (p < 0.05). As for root canal transportation, 2Shape reported significantly higher values compared to HyFlex CM in the cervical region independently from the coronal flaring. In the apical region, 2Shape caused significantly minor canal transportation when used with coronal flaring with compared with the absence of coronal flaring. Regarding the centralization, HyFlex CM showed higher values than 2Shape in the cervical, independently from coronal flaring. In the apical region, 2Shape with coronal flaring exhibited significant major centering ratio, compared with not. Within the limitations of this study, coronal flaring reduced canal transportation and improved centralization of the 2Shape files in the apical section while it had no significant influence on shaping ability of the HyFlex CM instruments. Coronal flaring could represent a valid strategy to improve the shaping ability of NiTi files knowing that its benefit could be influenced by the shaping file used

Munc13-4*rab27 complex tethers secretory lysosomes at the plasma membrane

Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are critical for the immune response against virus infections or transformed cells. They kill target cells via polarized exocytosis of lytic proteins from secretory lysosomes (SL). Rab27a and munc13-4 interact directly and are required for target cell killing. How they cooperate in the intricate degranulation process is not known. We identified critical residues in munc13-4 for rab27 interaction and tested binding mutants in several complementation assays. In a rat mast cell line we replaced endogenous munc13-4 with ectopically expressed munc13-4 constructs. Unlike wild type munc13-4, binding mutants fail to rescue β-hexosaminidase secretion. In accord, expression of binding mutants in CTL of Familial Hemophagocytic Lymphohistiocytosis type 3 patients, does not rescue CD107 appearance on the plasma membrane. Total Internal Reflection Fluorescence (TIRF) imaging shows that munc13-4*rab27a restricts motility of SL in the subapical cytoplasm. We propose that rab27*munc13-4 tethers SL to the plasma membrane, a requirement for formation of a cognate SNARE complex for fusion

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

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Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics

International audienceIn eukaryotic cells, membrane proteins play a crucial role. They fall into three categories: intrinsic proteins, extrinsic proteins, and proteins that are essential to the human genome (30% of which is devoted to encoding them). Hydrophobic interactions inside the membrane serve to stabilize integral proteins, which span the lipid bilayer. This review investigates a number of computational and experimental methods used to study membrane proteins. It encompasses a variety of technologies, including electrophoresis, X-ray crystallography, cryogenic electron microscopy (cryo-EM), nuclear magnetic resonance spectroscopy (NMR), biophysical methods, computational methods, and artificial intelligence. The link between structure and function of membrane proteins has been better understood thanks to these approaches, which also hold great promise for future study in the field. The significance of fusing artificial intelligence with experimental data to improve our comprehension of membrane protein biology is also covered in this paper. This effort aims to shed light on the complexity of membrane protein biology by investigating a variety of experimental and computational methods. Overall, the goal of this review is to emphasize how crucial it is to understand the functions of membrane proteins in eukaryotic cells. It gives a general review of the numerous methods used to look into these crucial elements and highlights the demand for multidisciplinary approaches to advance our understanding