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1. Identification of human host factors required for beta-defensin-2 expression in intestinal epithelial cells upon a bacterial challenge

Author: Weronika Wozniak, Emmanuel Sechet, Yong-Jun Kwon, Nathalie Aulner, Lionel Navarro, and Brice Sperandio
Subjects: Innate immunity, Human intestinal epithelial cell, Antimicrobial peptide, Beta-defensin-2, Gene regulation, Bacterial challenge, Medicine, Science
Abstract: Abstract The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.
Published: 2024
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2. An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus

Author: Marielle Cochet, François Piumi, Kamila Gorna, Noémie Berry, Gaëlle Gonzalez, Anne Danckaert, Nathalie Aulner, Odile Blanchet, Stéphan Zientara, Francesc Xavier Donadeu, Hélène Munier-Lehmann, Jennifer Richardson, Alexandra Benchoua, and Muriel Coulpier
Subjects: Equine, brain, neural progenitors, Flavivirus, antiviral, statin, Veterinary medicine, SF600-1100
Abstract: Abstract Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2′C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.
Published: 2024
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3. A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Author: Alexandra Maure, Emeline Lawarée, Francesco Fiorentino, Alexandre Pawlik, Saideep Gona, Alexandre Giraud-Gatineau, Matthew J G Eldridge, Anne Danckaert, David Hardy, Wafa Frigui, Camille Keck, Claude Gutierrez, Olivier Neyrolles, Nathalie Aulner, Antonello Mai, Mélanie Hamon, Luis B Barreiro, Priscille Brodin, Roland Brosch, Dante Rotili, and Ludovic Tailleux
Subjects: Biology (General), QH301-705.5
Abstract: Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.
Published: 2024
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4. LocalZProjector and DeProj: a toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images

Author: Sébastien Herbert, Léo Valon, Laure Mancini, Nicolas Dray, Paolo Caldarelli, Jérôme Gros, Elric Esposito, Spencer L. Shorte, Laure Bally-Cuif, Nathalie Aulner, Romain Levayer, and Jean-Yves Tinevez
Subjects: Image analysis, Morphology, 3D imaging, Tissue imaging, Biology (General), QH301-705.5
Abstract: Abstract Background Quantitative imaging of epithelial tissues requires bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common preprocessing step consists of projecting the acquired 3D volume on a 2D plane mapping the tissue surface. While segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in developmental and cell biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement. Results Here we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper, we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools. Conclusions We find that LocalZProjector performs well even in situations where the volume to project also contains unwanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.
Published: 2021
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5. Corrigendum: Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols

Author: Audrey Chansard, Nelly Dubrulle, Mathilde Poujol de Molliens, Pierre B. Falanga, Tharshana Stephen, Milena Hasan, Ger van Zandbergen, Nathalie Aulner, Spencer L. Shorte, Brigitte David-Watine, the Milieu Intérieur Consortium, Laurent Abel, Andres Alcover, Hugues Aschard, Kalla Astrom, Philippe Bousso, Pierre Bruhns, Ana Cumano, Caroline Demangel, Ludovic Deriano, James Di Santo, Françoise Dromer, Gérard Eberl, Jost Enninga, Jacques Fellay, Ivo Gomperts-Boneca, Serge Hercberg, Olivier Lantz, Hugo Mouquet, Etienne Patin, Sandra Pellegrini, Stanislas Pol, Antonio Rausell, Lars Rogge, Anavaj Sakuntabhai, Olivier Schwartz, Benno Schwikowski, Spencer Shorte, Frédéric Tangy, Antoine Toubert, Mathilde Trouvier, Marie-Noëlle Ungeheuer, Darragh Duffy, Matthew L. Albert, and Lluis Quintana-Murci
Subjects: immortalization, HSV-1, FACS, cytokines, human primary cells, NF-kB, Immunologic diseases. Allergy, RC581-607
Published: 2021
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6. Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols

Author: Audrey Chansard, Nelly Dubrulle, Mathilde Poujol de Molliens, Pierre B. Falanga, Tharshana Stephen, Milena Hasan, Ger van Zandbergen, Nathalie Aulner, Spencer L. Shorte, Brigitte David-Watine, the Milieu Intérieur Consortium, Laurent Abel, Andres Alcover, Hugues Aschard, Kalla Astrom, Philippe Bousso, Pierre Bruhns, Ana Cumano, Caroline Demangel, Ludovic Deriano, James Di Santo, Françoise Dromer, Gérard Eberl, Jost Enninga, Jacques Fellay, Ivo Gomperts-Boneca, Serge Hercberg, Olivier Lantz, Hugo Mouquet, Etienne Patin, Sandra Pellegrini, Stanislas Pol, Antonio Rausell, Lars Rogge, Anavaj Sakuntabhai, Olivier Schwartz, Benno Schwikowski, Spencer Shorte, Frédéric Tangy, Antoine Toubert, Mathilde Trouvier, Marie-Noëlle Ungeheuer, Darragh Duffy, Matthew L. Albert, and Lluis Quintana-Murci
Subjects: immortalization, HSV-1, FACS, cytokines, human primary cells, NF-κB, Immunologic diseases. Allergy, RC581-607
Abstract: The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our “Add-only” protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.
Published: 2021
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7. Targeting Macrophage Histone H3 Modification as a Leishmania Strategy to Dampen the NF-κB/NLRP3-Mediated Inflammatory Response

Author: Hervé Lecoeur, Eric Prina, Thibault Rosazza, Kossiwa Kokou, Paya N’Diaye, Nathalie Aulner, Hugo Varet, Giovanni Bussotti, Yue Xing, Geneviève Milon, Robert Weil, Guangxun Meng, and Gerald F. Späth
Subjects: Biology (General), QH301-705.5
Abstract: Summary: Aberrant macrophage activation during intracellular infection generates immunopathologies that can cause severe human morbidity. A better understanding of immune subversion strategies and macrophage phenotypic and functional responses is necessary to design host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response that is induced in primary and lesional macrophages infected by the parasite Leishmania amazonensis and dampens NF-κB and NLRP3 inflammasome activation. Subversion is amastigote-specific and characterized by a decreased expression of activating and increased expression of de-activating components of these pro-inflammatory pathways, thus revealing a regulatory dichotomy that abrogates the anti-microbial response. Changes in transcript abundance correlate with histone H3K9/14 hypoacetylation and H3K4 hypo-trimethylation in infected primary and lesional macrophages at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a Leishmania immune subversion strategy targeting host cell epigenetic regulation to establish conditions beneficial for parasite survival and open avenues for host-directed, anti-microbial drug discovery. : Lecoeur et al. demonstrate that the parasite Leishmania amazonensis modifies expression of NF-kB-related genes and prevents NLRP3 inflammasome activation of host macrophages in vitro and in vivo by changing histone H3 modifications at the promotor of pro-inflammatory genes. This mechanism of immune subversion opens avenues for host-directed, anti-leishmanial therapies. Keywords: Leishmania amazonensis, amastigotes, lesional macrophage, NF-κB, NLRP3 inflammasome, H3 acetylation, H3 methylation, epigenetics, histone
Published: 2020
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8. High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.

Author: Nathalie Aulner, Anne Danckaert, Eline Rouault-Hardoin, Julie Desrivot, Olivier Helynck, Pierre-Henri Commere, Hélène Munier-Lehmann, Gerald F Späth, Spencer L Shorte, Geneviève Milon, and Eric Prina
Subjects: Arctic medicine. Tropical medicine, RC955-962, Public aspects of medicine, RA1-1270
Abstract: Background/objectivesHuman leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.Methodology/principal findingsWe have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.Conclusions/significanceOur assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.
Published: 2013
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9. Intracellular bacteria encode inhibitory SNARE-like proteins.

Author: Fabienne Paumet, Jordan Wesolowski, Alejandro Garcia-Diaz, Cedric Delevoye, Nathalie Aulner, Howard A Shuman, Agathe Subtil, and James E Rothman
Subjects: Medicine, Science
Abstract: Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.
Published: 2009
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10. Immunometabolic profiling ofin vitroandex vivo Leishmania-infected macrophages (LIMs) reveals unique polarization and bioenergetic signatures

Author: Sheng Zhang, Hervé Lecoeur, Hugo Varet, Rachel Legendre, Nassim Mahtal, Caroline Proux, Nathalie Aulner, Spencer Shorte, Capucine Granjean, Philippe Bousso, Eric Prina, and Gerald F. Späth
Abstract: Macrophages are the major host cells of the protozoan parasiteLeishmaniain mammalian infection. These key innate immune cells display remarkable phenotypic plasticity ranging from pro-inflammatory M1 to anti-inflammatory M2 macrophages that can control infection and tissue homeostasis, respectively. It has been recognized thatLeishmaniaexploits macrophage phenotypic plasticity to establish chronic infection. However, the current notion that these parasites simply trigger an M2-like phenotype seems over-simplified considering the immunopathology observed during leishmaniasis, is which often characterized by a mixed Th1/Th2 immune response. Here we combined a series of systems-level analyses to shed new light on the phenotype ofLeishmania-infected macrophages (LIMs). Immunometabolic profiling by RNAseq, quantitative RT-qPCR, cytokine immunoassays, and bioenergetic flux analysis ofLeishmania amazonensis-infected bone marrow-derived macrophages (BMDMs) revealed a highly complex, mixed polarization phenotype and a unique bioenergetic signature.In vitroLIMs were characterized by (i) co-expression of both M1 and M2 markers at RNA and protein levels, (ii) expression changes of a unique set of inflammatory genes (e.g. RELA, IGFBP3, VEGFA), and (iii) modulation of metabolic gene expression, including up regulation of glycolytic genes. This LIMs-specific phenotype persisted for at least 30 days and was confirmedin vivoin lesion-isolated LIMs. Likewise,in vitroLIMs showed a mixed bioenergetic profile, increasing both glycolytic activity and oxidative phosphorylation (OXPHOS) compared to uninfected, nonpolarized macrophages (M0). Surprisingly, the elimination of intracellular parasites using the fast-acting, anti-leishmanial drug L-leucine-O-methyl ester (LME) enhanced both bioenergetic pathways to levels comparable to M1 (glycolysis) and M2 (OXPHOS) macrophages as soon as one hour after treatment, suggesting thatL. amazonensislimits in real-time energy metabolism and ATP production in LIMs. In conclusion,L. amazonensisinfection establishes a mixed, immunometabolomic polarization profile in infected BMDMs and lesional macrophages that on one hand can promote an M1-regulated Th1 response required for the recruitment of new host cells to infected tissues, while at the same time promoting an M2 phenotype that dampens pro-inflammatory immune-signaling and shields intracellular parasites from leishmanicidal Nitric Oxide (NO).
Published: 2022
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11. Identification of antiviral molecules against West-Nile virus by an approach combining cell imaging and equine neural cells derived from induced-pluripotent stem cells

Author: Marielle Cochet-Bernoin, François Piumi, Kamila Gorna, Gaelle Gonzalez, Anne Danckaert, Nathalie Aulner, Hélène Munier-Lehmann, Stephan Zientara, Xavier Donadeu, F., Alexandra Benchoua, Muriel Coulpier, Virologie UMR1161 (VIRO), École nationale vétérinaire d'Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-École nationale vétérinaire d'Alfort (ENVA), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], The Roslin Institute, CECS - I-STEM, Association française contre les myopathies (AFM-Téléthon), École nationale vétérinaire - Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), École nationale vétérinaire - Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP), Biotechnology and Biological Sciences Research Council (BBSRC), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), and Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon
Subjects: [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2021

12. LocalZProjector and DeProj: a toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images

Author: Nathalie Aulner, Spencer L. Shorte, Sébastien Herbert, Elric Esposito, Romain Levayer, Laure Bally-Cuif, Jean-Yves Tinevez, Jerome Gros, Paolo Caldarelli, Laure Mancini, Léo Valon, Nicolas Dray, Hub d'analyse d'images - Image Analysis Hub (Platform) (IAH), Institut Pasteur [Paris] (IP), Mort cellulaire et homéostasie des épithéliums / Cell death and epithelial homeostasis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Neurogénétique du Poisson zébré / Zebrafish Neurogenetics, Collège Doctoral, Sorbonne Université (SU), Régulation Dynamique de la Morphogénèse - Dynamic Regulation of Morphogenesis, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), This work was supported by the Labex REVIVE (Investissement d’Avenir, ANR-10-LABX-73), France BioImaging (Investissement d’Avenir, ANR-10–INSB–04) and the Région Île-de-France in the framework of DIM ELICIT. Work in the R. L. laboratory is supported by the Institut Pasteur (G5 starting package), a ERC starting grant (CoSpaDD, #758457), the 'Cercle FSER' and the CNRS (UMR 3738). L. V. is supported by a Post-doctoral grant 'Aide au 224 Retour en France' from the Fondation pour la Recherche Médicale (ARF20170938651) and a Marie Sklodowska-Curie postdoctoral fellowship (MechDeath, 789573). Work in the J. G. laboratory was supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013) / ERC Grant Agreement no. 337635. P. C. is sponsored by the Institut Pasteur and the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie (665807). Work in the L. B-C. laboratory was supported by the Ligue Nationale Contre le Cancer and the European Research Council (AdG 322936). L. M. was recipient of a PhD student fellowship from the Fondation pour la Recherche Médicale., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 758457,H2020-EU.1.1. - EXCELLENT SCIENCE - European Research Council (ERC),ERC-2017-STG,CoSpaDD(2018), European Project: 789573,FP7-IDEAS-ERC - Specific programme: 'Ideas' implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013),H2020-MSCA-IF-2017,MechDeath(2019), European Project: 337635,EC:FP7:ERC,ERC-2013-StG,LIMBCELLDYNAMICS(2014), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), European Project: 322936,EC:FP7:ERC,ERC-2012-ADG_20120314,SYSTEMATICS(2013), ANR-10-LABX-73), France BioImaging (Investissement d’ Avenir, ANR-10–INSB–04) and the Région Île-de-France in the framework of DIM ELICIT. Work in the R. L. laboratory is supported by the Institut Pasteur (G5 starting package), a ERC starting grant (CoSpaDD, #758457), the 'Cercle FSER' and the CNRS (UMR 3738). L. V. is supported by a Postdoctoral grant 'Aide au 224 Retour en France' from the Fondation pour la Recherche Médicale (ARF20170938651) and a Marie Sklodowska-Curie postdoctoral fellowship (MechDeath, 789573). Work in the J. G. laboratory was supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013) / ERC Grant Agreement no. 337635. P. C. is sponsored by the Institut Pasteur and the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie (665807). Work in the L. B-C. laboratory was supported by the Ligue Nationale Contre le Cancer and the European Research Council (AdG 322936). L. M. was recipient of a PhD student fellowship from the Fondation pour la Recherche Médicale., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Neurogénétique du Poisson zébré - Zebrafish Neurogenetics, Collège doctoral [Sorbonne universités], Sorbonne Université, Institut Pasteur [Paris], and Département de Biologie du Développement et Cellules souches - Department of Developmental and Stem Cell Biology
Subjects: Morphology, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, QH301-705.5, Computer science, [SDV]Life Sciences [q-bio], [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Signal, Tissue imaging, Image analysis, 03 medical and health sciences, Imaging, Three-Dimensional, 0302 clinical medicine, 3D imaging, Segmentation, Computer vision, Biology (General), Projection (set theory), Process (anatomy), 030304 developmental biology, Microscopy, 0303 health sciences, Plane (geometry), business.industry, Distortion (optics), Visibility (geometry), Range (mathematics), [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Artificial intelligence, business, Software, 030217 neurology & neurosurgery
Abstract: BackgroundQuantitative imaging of epithelial tissues prompts for bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common pre-processing step consists in projecting the acquired 3D volume on a 2D plane mapping the tissue surface. Indeed, while segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in Developmental and Cell Biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement.ResultsHere we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes, and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a user-friendly and configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools.ConclusionsWe find that LocalZProjector performs well even in situations where the volume to project also contains un-wanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.
Published: 2021
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13. A Role for Taok2 in Listeria monocytogenes Vacuolar Escape

Author: Alain Charbit, Jost Enninga, Nathalie Aulner, Noelia Lopez-Montero, Jason Ziveri, Steven Rolland, Javier Pizarro-Cerdá, Pascale Cossart, Camille Morel, Juan J. Quereda, Théodore Grenier, Anne Danckaert, Interactions Bactéries-Cellules (UIBC), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Universidad Cardenal Herrera-CEU (CEU-UCH), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Génomique Fonctionnelle de Lyon (IGFL), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Pasteur [Paris], Yersinia, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes, Sorbonne Paris Cité, Paris, France, Inflammasome NLRP3 – NLRP3 Inflammasome, Centre International de Recherche en Infectiologie - UMR (CIRI), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), This work was supported by Institut Pasteur, Université Paris Diderot, Agence Nationale de la Recherche (grant numbers ANR-15-CE15-0017 StopBugEntry to J. P. C., J. E., and A. C., and ANR-10-INSB-04-01, France-BioImaging to N. A.). J. J. Q. is supported by the Spanish Ministry of Science, Innovation and Universities (grant number RYC-2018-024985-I Ramón y Cajal contract). J. E. was supported by the European Research Council (consolidator grant EndoSubvert). P. B. I. is supported by the Région Ile de France (DIM1Health) and Groupement d’Intérêt Scientifique Infrastructures en Biologie Santé et Agronomie. The teams of P. C., J. P. C., N. A., and J. E. are members of Laboratory of Excellence Integrative Biology of Emerging Infectious Diseases., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 682809,EndoSubvert(2017), Producción Científica UCH 2022, UCH. Departamento de Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Charbit, Alain, Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes - - StopBugEntry2015 - ANR-15-CE15-0017 - AAPG2015 - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Common mechanisms of host membrane trafficking subversion by intracellular pathogens to rupture bacterial containing vacuoles - EndoSubvert - 2017-01-01 - 2021-12-31 - 682809 - VALID, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Producción Científica UCH 2020
Subjects: 0301 basic medicine, Cytoplasm, media_common.quotation_subject, Citoplasma, 030106 microbiology, Vacuole, Biology, medicine.disease_cause, Pathogenic bacteria, 03 medical and health sciences, Hemolysin Proteins, Cytosol, Listeria monocytogenes, Bacterial Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, medicine, Immunology and Allergy, Humans, Células epiteliales, vacuolar escape, Listeriosis, Kinase activity, Internalization, Pathogen, ComputingMilieux_MISCELLANEOUS, media_common, Epithelial cells, siRNA screen, Kinase, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, STE1-like kinase, 030104 developmental biology, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Bacterias patógenas, Vacuoles, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Taok2
Abstract: Este artículo se encuentra disponible en la siguiente URL: https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaa367/5862450 En esta investigación también participan: Nathalie Aulner, Anne Danckaert, Alain Charbit, Jost Enninga, Pascale Cossart y Javier Pizarro Cerdá. The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.
Published: 2021
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14. Unveiling inter individual variability of human fibroblast innate immune response using robust cell-based protocols

Author: Nelly Dubrulle, Nathalie Aulner, Milena Hasan, Tharshana Stephen, Brigitte David-Watine, Audrey Chansard, Mathilde Poujol de Molliens, Pierre Barthélémy Falanga, Ger van Zandbergen, and Spencer Louis Shorte
Subjects: education.field_of_study, Innate immune system, Population, Cell, Context (language use), Computational biology, Biology, Immune system, medicine.anatomical_structure, TLR3, medicine, education, Cell activation, Fibroblast
Abstract: The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies.A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our “Add-only” protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.
Published: 2020
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15. Fluorescence imaging host pathogen interactions: fifteen years benefit of hindsight…

Author: Nathalie Aulner, Audrey Salles, Pascal Roux, Spencer L. Shorte, Anne Danckaert, Julien Fernandes, Jean-Yves Tinevez, Marie-Anne Nicola, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris] (IP), We are grateful for support from the 7th Framework Program of the European Commission (LEISHDRUG project, 223414), the French Government (L’Agence nationale de la recherche (ANR)) programmes: Investissements d’Avenir programme (‘Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases’, grant ANR-10-LABX-62-IBEID and ‘Laboratoire Revive’, grant ANR 10-LBX-73-REVIVE), France BioImaging (FBI, grant ANR-10-INSB-04-01), the Région Ile de France (Domaine d’intêret majeur Innovative technologies for life sciences, DIM ELICIT) and the GIS IBiSA (Infrastructures en biologie santé et agronomie) and the Institut Pasteur., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 223414,EC:FP7:HEALTH,FP7-HEALTH-2007-B,LEISHDRUG(2008), and Institut Pasteur [Paris]
Subjects: 0301 basic medicine, Microbiology (medical), Fluorescence-lifetime imaging microscopy, MESH: Host-Pathogen Interactions, [SDV]Life Sciences [q-bio], ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, MESH: Automation, Laboratory, Biology, Microbiology, MESH: Containment of Biohazards, 03 medical and health sciences, 0302 clinical medicine, Optical imaging, MESH: Laboratories/organization & administration, MESH: Microscopy, Fluorescence/methods, Humans, Automation, Laboratory, MESH: Humans, Containment of Biohazards, MESH: Molecular Imaging/instrumentation, Data science, Molecular Imaging, 030104 developmental biology, Infectious Diseases, Microscopy, Fluorescence, Host-Pathogen Interactions, MESH: Microscopy, Fluorescence/instrumentation, Laboratories, MESH: Molecular Imaging/methods, Host (network), 030217 neurology & neurosurgery, Hindsight bias
Abstract: International audience; We consider in review current state-of-the-art fluorescence microscopy for investigating the host-pathogen interface. Our perspective is honed from years with literally thousands of microbiologists using the variety of imaging technologies available within our dedicated BSL2/BSL3 optical imaging research service facilities at the Institut Pasteur Paris founded from scratch in 2001. During fifteen years learning from the success and failures of introducing different fluorescence imaging technologies, methods, and technical development strategies we provide here a synopsis review of our experience to date and a synthesis of how we see the future in perspective for fluorescence imaging at the host-pathogen interface.
Published: 2018
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16. Targeting Macrophage Histone H3 Modification as a Leishmania Strategy to Dampen the NF-κB/NLRP3-Mediated Inflammatory Response

Author: Paya N’Diaye, Guangxun Meng, Nathalie Aulner, Eric Prina, Hugo Varet, Kossiwa Kokou, Robert Weil, Yue Xing, Thibault Rosazza, Gerald F. Späth, Geneviève Milon, Giovanni Bussotti, Hervé Lecoeur, Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Key Laboratory of Molecular Virology and Immunology [Shanghai, China], Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), This project was supported by a fund of the Institut Pasteur International Direction to the International Mixed Unit 'Inflammation and Leishmania Infection' and by the International Partnership Program (153831KYSB20190008) of the Chinese Academy of Sciences. The UtechS PBI/C2RT is part of the FranceBioImaging infrastructure, supported by the French National Research Agency (ANR-10-INSB-04-01, Investments for the Future) and is supported by Conseil de la Région Ile-de-France (Domaine d’Intérêt Majeur DIM1HEALTH) and Fondation Française pour la Recherche Médicale (Programme Grands Equipements)., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Parasitologie moléculaire et Signalisation, Key Laboratory of Molecular Virology & Immunology (LMVI), Pasteur International Mixed Unit 'Inflammation and Leishmania infection' (IMU-InflaLeish), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Innate Immunity [China], Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Leishmania Lab [Corée du sud], Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Technologie et Service BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], This project was supported by a fund of the Institut Pasteur International Direction to the International Mixed Unit 'Inflammation and Leishmania Infection' and by the International Partnership Program (153831KYSB20190008) of the Chinese Academy of Sciences. We would like to thank Jean-Marc Cavaillon for support and helpful discussions and Drs. Emmanuel Laplantine and Anastassia V. Komarova for providing antibodies. The UtechS PBI/C2RT is part of the FranceBioImaging infrastructure, supported by the French National Research Agency (ANR-10-INSB-04-01, Investments for the Future) and is supported by Conseil de la Région Ile-de-France (Domaine d’Intérêt Majeur DIM1HEALTH) and Fondation Française pour la Recherche Médicale (Programme Grands Equipements)., ANR-10-INSB-04-01,INSB,Investissements d'Avenir, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Varet, Hugo, and Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID
Subjects: 0301 basic medicine, [SDV.IMM] Life Sciences [q-bio]/Immunology, Inflammasomes, [SDV]Life Sciences [q-bio], H3 methylation, amastigotes, histone, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Biochemistry, Genetics and Molecular Biology, NF-κB, Histones, 03 medical and health sciences, Histone H3, chemistry.chemical_compound, 0302 clinical medicine, Immune system, Animals, Macrophage, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Epigenetics, lesional macrophage, lcsh:QH301-705.5, Leishmania, biology, epigenetics, Macrophages, NF-kappa B, H3 acetylation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, NLRP3 inflammasome, 3. Good health, Human morbidity, Cell biology, [SDV] Life Sciences [q-bio], 030104 developmental biology, Histone, chemistry, lcsh:Biology (General), biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, 030217 neurology & neurosurgery, Leishmania amazonensis
Abstract: Summary: Aberrant macrophage activation during intracellular infection generates immunopathologies that can cause severe human morbidity. A better understanding of immune subversion strategies and macrophage phenotypic and functional responses is necessary to design host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response that is induced in primary and lesional macrophages infected by the parasite Leishmania amazonensis and dampens NF-κB and NLRP3 inflammasome activation. Subversion is amastigote-specific and characterized by a decreased expression of activating and increased expression of de-activating components of these pro-inflammatory pathways, thus revealing a regulatory dichotomy that abrogates the anti-microbial response. Changes in transcript abundance correlate with histone H3K9/14 hypoacetylation and H3K4 hypo-trimethylation in infected primary and lesional macrophages at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a Leishmania immune subversion strategy targeting host cell epigenetic regulation to establish conditions beneficial for parasite survival and open avenues for host-directed, anti-microbial drug discovery. : Lecoeur et al. demonstrate that the parasite Leishmania amazonensis modifies expression of NF-kB-related genes and prevents NLRP3 inflammasome activation of host macrophages in vitro and in vivo by changing histone H3 modifications at the promotor of pro-inflammatory genes. This mechanism of immune subversion opens avenues for host-directed, anti-leishmanial therapies. Keywords: Leishmania amazonensis, amastigotes, lesional macrophage, NF-κB, NLRP3 inflammasome, H3 acetylation, H3 methylation, epigenetics, histone
Published: 2020
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17. Next-Generation Phenotypic Screening in Early Drug Discovery for Infectious Diseases

Author: Anne Danckaert, Spencer L. Shorte, Nathalie Aulner, JongEun Ihm, David Shum, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), We are grateful for support from the Seventh Framework Program of the European Commission (LEISHDRUG project, 223414), the French Government [L'Agence nationale de la recherche (ANR)] programs: Investissements d'Avenir programme ('Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases', grant ANR-10-LABX-62-IBEID and ‘Laboratoire Revive’, grant ANR 10-LBX-73-REVIVE), France BioImaging (FBI, grant ANR-10-INSB-04-01)), the Région Ile de France (Domaine d’intêret majeur Innovative technologies for life sciences, DIM ELICIT, and DIM1Health), the GIS IBiSA (Infrastructures en biologie santé et agronomie), the National Research Foundation (NRF) of Korea (NRF-2017M3A9G6068254 Global Network and Research Infrastructure to Infectious Diseases), the Gyeonggi Provincial Government, the Institut Pasteur Korea, and Institut Pasteur Paris., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 223414,EC:FP7:HEALTH,FP7-HEALTH-2007-B,LEISHDRUG(2008), and Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Drug discovery, Emerging technologies, Phenotypic screening, 030231 tropical medicine, Computational Biology, Computational biology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Communicable Diseases, 3. Good health, 03 medical and health sciences, Phenotype, 030104 developmental biology, 0302 clinical medicine, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Drug Discovery, Host-Pathogen Interactions, Molecular targets, Humans, Parasitology, Cells, Cultured
Abstract: International audience; Cell-based phenotypic screening has proven to be valuable, notably in recapitulating relevant biological conditions, for example, the host cell/pathogen niche. However, the corresponding methodological complexity is not readily compatible with high-throughput pipelines, and fails to inform either molecular target or mechanism of action, which frustrates conventional drug-discovery roadmaps. We review the state-of-the-art and emerging technologies that suggest new strategies for harnessing value from the complexity of phenotypic screening and augmenting powerful utility for translational drug discovery. Advances in cellular, molecular, and bioinformatics technologies are converging at a cutting edge where the complexity of phenotypic screening may no longer be considered a hinderance but rather a catalyst to chemotherapeutic discovery for infectious diseases. Phenotypic Screening for Infectious Disease Drug Discovery The term 'phenotype' (Figure 1) was coined originally in 1903 by the Danish botanist Wilhelm Johannsen [1-3] and emerged as foundational in experimental, theoretical, and fundamental biology juxtaposed with 'genotype'. However, unlike genotype, for which a definition arises from tangible minimal information and molecular typing, the term phenotype lacks clear definition [4], relying on semantic description, for example, 'morphology', 'behavior', 'appearance', 'structure' etc. Indeed, there is an entire literature on the meaning of 'phenotype' but, more than 100 years after its conceptualization, it was only in recent years that the discipline and tools enabling ontology (see Glossary) for high-throughput and high-dimensional phenotyping began to emerge [4-7]. Specifically, for microbiology (MicroO [6]), there are efforts to align with other communitybased efforts establishing standards for ontology in genetics (GO i), phenotype (PATO ii), smallmolecule chemical entities of biological interest (ChEBI iii), and PubChem iv. Indeed, such efforts are invigorated, in part, by the realization that the performance of powerful natural language processing with neural-networks, machine-learning, and deep-learning methods is wholly dependent on the underlying means for data extraction that are linked to such ontologies, and ultimately the ability to relate them [7]. For the purposes of the current article we use the term 'phenotype' in the restrictive context of phenotypic screening [8,9].
Published: 2019
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18. Leishmania targets the macrophage epigenome and dampens the NF-κB/NLRP3-mediated inflammatory response

Author: Nathalie Aulner, Y. Xing, Eric Prina, G. F. Spaeth, R. Weil, Giovanni Bussotti, Hugo Varet, Hervé Lecoeur, Thibault Rosazza, G. X. Meng, Kossiwa Kokou, and P. N'Diaye
Subjects: chemistry.chemical_compound, Histone H3, Immune system, chemistry, Intracellular parasite, Macrophage, NF-κB, Epigenetics, Epigenome, Biology, Cell biology, Human morbidity
Abstract: SummaryAberrant macrophage activation during intracellular infection generates important immunopathologies that can cause severe human morbidity. A better understanding of microbial immune subversion strategies and macrophage phenotypic and functional responses is a prerequisite for the design of novel, host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response induced in primary macrophages infected by the human parasite Leishmania amazonensis that prevents NF-κB and NLRP3 inflammasome activation. This unusual subversion is characterized by respectively suppression and induction of activating and de-activating components of the NF-κB and NLRP3 pathways. This dichotomic modulation was associated with histone H3 hypoacetylation at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a novel Leishmania immune subversion strategy targeting host cell epigenetic regulation to modulate the macrophage phenotype. Modulation of the macrophage epigenetic landscape establishes conditions beneficial for intracellular parasite survival, and opens interesting new venues for host-directed, anti-microbial drug discovery.
Published: 2019
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19. Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening

Author: Eric Prina, Nathalie Aulner, Suzanne Lamotte, Gerald F. Späth, Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], The authors would like to thank Dr. Julio Martin, head of Parasite Chemotherapy at GSK Diseases of the Developing World (GLAXOSMITHKLINE INVESTIGACIÓN Y DESARROLLO, S.L.) for providing the Leish-Box compounds. The UtechS PBI is grateful for support from the French Government (L’Agence nationale de la recherche (ANR)) programmes: Investissements d’Avenir programme (‘Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases’, grant ANR-10-LABX-62-IBEID and ‘Laboratoire Revive’, grant ANR 10-LBX-73-REVIVE), France BioImaging (FBI, grant ANR-10-INSB-04-01), the Région Ile de France (Domaine d’intêret majeur Innovative technologies for life sciences, DIM 1HEALTH) and the GIS IBiSA (Infrastructures en biologie santé et agronomie) and the Institut Pasteur. S.L., G.F.S. and E.P. are funded by Institut Pasteur and INSERM U1201 and were also supported by the FP7 A-ParaDDisE program funded under the European Union’s Seventh Framework Programme (grant agreement 602080) and the Agence Natioinale de Recherche (ANR) Pathomethylome project (ANR-15-CE12-0020-02). SL is a PhD student from the Université Paris Diderot, Sorbonne Paris Cité. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-15-CE12-0020,PATHO-METHYLOME,Rôle de la méthylation des lysines dans les interactions hôte-pathogene(2015), European Project: 602080,EC:FP7:HEALTH,FP7-HEALTH-2013-INNOVATION-1,A-PARADDISE(2014), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, Drug, THP-1 Cells, Phenotypic screening, media_common.quotation_subject, Leishmania mexicana, Antiprotozoal Agents, Drug Evaluation, Preclinical, Leishmaniasis, Cutaneous, Virulence, lcsh:Medicine, Computational biology, Biology, Article, 03 medical and health sciences, 0302 clinical medicine, Species Specificity, Animals, Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Author Correction, Amastigote, lcsh:Science, Cells, Cultured, media_common, Mice, Inbred BALB C, Multidisciplinary, Molecular Structure, Macrophages, lcsh:R, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Leishmania, biology.organism_classification, Phenotype, 3. Good health, 030104 developmental biology, Drug development, Leishmaniasis, Visceral, lcsh:Q, 030217 neurology & neurosurgery, Ex vivo, Leishmania donovani
Abstract: International audience; The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits. Here we present such a novel strategy based on physiologically relevant, ex vivo biology. We established high content phenotypic assays that combine primary murine macrophages and lesion-derived, virulent L. donovani and L. amazonensis amastigotes, which we applied to validate previously identified, anti-leishmanial hit compounds referred to as 'GSK Leish-Box'. Together with secondary screens using cultured promastigotes, our pipeline distinguished stage-and/or species-specific compounds, including 20 hits with broad activity at 10 µM against intracellular amastigotes of both viscerotropic and dermotropic Leishmania. Even though the GSK Leish-Box hits were identified by phenotypic screening using THP-1 macrophage-like cells hosting culture-derived L. donovani LdBob parasites, our ex vivo assays only validated anti-leishmanial activity at 10 µM on intra-macrophagic L. donovani for 23 out of the 188 GSK Leish-Box hits. In conclusion, our comparative approach allowed the identification of hits with broad anti-leishmanial activity that represent interesting novel candidates to be tested in animal models. Physiologically more relevant screening approaches such as described here may reduce the very high attrition rate observed during pre-clinical and clinical phases of the drug development process. Leishmaniases are neglected diseases caused by protozoan parasites of the genus Leishmania that are transmitted by the bite of female Phlebotomine sandflies. Almost 1 billion people are at risk of infection in close to 100 endemic countries throughout the tropical and subtropical regions of Africa, Asia, the Mediterranean countries and South and Central America 1. Leishmaniasis clinical manifestations range from self-healing cutaneous lesions with possible mucosal dissemination to severe visceral forms, causing death if untreated. In the absence of efficient reservoir and vector control strategies, and of preventive or therapeutic human vaccines , the mainstay of current intervention strategy to limit the disease is chemotherapy. Leishmaniases remain the only trypanosomatid diseases for which therapeutics are largely based on repurposed drugs, including anti-fungal (amphotericin B), anticancer (miltefosine), antibiotic (paromomycin) and antimalarial (sitamaquine) molecules 2,3. However, all current treatments are limited and show serious drawbacks, including high cost that are prohibitory for resource-limited countries, poor compliance due to constraining mode of administration 4 , poor safety with important adverse effects due to toxicity 5 , treatment failure and drug resistance 6. Similarly, current efforts to overcome the drug resistance by developing drug combination therapies 7,8 may fail in light of the identification of an L. infantum strain that gained resistance against antimony and amphotericin B 9. Multi-drug resistance was also documented in Indian field isolates 10 , and was confirmed experimentally by the in vitro selection of L. donovani parasites showing resistance to different drug combinations and even cross-resistance to unrelated drugs 11. Combination therapy therefore may be of only limited use, raising important concerns on the current
Published: 2019
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20. Author Correction: Discovery of novel hit compounds with broad activity against visceral and cutaneous Leishmania species by comparative phenotypic screening

Author: Gerald F. Späth, Nathalie Aulner, Eric Prina, and Suzanne Lamotte
Subjects: Multidisciplinary, Phenotypic screening, lcsh:R, ComputingMethodologies_DOCUMENTANDTEXTPROCESSING, lcsh:Medicine, lcsh:Q, Computational biology, Biology, Leishmania species, lcsh:Science
Abstract: A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
Published: 2019
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21. Assessing Vacuolar Escape of Listeria Monocytogenes

Author: Javier Pizarro-Cerdá, Spencer L. Shorte, Anne Danckaert, Martin Sachse, Théodore Grenier, Damien Balestrino, Jennifer Fredlund, Pascale Cossart, Nathalie Aulner, Jost Enninga, Juan J. Quereda, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), Laboratoire Microorganismes : Génome et Environnement (LMGE), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Centre National de la Recherche Scientifique (CNRS), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Imagopole (CITECH), Work in the laboratory of P.C. is supported by the Institut Pasteur (Unité des Interactions Bactéries-Cellules), the Institut National de la Santé et de la Recherche Médicale (Unité 604), the Institut National de la Recherche Agronomique (Unité Sous Contrat 2020), the Fondation Les Mousquetaires, and an European Research Council Advanced Grant (BacEpiCell 670823). The Imagopole is part of the FranceBioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, 'Investments for the Future') and is grateful to supports by Conseil de la Region Ile-de-France (program Sesame 2007, project Imagopole to S.S.) and from the Fondation Française pour la Recherche Médicale (FRM, Programme Grands Equipements to N.A.). J.E. is supported by the Institut Pasteur (PTR-460), by the Institut Pasteur Carnot-MIE program and by a European Research Council Starting Grant (RuptEffects 261166)., Collin M, We thank Servier Medical Art (http://www.servier.com/Powerpoint-image-bank) for providing drawings used in Fig. 1., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris], Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS)-Université d'Auvergne - Clermont-Ferrand I (UdA), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Dynamique des Interactions Hôte-Pathogène, ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-15-CE15-0017,StopBugEntry,Novel common host targets during bacterial invasion of humans(2015), ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), vicente, marie-therese, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes - - StopBugEntry2015 - ANR-15-CE15-0017 - AAPG2015 - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, and Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID
Subjects: 0301 basic medicine, Phagocytosis, media_common.quotation_subject, [SDV]Life Sciences [q-bio], 030106 microbiology, education, Vacuole, Biology, medicine.disease_cause, Microbiology, 03 medical and health sciences, Listeria monocytogenes, medicine, Fluorescence Resonance Energy Transfer, Internalization, Pathogen, health care economics and organizations, media_common, Correlative light/electron microcopy (CLEM), CCF4, Listeriolysin O, Biological Transport, Förster resonance energy transfer (FRET) microscopy, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, [SDV] Life Sciences [q-bio], Microscopy, Electron, 030104 developmental biology, Förster resonance energy transfer, Microscopy, Fluorescence, Phospholipases, Vacuoles, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract: International audience; Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.
Published: 2016
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22. Screening out irrelevant cell-based models of disease

Author: Spencer L. Shorte, Nathalie Aulner, Carina von Schantz, Peter Horvath, Gerardo Turcatti, Marc Bickle, María C. Montoya, Päivi Östling, Neil O. Carragher, Daniel Ebner, Vilja Pietiäinen, Elaine Del Nery, Anthony Davies, and Leo S. Price
Subjects: 0301 basic medicine, Induced Pluripotent Stem Cells, Cell Culture Techniques, Drug Evaluation, Preclinical, Translational research, Drug development, Disease, Bioinformatics, Models, Biological, 03 medical and health sciences, Genome editing, Multidisciplinary approach, High-Throughput Screening Assays, Drug Discovery, Medicine, Animals, Humans, Induced pluripotent stem cell, Cell Line, Transformed, Pharmacology, Drug discovery, business.industry, Small molecules, General Medicine, Data science, 3. Good health, 030104 developmental biology, Pharmaceutical Preparations, Drug screening, business
Abstract: The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.
Published: 2016
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23. Discovery of novel small molecule cell type-specific enhancers of NF-κB nuclear translocation

Author: Alison Rinderspacher, Michael Wyler, Stephan C. Schürer, Yuli Xie, Nathalie Aulner, Dusica Vidovic, Udo Toebben, Lars Branden, Gangli Gong, Yan Feng, Deborah H. Smith, Zhengxiang Zhu, Donald W. Landry, Yidong Liu, Shi Xian Deng, Yufei Tang, and Caty Chung
Subjects: Leupeptins, Clinical Biochemistry, Tetrazoles, Pharmaceutical Science, Chromosomal translocation, Biochemistry, Jurkat cells, Jurkat Cells, chemistry.chemical_compound, Nitriles, Drug Discovery, Quinazoline, Combinatorial Chemistry Techniques, Humans, Sulfones, Enhancer, Molecular Biology, Molecular Structure, Activator (genetics), Organic Chemistry, NF-kappa B, NF-κB, Small molecule, Cell biology, chemistry, Sulfoxides, embryonic structures, Quinazolines, cardiovascular system, Molecular Medicine, Signal transduction
Abstract: An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.
Published: 2009
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24. Pharmacological assessment defines Leishmania donovani casein kinase 1 as a drug target and reveals important functions in parasite viability and intracellular infection

Author: Olivier Leclercq, Emilie Durieu, Cedric Notredame, Nathalie Aulner, Laurent Meijer, Eric Prina, Jean-François Taly, Pascale Pescher, Gerald F. Späth, Najma Rachidi, Parasitologie moléculaire et Signalisation, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Phophorylation de protéines et Pathologies Humaines (P3H), Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre for Genomic Regulation (CRG), Universitat Pompeu Fabra [Barcelona] (UPF), Imagopole (CITECH), Institut Pasteur [Paris] (IP), Immunophysiologie et Parasitisme, This work was supported by the 7th Framework Program of the European Commission through grants to the LEISHDRUG project (223414), ANR-11-RPIB-0016 TRANSLEISH, the French Government's Investissements d'Avenir program Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (grant no. ANR-10-LABX-62-IBEID), the Conseil de la Région Ile-de-France (program S.E.S.A.M.E. 2007 Imagopole to Spencer L. Shorte) in the framework of France bioimaging (grant no. ANR-10-INSB-04-01, Investments for the Future, to Imagopole), the Fondation Française pour la Recherche Médicale (FRM, Grands Équipements program, to Nathalie Aulner), and Chemical Library projects I06-222/R and I09-1739/R to Hélène Munier-Lehmann., ANR-11-RPIB-0016,TRANSLEISH,Découverte de nouvelles protéines kinases chez Leishmania donovani à partir des inhibiteurs tête de série issus d'un criblage phénotypique(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 223414,EC:FP7:HEALTH,FP7-HEALTH-2007-B,LEISHDRUG(2008), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]
Subjects: Indoles, Leishmania donovani, Biology, Phloroglucinol, Inbred C57BL, 03 medical and health sciences, Mice, Casein Kinase I, Cricetinae, Animals, Pharmacology (medical), Experimental Therapeutics, Protein kinase A, Amastigote, Leishmaniasis, Conserved Sequence, 030304 developmental biology, Pharmacology, Visceral, 0303 health sciences, 030306 microbiology, Kinase, Macrophages, Imidazoles, Leishmania, biology.organism_classification, Isoquinolines, Trypanocidal Agents, 3. Good health, Mice, Inbred C57BL, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, Benzamides, Leishmaniasis, Visceral, Female, Casein kinase 1, Sequence Alignment, Intracellular
Abstract: Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the disease-relevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.
Published: 2014
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25. From drug screening to target deconvolution: A target-based drug discovery pipeline using Leishmania casein kinase 1 isoform 2 to identify compounds with anti-leishmanial activity

Author: Laurent Meijer, Gerald F. Späth, Alexios-Leandros Skaltsounis, Nicolas Gaboriaud-Kolar, Nathalie Aulner, Olivier Leclercq, Konstantina Vougogiannopoulou, Audrey Defontaine, Nassima Oumata, Hervé Galons, Emilie Durieu, Eric Prina, Najma Rachidi, Sandrine Ruchaud, Joo Hwan No, Phophorylation de protéines et Pathologies Humaines (P3H), Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur [Paris] (IP), Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), ManRos Therapeutics, Department of Pharmacognosy and Chemistry of Natural Products, National and Kapodistrian University of Athens (NKUA), Imagopole (CITECH), Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This study was supported by the 7th Framework Program of the European Commission through grants to the LEISHDRUG project (223414), by ANR-11-RPIB-0016 TRANSLEISH, and by the French Government's Investissements d'Avenir Program Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (grant ANR-10-LABX-62-IBEID). The Imagopole-CiTech is part of the FranceBioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, Investments for the Future) and is supported by the Conseil de la Region Ile-de-France (program Sesame 2007, project Imagopole, S. Shorte) and the Fondation Française pour la Recherche Médicale (Programme Grands Equipements [N.A.]), ANR-11-RPIB-0016,TRANSLEISH,Découverte de nouvelles protéines kinases chez Leishmania donovani à partir des inhibiteurs tête de série issus d'un criblage phénotypique(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 223414,EC:FP7:HEALTH,FP7-HEALTH-2007-B,LEISHDRUG(2008), Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Parasitologie moléculaire et Signalisation, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), National and Kapodistrian University of Athens = University of Athens (NKUA | UoA), Institut Pasteur de Corée, Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), ANR-11-RPIB-0016,TRANSLEISH,Découverte de nouvelles protéines kinases chez Leishmania donovani à partir des inhibiteurs tête de série issus d’un criblage phénotypique(2011), ANR-10-LABX-0062/10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Station biologique de Roscoff [Roscoff] (SBR), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, 030106 microbiology, Antiprotozoal Agents, Biology, Cell Line, 03 medical and health sciences, Casein Kinase I, Drug Discovery, Animals, Humans, Protein Isoforms, [CHIM]Chemical Sciences, Pharmacology (medical), Amastigote, Leishmania, Pharmacology, Kinase, Drug discovery, Intracellular parasite, Macrophages, Biological activity, biology.organism_classification, 3. Good health, 030104 developmental biology, Infectious Diseases, Biochemistry, Casein kinase 1
Abstract: Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.
Published: 2016
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26. Sequence characteristics of functional siRNAs

Author: Peter D. Kelly, Urs Rutishauser, Nathalie Aulner, Thomas U. Mayer, David Shum, Andrzej Zatorski, Bernd Jagla, Ouathek Ouerfelli, Dino A. De Angelis, Allen Volchuk, James E. Rothman, and Da Song
Subjects: Genetics, Gene knockdown, Small interfering RNA, Base Sequence, Bioinformatics, Genome, Human, Sequence Analysis, RNA, Sequence analysis, Trans-acting siRNA, Computational Biology, Gene Expression, RNA, Biology, Sense strand, RNA interference, Protein Biosynthesis, Gene Targeting, Humans, RNA Interference, RNA, Small Interfering, Molecular Biology, Genetic screen
Abstract: RNA interference in mammalian cells is actively used to conduct genetic screens, to identify and to validate targets, and to elucidate regulators and modifiers of cellular pathways. To ensure the specificity and efficacy of the active 21mer siRNA molecules, it is pertinent to develop a strategy for their rational design. Here we show that most functional siRNAs have characteristic sequence features. We tested 601 siRNAs targeting one exogenous and three endogenous genes. The efficacy of the siRNAs was determined at the protein level. Using a decision tree algorithm in combination with information analysis, our analyses revealed four sets of rules with a mean knockdown efficacy ranging from 60% to 73%. (To distinguish between percentages used to describe the quality of an siRNA and the percentages used to describe parts of data sets we underlined the former throughout this paper.) The best rule comprises an A/U at positions 10 and 19, a G/C at position 1, and more than three A/Us between positions 13 and 19, in the sense strand of the siRNA sequence. Using these rules, there is a 99.9% chance of designing an effective siRNA in a set of three with more than 50% knockdown efficiency in a biological readout.
Published: 2005
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27. The AT-Hook Protein D1 Is Essential for Drosophila melanogaster Development and Is Implicated in Position-Effect Variegation

Author: Sam Janssen, Emmanuel Käs, Caroline Monod, Nathalie Aulner, Alhousseynou Sall, Ulrich K. Laemmli, Denis Jullien, Olivier Cuvier, and Guillaume Mandicourt
Subjects: Male, X Chromosome, Heterochromatin, Molecular Sequence Data, Genes, Insect, DNA, Satellite, Gametogenesis, Retinoblastoma-like protein 1, Animals, Drosophila Proteins, Constitutive heterochromatin, Heterochromatin assembly, Molecular Biology, In Situ Hybridization, Variegation, Base Composition, Eye Color, biology, fungi, Gene Expression Regulation, Developmental, Cell Biology, Position-effect variegation, biology.organism_classification, Immunohistochemistry, DNA Dynamics and Chromosome Structure, Molecular biology, DNA-Binding Proteins, Drosophila melanogaster, DNA Transposable Elements, Female, Photoreceptor Cells, Invertebrate, Drosophila Protein
Abstract: We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm(3) AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (w(m4h)) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm(3) satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation.
Published: 2002
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28. Shigella subverts the host recycling compartment to rupture its vacuole

Author: Spencer L. Shorte, Allon Weiner, Nora Mellouk, Nathalie Aulner, Christine Schmitt, Anne Danckaert, Michael Elbaum, Jost Enninga, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Imagopole (CITECH), Institut Pasteur [Paris] (IP), Microscopie Ultrastructurale (Plate-forme), Department of Materials and Interfaces [Rehovot, Israël], Weizmann Institute of Science [Rehovot, Israël], Imagerie Dynamique (Plate-Forme) (PFID), This work was supported by the Institut Pasteur (PTR 460), by a fellowship from the Fondation pour la Recherche Médicale (FRM) to N.M., by fellowships from the Pasteur-Weizmann Council, EMBO and the FRM to A.W., and by a grant from the Region Ile de France to J.E. and G. Tran Van Nhieu (DIM-Malinf). Electron microscopy was performed in the Irving and Cherna Moscowitz Center for Nano and Bio-nano Imaging of the Weizmann Institute. J.E. and S.L.S. are members of the LabEx consortium IBEID. The Imagopole is part of the France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, 'Investments for the future'). It is further supported by the Conseil de la Region Ile-de-France (Sesame 2007, project Imagopole) and from the FRM (Programme Grands Equipements) (to N.A.). J.E. is supported by an ERC starting grant (Rupteffects, number 261166), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 261166, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]
Subjects: Cancer Research, Cytoplasm, Virulence Factors, media_common.quotation_subject, [SDV]Life Sciences [q-bio], Phosphatase, Mutant, Phosphoinositides, Vacuole, vacuolar rupture, Biology, medicine.disease_cause, Microbiology, Gene Knockout Techniques, Bacterial Proteins, Immunology and Microbiology(all), Virology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Shigella, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Internalization, Molecular Biology, Actin, media_common, Vesicle, High-content screening, Epithelial Cells, Intracellular Membranes, Endocytosis, Phosphoric Monoester Hydrolases, Cell biology, Cytosol, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, rab GTP-Binding Proteins, Host-Pathogen Interactions, Vacuoles, Parasitology, Rab-GTPase
Abstract: International audience; Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion.
Published: 2014
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29. Induction of Early Transcription in One-Cell Mouse Embryos by Microinjection of the Nonhistone Chromosomal Protein HMG-I

Author: Christine Bouniol-Baly, Caroline Monod, Nathalie Beaujean, Nathalie Aulner, Karima Kissa, Emmanuel Käs, Pascale Debey, Denis Jullien, Claudine Amirand, Institut National de la Recherche Agronomique (INRA), Laboratoire associé dynamique nucleaire et développement, ProdInra, Migration, Biologie du développement et reproduction (BDR), Centre National de la Recherche Scientifique (CNRS)-École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes (LBMCE), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), International Institute of Molecular and Cell Biology [Warsaw] (IIMCB), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], Laboratoire de physicochimie biomoléculaire et cellulaire (LPBC), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Sorbonne Paris Nord, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, Microinjections, Transcription, Genetic, Zygote, [SDV]Life Sciences [q-bio], Biology, Chorionic Gonadotropin, scaffold-associated regions, Mice, 03 medical and health sciences, 0302 clinical medicine, early embryonic transcription, Transcription (biology), AT hook, Animals, HMGA1a Protein, [SDV.BDD]Life Sciences [q-bio]/Development Biology, nuclear compartments, Microinjection, Molecular Biology, ComputingMilieux_MISCELLANEOUS, colocalization software image analysis, 030304 developmental biology, Cell Nucleus, 0303 health sciences, DNA synthesis, Ovary, High Mobility Group Proteins, DNA replication, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Embryo, Cell Biology, Cell cycle, Molecular biology, Chromatin, DNA-Binding Proteins, Mice, Inbred C57BL, [SDV] Life Sciences [q-bio], Microscopy, Fluorescence, Mice, Inbred CBA, Oocytes, Maternal to zygotic transition, Female, HMG-I, 030217 neurology & neurosurgery, Transcription Factors, Developmental Biology
Abstract: International audience; In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. We propose that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state.
Published: 2000
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30. Potent inhibitors of Huntingtin protein aggregation in a cell-based assay

Author: Maria Laura Cremona, Ai Yamamoto, Udo Többen, Dusica Vidovic, Yuli Xie, Nathalie Aulner, Donald W. Landry, Caty Chung, Lars Branden, Gangli Gong, Katherine M. Myers, Alison Rinderspacher, Stephan C. Schürer, Yidong Liu, Monique Andersen, and Shi Xian Deng
Subjects: Huntingtin, Chemistry, Pharmaceutical, High-throughput screening, Clinical Biochemistry, Drug Evaluation, Preclinical, Pharmaceutical Science, Nerve Tissue Proteins, Biochemistry, Article, Inhibitory Concentration 50, Structure-Activity Relationship, chemistry.chemical_compound, Drug Discovery, Quinazoline, Huntingtin Protein, Combinatorial Chemistry Techniques, Humans, Structure–activity relationship, Nuclear protein, Molecular Biology, Molecular Structure, Drug discovery, Chemistry, Organic Chemistry, Nuclear Proteins, Small molecule, Models, Chemical, Drug Design, Quinazolines, Molecular Medicine, Peptides
Abstract: A quinazoline that decreases polyglutamine aggregate burden in a cell-based assay was identified from a high-throughput screen of a chemical-compound library, provided by the NIH Molecular Libraries Small Molecule Repository (MLSMR). A structure and activity study yielded leads with submicromolar potency.
Published: 2009
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31. The Drosophila poly(A)-binding protein II is ubiquitous throughout Drosophila development and has the same function in mRNA polyadenylation as its bovine homolog in vitro

Author: Uwe Kühn, Nathalie Aulner, Henri-Marc Bourbon, Béatrice Benoit, Elmar Wahle, Annette Nemeth, and Martine Simonelig
Subjects: DNA, Complementary, Polyadenylation, Blotting, Western, Molecular Sequence Data, Gene Expression, RNA-binding protein, Biology, Poly(A)-Binding Proteins, Mice, MRNA polyadenylation, Poly(A)-binding protein, Genetics, Animals, Humans, Amino Acid Sequence, RNA, Messenger, Cloning, Molecular, Peptide sequence, Base Sequence, Sequence Homology, Amino Acid, RNA-Binding Proteins, biology.organism_classification, Molecular biology, Poly(A)-Binding Protein II, Cell biology, Drosophila melanogaster, biology.protein, Insect Proteins, Cattle, Rabbits, Poly A, Drosophila Protein, Research Article
Abstract: The poly(A)-binding protein II (PABP2) is one of the polyadenylation factors required for proper 3'-end formation of mammalian mRNAs. We have cloned Pabp2, the gene encoding the Drosophila homolog of mammalian PABP2, by using a molecular screen to identify new Drosophila proteins with RNP-type RNA-binding domains. Sequence comparison of PABP2 from Drosophila and mammals indicates that the most conserved domains are the RNA-binding domain and a coiled-coil like domain which could be involved in protein-protein interactions. Pabp2 produces four mRNAs which result from utilization of alternative poly(A) sites and encode the same protein. Using an antibody raised against Drosophila PABP2, we show that the protein accumulates in nuclei of all transcriptionally active cells throughout Drosophila development. This is consistent with a general role of PABP2 in mRNA polyadenylation. Analysis of Drosophila PABP2 function in a reconstituted mammalian polyadenylation system shows that the protein has the same functions as its bovine homolog in vitro : it stimulates poly(A) polymerase and is able to control poly(A) tail length.
Published: 1999
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32. The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors

Author: Nadine Tarantino, Nathalie Aulner, Sara Ortica, Neetu Gupta-Rossi, Alain Israël, Signalisation Moléculaire et Activation Cellulaire (SMAC), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Imagopole (CITECH), Institut Pasteur [Paris], The Imagopole was sponsored by the Soutien aux Equipes Scientifiques pour l'Acquisition de Moyens Expérimentaux (SESAME) 2007 program, Conseil de la Région Ile‐de‐France, and by a grant from the Fondation pour la Recherche Médicale (FRM). S.O. was supported by grants from the Domaine d'Intérêt Majeur (DIM)‐Stem Cells and Cell Therapy (STEM)‐Pôle (Ile‐de‐France) and the FRM. Notch2 (Artavanis‐Tsakonas) antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the U.S. National Institute of Child Health and Human Development and maintained by the University of Iowa (Iowa City, IA, USA)., The authors thank Mary Weiss and Hélène Strick‐Marchand (Institut Pasteur, Paris, France) for the kind gift of bipotential mouse embryonic liver cells and helpful discussions. The authors are grateful to M. Bocchetta (Loyola University Medical Center, Chicago, IL, USA), I. Screpanti (Sapienza University of Rome, Rome, Italy), J. Kitajewski (Columbia University Medical Center, New York, NY, USA), Y. Jacob and E. Birlouez (Institut Pasteur, Paris, France), and M. X. Ilagan (Washington University School of Medicine, St. Louis, MO, USA) for constructs. The authors thank S. Cairo, P. Falanga, P. Mourikis, S. Yennek, F. Logeat, D. Ndiaye, and D. Kachaner (Institut Pasteur) for materials and advice. The authors especially thank P. H. Commere and A. Danckaert (Imagopole, Institut Pasteur). The authors acknowledge the Imagopole‐Plateforme d'Imagerie Dynamique (PFID) France–BioImaging infrastructure, supported by the French National Research Agency (ANR 10‐INSB‐04‐01, Investments for the Future), for advice and access to the Opera system., Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)
Subjects: MESH: Liver / metabolism, Notch signaling pathway, Fluorescent Antibody Technique, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Real-Time Polymerase Chain Reaction, Biochemistry, MESH: Liver / cytology, Cell Line, Mice, MESH: Receptors, Notch / genetics, hepatoblasts, MESH: Hepatocytes / cytology, MESH: Cell Proliferation, Genetics, Animals, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Progenitor cell, paralogs, Receptor, Molecular Biology, MESH: Mice, MESH: Fluorescent Antibody Technique, polyploidy, Cell Proliferation, Receptors, Notch, Cell growth, Chemistry, Kinase, MESH: Real-Time Polymerase Chain Reaction, Stem Cells, Regeneration (biology), MESH: Receptors, Notch / metabolism, bipotential stem cells, Cell Differentiation, Anatomy, MESH: Cell Differentiation / physiology, Embryonic stem cell, MESH: Cell Differentiation / genetics, Cell biology, MESH: Cell Line, Liver, Apoptosis, MESH: Stem Cells / metabolism, Hepatocytes, MESH: Stem Cells / cytology, MESH: Hepatocytes / metabolism, CDK inhibitors, Biotechnology
Abstract: International audience; The Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ-secretase inhibitor, at 50 μM for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors.
Published: 2013
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33. HIV-1 Nef Inhibits Ruffles, Induces Filopodia, and Modulates Migration of Infected Lymphocytes

Author: Christophe Machu, Dominika Rudnicka, Olivier Renaud, Cinzia Nobile, Françoise Porrot, Claire Hivroz, Nathalie Aulner, Milena Hasan, Nathalie Sol-Foulon, Marie-Christine Prévost, Olivier Schwartz, Virus et Immunité, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Immunité et cancer ( U932 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut Curie-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Centre d'immunologie humaine ( CIH ), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Imagopole ( CITECH ), Institut Pasteur [Paris], Génétique du Développement et Cancer, Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -INSTITUT CURIE-Centre National de la Recherche Scientifique ( CNRS ), Microscopie électronique (Plate-forme), program Sesame 2007, project Imagopde, Spencer Shorte FP6 contract, INTRAPATH program, ANR : FP7 contract 201412,FP7 contract 201412, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Immunité et cancer (U932), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d'immunologie humaine (CIH), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagopole (CITECH), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), FP7 contract 201412, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie-Centre National de la Recherche Scientifique (CNRS), ANR: FP7 contract 201412,FP7 contract 201412, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Chemokine, [ SDV.MP.PAR ] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Immunology, Motility, HIV Infections, Biology, Microbiology, Flow cytometry, 03 medical and health sciences, Imaging, Three-Dimensional, 0302 clinical medicine, Cell Movement, Virology, medicine, Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Lymphocytes, Pseudopodia, nef Gene Products, Human Immunodeficiency Virus, Cytoskeleton, Cell Shape, 030304 developmental biology, 0303 health sciences, medicine.diagnostic_test, virus diseases, Cell migration, Flow Cytometry, Actin cytoskeleton, Virus-Cell Interactions, 3. Good health, Cell biology, 030220 oncology & carcinogenesis, Insect Science, HIV-1, biology.protein, Cell Surface Extensions, Filopodia
Abstract: The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV withnefdeleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.
Published: 2010
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34. Cell-based assays to probe the ERK MAP kinase pathway in endothelial cells

Author: Michael R, Wyler, Deborah H, Smith, Eftihia, Cayanis, Udo, Többen, Nathalie, Aulner, and Thomas, Mayer
Subjects: Mitogen-Activated Protein Kinase 1, Umbilical Veins, Mitogen-Activated Protein Kinase 3, Humans, Tetradecanoylphorbol Acetate, Biological Assay, Endothelium, Vascular, Phosphorylation, Cells, Cultured, Protein Kinase C, Signal Transduction
Abstract: To understand signaling pathways in mammalian cells, cell-based assays are relatively new and extremely powerful tools. We have developed a battery of phenotypic assays to study signaling; two of them are described in detail in this chapter. A subset of these assays monitors mitogen-activated protein (MAP) kinase pathways. MAP kinases are principal regulators of fundamental processes in mammalian cells, including growth, cell division, differentiation, stress responses, and neoplastic transformation. Here we describe two cell-based assays querying the function of ERK (extracellular signal regulated kinase), one of the three principal MAP kinases in mammalian cells. We selected human umbilical vein endothelial cells (HUVECs), a primary cell type, because they show a very dynamic response to various activators. Both assays are phenotypic assays and use well-established phosphorylation-specific primary antibodies to study activation. Fluorochrome-coupled secondary antibodies were used to label phosphorylated target proteins; images were captured with the INCell Analyzer 3000 and analyzed with the INCell Analyzer 3000 software. The first of these two assays monitors phosphorylation of ERK1/2, while the second assay monitors activation of the transcription factor CREB (cAMP response element-binding protein). The assays described in this chapter cover major checkpoints of the ERK signaling pathway: (1) MAP kinase activation and (2) subsequent transcription factor activation. Both assays exhibit robust performance and can easily be used for high-throughput screening.
Published: 2009

35. Cell-Based Assays to Probe the ERK MAP Kinase Pathway in Endothelial Cells

Author: Thomas U. Mayer, Michael Wyler, Udo Többen, Deborah H. Smith, Nathalie Aulner, and Eftihia Cayanis
Subjects: MAPK/ERK pathway, MAP kinase kinase kinase, biology, Akt/PKB signaling pathway, Chemistry, Mitogen-activated protein kinase, biology.protein, Neoplastic transformation, Cyclin-dependent kinase 9, Signal transduction, Mitogen-activated protein kinase kinase, Cell biology
Abstract: To understand signaling pathways in mammalian cells, cell-based assays are relatively new and extremely powerful tools. We have developed a battery of phenotypic assays to study signaling; two of them are described in detail in this chapter. A subset of these assays monitors mitogen-activated protein (MAP) kinase pathways. MAP kinases are principal regulators of fundamental processes in mammalian cells, including growth, cell division, differentiation, stress responses, and neoplastic transformation. Here we describe two cell-based assays querying the function of ERK (extracellular signal regulated kinase), one of the three principal MAP kinases in mammalian cells. We selected human umbilical vein endothelial cells (HUVECs), a primary cell type, because they show a very dynamic response to various activators. Both assays are phenotypic assays and use well-established phosphorylation-specific primary antibodies to study activation. Fluorochrome-coupled secondary antibodies were used to label phosphorylated target proteins; images were captured with the INCell Analyzer 3000 and analyzed with the INCell Analyzer 3000 software. The first of these two assays monitors phosphorylation of ERK1/2, while the second assay monitors activation of the transcription factor CREB (cAMP response element-binding protein). The assays described in this chapter cover major checkpoints of the ERK signaling pathway: (1) MAP kinase activation and (2) subsequent transcription factor activation. Both assays exhibit robust performance and can easily be used for high-throughput screening.
Published: 2009
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36. Intracellular bacteria encode inhibitory SNARE-like proteins

Author: Jordan Wesolowski, Agathe Subtil, James E. Rothman, Howard A. Shuman, Alejandro Garcia-Diaz, Cédric Delevoye, Fabienne Paumet, Nathalie Aulner, Departement of Microbiology and Immunology, Thomas Jefferson University, Department of Cell Biology [New Haven], Yale School of Medicine [New Haven, Connecticut] (YSM)-Howard Hughes Medical Institute (HHMI), Structure et compartimentation membranaire, Compartimentation et dynamique cellulaires (CDC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Imagopole (CITECH), Institut Pasteur [Paris] (IP), Department of Microbiology, Columbia University Irving Medical Center (CUIMC), Biologie des Interactions Cellulaires, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Yale University School of Medicine-Howard Hughes Medical Institute (HHMI), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Endocytic cycle, lcsh:Medicine, Legionella, Biology, Biochemistry, Models, Biological, Cell Line, Infectious Diseases/Bacterial Infections, 03 medical and health sciences, Cell Biology/Membranes and Sorting, Bacterial Proteins, Animals, Immunology/Cellular Microbiology and Pathogenesis, Chlamydia, lcsh:Science, Structural motif, 030304 developmental biology, Phagosome, Glycoproteins, 0303 health sciences, Multidisciplinary, Cell fusion, Microscopy, Confocal, Bacteria, 030306 microbiology, Intracellular parasite, lcsh:R, Lipid bilayer fusion, Biological Transport, Phosphoproteins, Endocytosis, Cell biology, Rats, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Membrane protein, Liposomes, lcsh:Q, Microbiology/Cellular Microbiology and Pathogenesis, SNARE Proteins, Intracellular, Research Article
Abstract: International audience; Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.
Published: 2009
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37. Cell‐Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation

Author: Peter D. Kelly, Geoffrey Barger, Bernd Jagla, Nathalie Aulner, Thomas U. Mayer, Deborah H. Smith, Michael Wyler, Matthew Beard, Lars Branden, James E. Rothman, and Udo Többen
Subjects: Drug discovery, Cell, Inflammation, Biology, Phenotype, Molecular biology, Cell biology, medicine.anatomical_structure, Cytoplasm, medicine, Tumor necrosis factor alpha, medicine.symptom, Signal transduction, Transcription factor
Abstract: Cell‐based assays are powerful tools for drug discovery and provide insight into complex signal transduction pathways in higher eukaryotic cells. Information gleaned from assays that monitor a cellular phenotype can be used to elucidate the details of a single pathway and to establish patterns of cross talk between pathways. By selecting the appropriate cell model, cell‐based assays can be used to understand the function of a specific cell type in a complex disease process such as inflammation. We have used human umbilical vein endothelial cells to establish three cell‐based, phenotypic assays that query different stages of a major signaling pathway activated in inflammation. One assay analyzes the tumor necrosis factor α (TNFα)‐induced translocation of the transcription factor NF‐ κ B from the cytoplasm into the nucleus 20 min after stimulation with TNFα. Two more assays monitor the expression of E‐selectin and VCAM‐1, 4 and 24 h after stimulation with TNFα. Indirect immunofluorescence and high‐throughput automated microscopy were used to analyze cells. Imaging was performed with the IN Cell Analyzer 3000. All assays proved to be highly robust. Z ′ values between 0.7 and 0.8 make each of the three assays well suited for use in high‐throughput screening for drug or probe discovery.
Published: 2006
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38. Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene

Author: Maria Korom, Andrew Martens, Nathalie Aulner, Dale Dorsett, and Robert A. Rollins
Subjects: Male, Transcriptional Activation, Saccharomyces cerevisiae Proteins, Chromosomal Proteins, Non-Histone, Cell Cycle Proteins, Nerve Tissue Proteins, Biology, SMC1A, Chromatids, ESCO2, Fungal Proteins, Animals, Drosophila Proteins, Wings, Animal, Molecular Biology, Cohesin loading, Genetics, Transcriptional Regulation, Cell Nucleus, Homeodomain Proteins, Cohesin, Nuclear Proteins, NIPBL, Cell Biology, Phosphoproteins, Establishment of sister chromatid cohesion, DNA-Binding Proteins, Drosophila melanogaster, Enhancer Elements, Genetic, Phenotype, Chromatid, Female, RNA Interference, Separase, biological phenomena, cell phenomena, and immunity, Transcription Factors
Abstract: The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication.
Published: 2004

39. Modification of position-effect variegation by competition for binding to Drosophila satellites

Author: Caroline Monod, Emmanuel Käs, Olivier Cuvier, and Nathalie Aulner
Subjects: Heterochromatin, Blotting, Western, Chromosomal translocation, Biology, DNA, Satellite, Biochemistry, chemistry.chemical_compound, Genetics, Animals, Deoxyribonuclease I, Transgenes, Binding site, Molecular Biology, Pericentric heterochromatin, Crosses, Genetic, Variegation, Binding Sites, Eye Color, fungi, Scientific Reports, DNA, Position-effect variegation, Recombinant Proteins, Cell biology, chemistry, Microscopy, Fluorescence, Drosophila, Electrophoresis, Polyacrylamide Gel
Abstract: White-mottled (w(m4)) position-effect variegation (PEV) arises by translocation of the white gene near the pericentric AT-rich 1.688 g/cm3 satellite III (SATIII) repeats of the X chromosome of Drosophila. The natural and artificial A*T-hook proteins D1 and MATH20 modify w(m4) PEV in opposite ways. D1 binds SATIII repeats and enhances PEV, presumably via a recruitment of protein partners, whereas MATH20 suppresses it. We show that D1 and MATH20 compete for binding to identical sites of SATIII repeats in vitro and that conditional MATH20 expression results in a displacement of D1 from pericentric heterochromatin in vivo. In the presence of intermediate levels of MATH20, we show that this displacement becomes selective for SATIII repeats. These results strongly suggest that the suppression of w(m4) PEV by MATH20 is due to a displacement of D1 from its preferred binding sites and provide additional support for a direct role of D1 in the assembly of AT-rich heterochromatin.
Published: 2002

40. A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape

Author: Damien Balestrino, Pascale Cossart, Jost Enninga, Javier Pizarro-Cerdá, Anne Danckaert, Alexandre Bobard, Juan J. Quereda, Spencer L. Shorte, Nathalie Aulner, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Imagopole (CITECH), Institut Pasteur [Paris] (IP), This work was supported by the Institut Pasteur (PTR460 grant to J.P.-C. and J.E.), Institut National de la Santé et de la Recherche Médicale (Unité 604), Institut National de la Recherche Agronomique (Unité Sous contract 2020), Fondation Les Mousquetaires, and European Research Council Advanced Grant (233348 MODELIST and 670823 BacCellEpi). The Imagopole is part of the France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, Investments for the Future) and received additional support from the Conseil de la Region Ile-de-France (program Sesame 2007, project Imagopole [S.S.]) and from the Fondation Française pour la Recherche Médicale (FRM) Programme Grands Equipements [N.A.]). P.C. is a Howard Hughes Medical Institute Senior International Research Scholar. J.E. is supported by an ERC starting grant (Rupteffects, 261166) and by the Institut Pasteur Carnot-MIE program. P.C. and J.E. acknowledge support from LabEx IBEID, ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 261166,EC:FP7:ERC,ERC-2010-StG_20091118,RUPTEFFECTS(2011), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), European Project: 233348,EC:FP7:ERC,ERC-2008-AdG,MODELIST(2009), vicente, marie-therese, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Revealing the mechanism of host membrane rupture by invasive pathogens and its role in triggering the immune response - RUPTEFFECTS - - EC:FP7:ERC2011-03-01 - 2016-02-29 - 261166 - VALID, Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID, Understanding the infection by the bacterium Listeria monocytogenes as a way to address key issues in biology - MODELIST - - EC:FP7:ERC2009-06-01 - 2015-05-31 - 233348 - VALID, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Dynamique des Interactions Hôte-Pathogène, ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), and ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010)
Subjects: 0301 basic medicine, Cytoplasm, Listeria, mammalian-cells, real-time, Vacuole, dynamic interplay, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, medicine.disease_cause, Applied Microbiology and Biotechnology, Heterocyclic Compounds, 4 or More Rings, Bacterial cell structure, beta-Lactamases, Microbiology, 03 medical and health sciences, Listeria monocytogenes, Bacterial Proteins, medicine, Methods, Fluorescence Resonance Energy Transfer, Humans, host-pathogen interactions, bacterial, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Ecology, biology, Intracellular parasite, biology.organism_classification, infection, 030104 developmental biology, Microscopy, Fluorescence, Host cell cytoplasm, Vacuoles, protein, Intracellular, Food Science, Biotechnology, HeLa Cells
Abstract: Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua , thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes .
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41. The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants

Author: Stacy Gellenoncourt, Nell Saunders, Rémy Robinot, Lucas Auguste, Maaran Michael Rajah, Jerôme Kervevan, Raphaël Jeger-Madiot, Isabelle Staropoli, Cyril Planchais, Hugo Mouquet, Julian Buchrieser, Olivier Schwartz, Lisa A. Chakrabarti, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université de Nîmes (UNIMES), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), This work was supported by the Urgence COVID-19 Fundraising Campaign of Institut Pasteur (COROCHIP, PFR4, and PFR7 projects) and Agence Nationale de Recherche sur le SIDA et les Maladies Infectieuses Emergentes (ANRS-MIE, project 19206) (L.A.C.) and by the Pasteur Institute, the European Health Emergency Preparedness and Response Authority (HERA), Urgence COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche Médicale (FRM), ANRS-MIE, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2, ANR Coronamito, and IDISCOVR (O.S.). S.G. is the recipient of an MESR/Ecole Doctorale BioSPC, Université Paris Cité, fellowship, R.R. is the recipient of a Sidaction fellowship, and J.K. is the recipient of an ANRS fellowship, We thank Nathalie Aulner and the UTechS Photonic BioImaging (UPBI) core facility of Institut Pasteur, a member of the France BioImaging network, for training on the Opera imaging system. We thank Jacob Seeler for help with gel image acquisition and all the members of the Virus and Immunity Unit for reagents and helpful discussions. The following reagent was obtained through BEI Resources, NIAID, NIH: SARS-related coronavirus 2, Wuhan-Hu-1 spike-pseudotyped lentiviral kit V2, NR-53816., ANR-21-CO14-0007,CoronaMito,Conséquences de l'infection par le SRAS-CoV-2 sur la fonction mitochondriale(2021), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020)
Subjects: epistasis, variants, MESH: Humans, MESH: Mutation, SARS-CoV-2, [SDV]Life Sciences [q-bio], Immunology, COVID-19, proteolytic cleavage, MESH: Spike Glycoprotein, Coronavirus, spike, Microbiology, Virology, Insect Science, Mutation, Spike Glycoprotein, Coronavirus, Humans, MESH: COVID-19, MESH: SARS-CoV-2
Abstract: SARS-CoV-2 remained genetically stable during the first three months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide, and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes.AUTHOR SUMMARYThe successive emergence of SARS-CoV-2 variants is fueling the COVID pandemic, thus causing a major and persistent public health issue. The parameters involved in the emergence of variants with higher pathogenic potential remain incompletely understood. The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions, and resulting in viral particles with higher infectious capacity. The Alpha and the Delta variants that spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic when compared to the original SARS-CoV-2 strain. Interestingly, Alpha and Delta both carried mutations in a spike cleavage site that needs to be processed by cellular proteases prior to viral entry. The cleavage site mutations P681H/R made the Alpha and Delta spikes more efficient at viral fusion, by generating a higher fraction of cleaved spikes subunits S1 and S2. We show here that the early D614G mutation and the late P681H/R mutations act synergistically to increase the fusion capacity of SARS-CoV-2 variants. Specifically, viruses with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite to the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.
Published: 2022
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42. Translocated Legionella pneumophila small RNAs mimic eukaryotic microRNAs targeting the host immune response

Author: Tobias Sahr, Pedro Escoll, Christophe Rusniok, Sheryl Bui, Gérard Pehau-Arnaudet, Gregory Lavieu, Carmen Buchrieser, Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Matière et Systèmes Complexes (MSC), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), Work in the C.B. laboratory was financed by the grant n° ANR-20-CE15-0021-01-BIOEV, the Institut Pasteur, the Fondation pour la Recherche Médicale (FRM) grant N° EQU201903007847 and the grant n°ANR-10-LABX-62-IBEID. For electron microscopy experiments M. Nilges and the Equipex CACSICE (n° ANR-11-EQPX-0008) provided funding for the Falcon II direct detector. G.L. lab is funded by ANR-20-CE15-0021-02, ANR-19-CE18-0020-03, and ANR-18-IDEX-0001, IdEx Université de Paris. S.B. is a recipient of a PhD fellowship from PSL University. The funders, other than the authors, did not play any role in the study or in the preparation of the article or decision to publish., We thank Monica Rolando for critically reading of the manuscript and helpful discussions and Nathalie Aulner and Anne Danckaert, Photonic BioImaging (PBI) UTechS for support., T.S., P.E., S.B., and G.L. performed the experiments, C.R. performed the bioinformatics analyses, GPA performed the electron and cryoTEM experiments, T.S., P.E., and C.B. designed the experiments, T.S. and C.B. wrote the article, C.B. supervised the work and acquired funds., ANR-20-CE15-0021,BioEV,Biogenèse, trafic et rôles des vésicules extracllulaires de Legionella pneumophila au cours de l'infection(2020), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), ANR-19-CE18-0020,EXCELLDISC,Vésicules Extracellulaires et thérapie du disque intervertébral(2019), and ANR-18-IDEX-0001,Université de Paris,Université de Paris(2018)
Subjects: MESH: Signal Transduction, Science, General Physics and Astronomy, MESH: Legionnaires' Disease, MESH: DEAD Box Protein 58, General Biochemistry, Genetics and Molecular Biology, Cell Line, Legionella pneumophila, MESH: Interleukin-1 Receptor-Associated Kinases, Extracellular Vesicles, Bacterial Proteins, Humans, Receptors, Immunologic, MESH: Extracellular Vesicles, MESH: Bacterial Proteins, MESH: Receptors, Immunologic, MESH: Humans, Multidisciplinary, MESH: Host-Pathogen Interactions, Eukaryota, General Chemistry, bacterial infections and mycoses, MESH: Legionella pneumophila, Immunity, Innate, MESH: Cell Line, respiratory tract diseases, MicroRNAs, MESH: Eukaryota, Interleukin-1 Receptor-Associated Kinases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Host-Pathogen Interactions, DEAD Box Protein 58, bacteria, MESH: Immunity, Innate, Legionnaires' Disease, MESH: MicroRNAs, Signal Transduction
Abstract: Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe form of pneumonia in humans, a phenotype evolved through interactions with aquatic protozoa in the environment. Here, we show that L. pneumophila uses extracellular vesicles to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways. The bacterial sRNA RsmY binds to the UTR of ddx58 (RIG-I encoding gene) and cRel, while tRNA-Phe binds ddx58 and irak1 collectively reducing expression of RIG-I, IRAK1 and cRel, with subsequent downregulation of IFN-β. Thus, RsmY and tRNA-Phe are bacterial trans-kingdom regulatory RNAs downregulating selected sensor and regulator proteins of the host cell innate immune response. This miRNA-like regulation of the expression of key sensors and regulators of immunity is a feature of L. pneumophila host-pathogen communication and likely represents a general mechanism employed by bacteria that interact with eukaryotic hosts.
Published: 2022
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43. Release of infectious virus and cytokines in nasopharyngeal swabs from individuals infected with non-alpha or alpha SARS-CoV-2 variants: an observational retrospective study

Author: Monel, Blandine, Planas, Delphine, Grzelak, Ludivine, Smith, Nikaïa, Robillard, Nicolas, Staropoli, Isabelle, Goncalves, Pedro, Porrot, Françoise, Guivel-Benhassine, Florence, Guinet, Nathalie Demory, Rodary, Julien, Puech, Julien, Euzen, Victor, Bélec, Laurent, Orvoen, Galdric, Nunes, Léa, Moulin, Véronique, Fourgeaud, Jacques, Wack, Maxime, Imbeaud, Sandrine, Campagne, Pascal, Duffy, Darragh, Di Santo, James, Bruel, Timothée, Péré, Hélène, Veyer, David, Schwartz, Olivier, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Université Paris Cité (UPCité), Immunologie Translationnelle - Translational Immunology lab, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Immunité Innée - Innate Immunity, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Paris-Centre de Recherche Cardiovasculaire (PARCC (UMR_S 970/ U970)), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Hôpital Vaugirard-Gabriel Pallez, Hôpital Corentin Celton [Issy-les-Moulineaux], Laboratoire de Virologie [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Work in OS lab is funded by Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR, Fondation pour la Recherche Médicale. DP is supported by the Vaccine Research Institute. LG is supported by the French Ministry of Higher Education, Research and Innovation. DD and JDS are funded by the ANR-20-COVI-0053 grant., We thank Maaran Michael Rajah, Nicoletta Casartelli, Michael White and Jeremy Dufloo for discussion and critical reading of the manuscript. We thank AGEPS, Assistance Publique - Hôpitaux de Paris, for providing the RDTs. We thank the patients who participated in the study, the clinical staff involved in their management, Théo Hirsch for stimulating discussions. We thank members of the Virus and Immunity Unit for discussion and help, Nathalie Aulner and the UtechS Photonic BioImaging (UPBI) core facility (Institut Pasteur), a member of the France BioImaging network, for image acquisition and analysis, and Cyril Planchais & Hugo Mouquet for the kind gift of mAb48., ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020), ANR-20-COVI-0053,CoVarImm,Variation de la réponse immune systémique et muqueuse pendant l'infection par le SRAS-CoV-2 et la convalescence(2020), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Vaccine Research Institute (VRI), Institut Pasteur [Paris]-Université Paris Cité (UPCité), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), École pratique des hautes études (EPHE), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP)
Subjects: Adult, Male, Nasal swabs, Medicine (General), variants, Research paper, SARS-CoV-2, COVID-19, Middle Aged, Antibodies, Viral, Immunoglobulin A, infectious particles, R5-920, Immunoglobulin G, Nasopharynx, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Medicine, Cytokines, Humans, Female, Aged, Retrospective Studies
Abstract: Background: The dynamics of SARS-CoV-2 alpha variant shedding and immune responses at the nasal mucosa remain poorly characterised. Methods: We measured infectious viral release, antibodies and cytokines in 426 PCR+ nasopharyngeal swabs from individuals harboring non-alpha or alpha variants. Findings: With both lineages, viral titers were variable, ranging from 0 to >106 infectious units. Rapid antigenic diagnostic tests were positive in 94% of samples with infectious virus. 68 % of individuals carried infectious virus within two days after onset of symptoms. This proportion decreased overtime. Viable virus was detected up to 14 days. Samples containing anti-spike IgG or IgA did not generally harbor infectious virus. Ct values were slightly but not significantly lower with alpha. This variant was characterized by a fast decrease of infectivity overtime and a marked release of 13 cytokines (including IFN-b, IP-10 and IL-10). Interpretation: The alpha variant displays modified viral decay and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection. Funding: This retrospective study has been funded by Institut Pasteur, ANRS, Vaccine Research Institute, Labex IBEID, ANR/FRM and IDISCOVR, Fondation pour la Recherche Médicale.
Published: 2021
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44. SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation

Author: Mathieu Hubert, Jérémy Dufloo, Rémy Robinot, Hugo Mouquet, Olivier Schwartz, Ludivine Grzelak, Nell Saunders, Elodie Bishop, Françoise Porrot, Delphine Planas, Maaran Michael Rajah, Julian Buchrieser, Lisa A. Chakrabarti, Marija Zivaljic, Alice Bongers, Stacy Gellenoncourt, Florence Guivel-Benhassine, Cyril Planchais, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Vaccine Research Institute (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Sorbonne Université (SU), Neurobiologie intégrative des Systèmes cholinergiques / Integrative Neurobiology of Cholinergic Systems (NISC), Institut Pasteur [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Ecole doctorale Cerveau Cognition et Comportement [Paris] (ED 158 - 3C), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Work in OS lab is funded by Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Fondation Pour la Recherche Médicale (FRM), Labex IBEID (ANR-10-LABX62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2, ANR CoronaMito AAP RA-COVID-19 V14, and IDISCOVR. Work in UPBI is funded by grant ANR-10-INSB-04-01 and Région Ile-de-France program DIM1-Health. MMR and MZ are supported by the Pasteur-Paris University (PPU) International Doctoral Program. MMR is also supported by Institut Pasteur Department of Virology 'Bourse de Soudure' fellowship. DP is supported by the Vaccine Research Institute. LG is supported by the French Ministry of Higher Education, Research and Innovation. EB is supported by the Medecine-Sciences ENS-PSL Program. HM laboratory is funded by the Institut Pasteur, the Milieu Intérieur Program (ANR-10-LABX-69- 01), the INSERM, REACTing, EU (RECOVER), and Fondation de France (#00106077) grants., We thank members of the Virus and Immunity Unit for helpful discussions, Dr. Nicoletta Casartelli for her critical reading of the manuscript, and Nathalie Aulner and the UtechS Photonic BioImaging (UPBI) core facility (Institut Pasteur), a member of the France BioImaging network, for image acquisition and analysis support., ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020), ANR-21-CO14-0007,CoronaMito,Conséquences de l'infection par le SRAS-CoV-2 sur la fonction mitochondriale(2021), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Vaccine Research Institute [Créteil, France] (VRI), Institut Pasteur [Paris] (IP)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ziani, Isma, Laboratoires d'excellence - Initiative for the creation of a Vaccine Research Institute - - VRI2010 - ANR-10-LABX-0077 - LABX - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Protéomique du SARS-CoV-2 - - PROTEO-SARS-CoV-22020 - ANR-20-COVI-0059 - COVID-19 - VALID, Conséquences de l'infection par le SRAS-CoV-2 sur la fonction mitochondriale - - CoronaMito2021 - ANR-21-CO14-0007 - COVID-19 - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, and Laboratoires d'excellence - GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE - - MILIEU INTERIEUR2010 - ANR-10-LABX-0069 - LABX - VALID
Subjects: fusion, coronavirus, MESH: Spike Glycoprotein, Coronavirus, MESH: Angiotensin-Converting Enzyme 2, medicine.disease_cause, Virus Replication, Giant Cells, SARS‐CoV‐2, MESH: Antibodies, Monoclonal, MESH: Giant Cells, MESH: Chlorocebus aethiops, Chlorocebus aethiops, MESH: Animals, Receptor, Coronavirus, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Syncytium, Strain (chemistry), General Neuroscience, Antibodies, Monoclonal, Articles, Transmembrane protein, Microbiology, Virology & Host Pathogen Interaction, Cell biology, MESH: HEK293 Cells, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Spike Glycoprotein, Coronavirus, MESH: Caco-2 Cells, Angiotensin-Converting Enzyme 2, MESH: Mutation, medicine.drug_class, Immunology, Alpha (ethology), MESH: Vero Cells, Biology, Monoclonal antibody, General Biochemistry, Genetics and Molecular Biology, Article, Cell Line, medicine, Animals, Humans, MESH: SARS-CoV-2, Beta (finance), syncytia, Molecular Biology, Vero Cells, MESH: Humans, General Immunology and Microbiology, SARS-CoV-2, MESH: Virus Replication, spike, MESH: Cell Line, HEK293 Cells, Mutation, Caco-2 Cells
Abstract: Severe COVID‐19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS‐CoV‐2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco‐2, Calu‐3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon‐induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS‐CoV‐2 emerging variants display enhanced syncytia formation., Spike protein mutations expressed by emerging SARS‐CoV‐2 variants‐of‐concern differentially affect host cell‐to‐cell fusion, ACE2 receptor binding, and antibody escape.
Published: 2021
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45. High-throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

Author: Cretin, Emma, Lopes, Priscilla, Vimont, Elodie, Tatsuta, Takashi, Langer, Thomas, Gazi, Anastasia, Sachse, Martin, Yu‐Wai‐Man, Patrick, Reynier, Pascal, Wai, Timothy, Cretin, Emma [0000-0002-8423-233X], Tatsuta, Takashi [0000-0003-0003-0587], Langer, Thomas [0000-0003-1250-1462], Gazi, Anastasia [0000-0002-2922-3625], Sachse, Martin [0000-0001-5981-9166], Yu‐Wai‐Man, Patrick [0000-0001-7847-9320], Reynier, Pascal [0000-0003-0802-4608], Wai, Timothy [0000-0002-6770-6222], Apollo - University of Cambridge Repository, Biologie mitochondriale – Mitochondrial biology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Max planck Institute for Biology of Ageing [Cologne], Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), University of Cambridge [UK] (CAM), Moorfields Eye Hospital [London], UCL-Institute of Ophthalmology, University College of London [London] (UCL), MitoVasc - Physiopathologie Cardiovasculaire et Mitochondriale (MITOVASC), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM), T.W. is supported by the European Research Council (ERC) Starting Grant No. 714472 (Acronym 'Mitomorphosis') and ATIP-AVENIR (INSERM/CNRS). E.C. is supported by a PhD scholarship from the French Ministry of Higher Education, Research, and Innovation (Ministère français de lʼEnseignement supérieur, de la Recherche et de lʼInnovation). T.L. was supported by funds of the German Research Council (CRC1218, project number 269925409. P.YWM. is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (UK) and also receives funding from Fight for Sight (UK), Moorfields Eye Charity, the Isaac Newton Trust (UK), the Addenbrooke's Charitable Trust, the National Eye Research Centre (UK), the International Foundation for Optic Nerve Disease (IFOND), the UK National Institute of Health Research (NIHR) as part of the Rare Diseases Translational Research Collaboration, the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014), and the NIHR Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health., We thank Kristin Tsuo and Vincent Guillemot for statistical assistance in R, Etienne Kornobis for Illumina sequencing, and Pierre-Henri Commere and Sandrine Schmutz for flow cytometry services at the Institut Pasteur. Imaging on the Opera Phenix, funded by the Région Ile-de-France program DIM1-Health, was facilitated by Nathalie Aulner. We thank Sylvie Fabrega of the Viral Vector for Gene Transfer core facility of Structure Fédérative de Recherche Necker, Université de Paris for lentiviral particle synthesis. We thank Michael Ryan for providing MEFs lacking MiD49/MiD51/Mff, Nils-Göran Larsson for providing mitoYFP mice, and Guangwei Du for plasmids. We thank Arnaud Echard for critical reading of the manuscript and Marie Lemesle for excellent administrative assistance., European Project: 714472,ERC-2016-STG,Mitomorphosis(2017), Yu-Wai-Man, Patrick [0000-0001-7847-9320], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université de Paris (UP), Institut Pasteur [Paris], Faculty of Brain Sciences of University College London (UCL), and Physiopathologie Cardiovasculaire et Mitochondriale (MITOVASC)
Subjects: Medicine (General), phospholipid metabolism, QH426-470, DNA, Mitochondrial, OPA1, Article, high‐throughput screening, GTP Phosphohydrolases, EMBO57, R5-920, EMBO16, Optic Atrophy, Autosomal Dominant, Genetics, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], High-throughput screening, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Articles, Fibroblasts, genetic modifiers, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, mitochondrial dynamics, High-Throughput Screening Assays, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, EMBO27, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology
Abstract: International audience; Mutations in OPA1 cause autosomal dominant optic atrophy (DOA) as well as DOA+, a phenotype characterized by more severe neurological deficits. OPA1 deficiency causes mitochondrial fragmentation and also disrupts cristae, respiration, mitochondrial DNA (mtDNA) maintenance, and cell viability. It has not yet been established whether phenotypic severity can be modulated by genetic modifiers of OPA1. We screened the entire known mitochondrial proteome (1,531 genes) to identify genes that control mitochondrial morphology using a first-in-kind imaging pipeline. We identified 145 known and novel candidate genes whose depletion promoted elongation or fragmentation of the mitochondrial network in control fibroblasts and 91 in DOA+ patient fibroblasts that prevented mitochondrial fragmentation, including phosphatidyl glycerophosphate synthase (PGS1). PGS1 depletion reduces CL content in mitochondria and rebalances mitochondrial dynamics in OPA1-deficient fibroblasts by inhibiting mitochondrial fission, which improves defective respiration, but does not rescue mtDNA depletion, cristae dysmorphology, or apoptotic sensitivity. Our data reveal that the multifaceted roles of OPA1 in mitochondria can be functionally uncoupled by modulating mitochondrial lipid metabolism, providing novel insights into the cellular relevance of mitochondrial fragmentation.
Published: 2021
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46. Rapid decline of neutralizing antibodies against SARS-CoV-2 among infected healthcare workers

Author: Marot, Stéphane, Malet, Isabelle, Leducq, Valentin, Zafilaza, Karen, Sterlin, Delphine, Planas, Delphine, Gothland, Adélie, Jary, Aude, Dorgham, Karim, Bruel, Timothée, Attali, Valérie, Baresse, Isabelle, Beurton, Alexandra, Boddaert, Jacques, Bourmaleau, Julie, Catala, Martin, Demoule, Alexandre, Dunoyer, Violaine, Esteban-Amarilla, Cristina, Hausfater, Pierre, Haziot, Noémie, Ip, Queyras, Kubis, Nathalie, Lhoest, Laurence, Lubetzki, Catherine, Marion, Fabienne, Morawiec, Elise, Mourtada, Leila, Orcel, Brigitte, Morelot-Panzini, Capucine, Raux, Mathieu, Reinhard, Christophe, Riquier, Claire, Roubertier, Xavier, Weiss, Nicolas, Zalc, Bernard, Burrel, Sonia, Boutolleau, David, Schwartz, Olivier, Gorochov, Guy, Calvez, Vincent, Marcelin, Anne-Geneviève, Institut Pierre Louis d'Epidémiologie et de Santé Publique (iPLESP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Vaccine Research Institute (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), The AGM laboratory is funded by Agence Nationale de Recherches sur le SIDA et les hépatites virales (AC43, Medical Virology), the SARS-CoV-2 Program of the Faculty of Medicine of Sorbonne University, the Agence Nationale de la Recherche. The GG laboratory is supported by the Fondation de France, 'Tous unis contre le virus' framework Alliance (Fondation de France, AP-HP, Institut Pasteur) in collaboration with the Agence Nationale de la Recherche (ANR Flash COVID19 program) and by the SARS-CoV-2 Program of the Faculty of Medicine of Sorbonne University ICOViD programs. The OS laboratory is funded by Institut Pasteur, the ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), 'TIMTAMDEN' ANR-14-CE14-0029, 'CHIKV-Viro-Immuno' ANR-14-CE14-0015-01, the Gilead HIV cure program, ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR, and Fondation pour la Recherche Médicale. We thank Nathalie Aulner and the UtechS Photonic BioImaging (UPBI) core facility (Institut Pasteur), a member of the France BioImaging network, for image acquisition and analysis., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-14-CE14-0029,TIMTAMDEN,Rôle des récepteurs TIM et TAM dans l'infection des cellules cibles par le virus de la dengue(2014), ANR-14-CE14-0015,CHIKV-Viro-Immuno,Multiplication et Relation avec l'hôte du virus Chikungunya(2014), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Vaccine Research Institute [Créteil, France] (VRI), Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), HAL-SU, Gestionnaire, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Laboratoires d'excellence - Initiative for the creation of a Vaccine Research Institute - - VRI2010 - ANR-10-LABX-0077 - LABX - VALID, Appel à projets générique - Rôle des récepteurs TIM et TAM dans l'infection des cellules cibles par le virus de la dengue - - TIMTAMDEN2014 - ANR-14-CE14-0029 - Appel à projets générique - VALID, and Appel à projets générique - Multiplication et Relation avec l'hôte du virus Chikungunya - - CHIKV-Viro-Immuno2014 - ANR-14-CE14-0015 - Appel à projets générique - VALID
Subjects: 0301 basic medicine, Immunoglobulin A, Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Science, General Physics and Astronomy, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Article, General Biochemistry, Genetics and Molecular Biology, Persistence (computer science), 03 medical and health sciences, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Medicine, Infection control, 030212 general & internal medicine, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Multidisciplinary, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, business.industry, SARS-CoV-2, General Chemistry, biochemical phenomena, metabolism, and nutrition, 3. Good health, Vaccination, Titer, 030104 developmental biology, Viral infection, Immunology, biology.protein, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, sense organs, Antibody, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: There are only few data concerning persistence of neutralizing antibodies (NAbs) among SARS-CoV-2-infected healthcare workers (HCW). These individuals are particularly exposed to SARS-CoV-2 infection and at potential risk of reinfection. We followed 26 HCW with mild COVID-19 three weeks (D21), two months (M2) and three months (M3) after the onset of symptoms. All the HCW had anti-receptor binding domain (RBD) IgA at D21, decreasing to 38.5% at M3 (p, The humoral immune response to SARS-CoV-2 infection is not yet fully understood. Here, Marot et al. monitor the longitudinal profile and neutralizing activity of IgG, IgA, and IgM among 26 healthcare workers and provide evidence for a short-lasting humoral immune protection due to a decrease of neutralizing antibody titers within 3 months.
Published: 2021
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47. Mitochondrial Dynamics and Activity in Legionella-Infected Cells

Author: Carmen Buchrieser, Ok-Ryul Song, Pedro Escoll, Priscille Brodin, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Work in C.B. laboratory is financed by Institut Pasteur and ANR (grant no. ANR-10-LABX-62-IBEID). P.E. is funded by the Fondation pour la Recherche Médicale (FRM) project DEQ20120323697., P.B. and O.R.S. received financial support from the European Community (ERC-STG INTRACELLTBno. 260901, MM4TB no. 260872), the ANR (grant no. ANR-10-EQPX-04-01), and the Région Nord Pas de Calais (convention no. 12000080)., Carmen Buchrieser, Hubert Hilbi, We thank Nathalie Aulner and the Imagopole-CiTech (part of France-BioImaging supported by ANR grant no. ANR-10-INSB-04-01, Conseil de la Région Ile-de-France, FRM), and the Technology Core of the Center for Translational Science (CRT) at Institut Pasteur, for technical support during the setup of the methods presented here., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Host mitochondria, Bioenergetics, Legionella, Mitochondrial respiration assay, MESH: Host-Pathogen Interactions, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Mitochondria / metabolism, Legionella pneumophila, Microbiology, 03 medical and health sciences, Living-cell assays, MESH: Data Analysis, MESH: Macrophages / metabolism, MitoTracker dyes, MESH: Macrophages / microbiology, 030304 developmental biology, MESH: Legionellosis / microbiology, MESH: Mitochondrial Dynamics, 0303 health sciences, Mitochondrial morphology, MESH: Humans, biology, 030306 microbiology, Mitochondrial fission, MESH: Molecular Imaging / methods, biology.organism_classification, Seahorse, Mitochondrial respiration, MESH: Image Processing, Computer-Assisted, MESH: Legionellosis / metabolism, MESH: Legionella / physiology, High-content microscopy, Mitochondrial dynamics, MESH: Cell Respiration, MESH: Cells, Cultured
Abstract: International audience; The study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages.
Published: 2019
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