UPR 9022 - IBMC : Réponse immunitaire et développement chez les insectes

15 rue René Descartes,
67084 Strasbourg Cedex
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Elena LEVASHINA

	UPR 9022 du CNRS Institut de Biologie Moléculaire et Cellulaire 15, rue René Descartes 67084 Strasbourg Cédex, FRANCE email: E.Levashina@ibmc.u-strasbg.fr Tel: 03 88 41 70 18 Fax: 03 88 60 69 22
Née le	29 Mars 1965 à Leningrad, URSS
NATIONALITE	Russe

FORMATION
Décembre 2001	Habilitation à Diriger des Recherches Titre: "Réponse immunitaire chez Diptera" Université Louis Pasteur, Strasbourg, France
Octobre 1994	Docteur en Génétique Titre: "Séection cellulaire in vitro pour le développement de plantes résistantes aux insectes phytosterol-dépendant" Department of Genetics and Breeding, St.Petersburg State University, Russia (Directeur de thèse: Prof. L.A.Lutova)
Juin 1988	Diplomé en Génétique des Plantes, St.Petersburg State University, Russia

EXPERIENCE PROFESSIONNELLE
Octobre 2006	Directeur de Recherche 2 du CNRS Institut de Biologie Moléculaire et Cellulaire (IBMC), Strasbourg, France, UPR 9022, Directeur – Prof. J.-M. Reichhart Domaine de Recherche: Analyse functionelle de la réponse immunitaire du moustique à l'infection au Plasmodium
Octobre 2002	Chargée de Recherche 1 du CNRS Institut de Biologie Moléculaire et Cellulaire (IBMC), Strasbourg, France, UPR 9022, Directeur – Prof. Jules A. Hoffmann Domaine de Recherche: Analyse functionelle de la réponse immunitaire du moustique à l'infection au Plasmodium
2001 - Octobre 2002	Staff Scientist European Molecular Biology Laboratory (EMBL) Heidelberg, Allemagne Directeur: Prof. Fotis C. Kafatos Sujet de recherche: Analyse fonctionelle des gènes de la réponse immunitaire chez le moustique
1999 - 2001	Bourse Postdoctorale European Molecular Biology Laboratory (EMBL) Heidelberg, Allemagne Directeur: Prof. Fotis C. Kafatos Sujet de recherche: Rôle des protéines à fonction thioester dans l'immunité de l'insecte vecteur Anopheles gambiae
1995 - 1998	Bourse d'étude Institut de Biologie Moleculaire et Cellulaire du CNRS Universite Louis Pasteur, Strasbourg, France UPR 9022 - Réponse Immunitaire et Développement chez les Insectes Directeur: Prof. Jules A. Hoffmann, Tuteur: Prof. Jean-Marc Reichhart Sujet de recherche: Régulation de la réponse immunitaire chez Drosophila melanogaster
1992 - 1995	Recherche Scientifique St.Petersburg State University, St.Petersburg, Russia Department of Genetics and Breeding Directeur: Prof. Sergey G. Inge-Vechtomov, Tuteur: Prof. Ludmila A. Lutova Sujet de recherche: - Charactérisation moléculaire des souches du radis (Raphanus sativus L.), présentant des habilités différentes à la formation de tumeurs; - développement de nouvelles stratégies pour la protection des plantes contre des parasites.
1991 - 1992	Bourse d'étude Université Louis Pasteur, Strasbourg, France Laboratoire de Biologie Generale, Tuteur: Prof. Jean-Marc Reichhart Sujet de recherche: Clonage moléculaire études de l'expression de la metchnikowin, peptide antimicrobien chez Drosophila

RECOMPENSES
2003	Donation Principale de la Fondation Schlumberger pour l’Education et la Recherche (FSER) pour la Création d’un Laboratoire
2004	EMBO Young Investigator Programme Award
2004	Programme “Avenir” de l’Insitut National de la Santé et de la Recherche Médicale (INSERM)

CONTRATS ET BOURSES DE RECHERCHE
Septembre 2005 – Août 2010	International Research Scholar of Howard Hughes Medical Institute (HHMI)
Octobre 2001 - Septembre 2004	Staff scientist Contract with EMBL
Janvier 1999 - Décembre 2000	EMBO Long Term Fellowship
Janvier 1992 - Mai 1992	UNESCO Short Term Fellowship, Laboratoire d'Enzymologie et Biochimie Végétale, Institut de Biologie Moléculaire des Plantes du CNRS, Universite Louis Pasteur, Strasbourg, France

Publications de Elena LEVASHINA

Titre, Autheur(s)	Journal, Références
Silencing of genes and alleles by RNAi in Anopheles gambiae. Lamacchia M, Clayton JR, Wang-Sattler R, Steinmetz LM, Levashina EA, Blandin SA. Abstract : Anopheles gambiae mosquitoes are the major vectors of human malaria parasites. However, mosquitoes are not passive hosts for parasites, actively limiting their development in vivo. Our current understanding of the mosquito antiparasitic response is mostly based on the phenotypic analysis of gene knockdowns obtained by RNA interference (RNAi), through the injection or transfection of long dsRNAs in adult mosquitoes or cultured cells, respectively. Recently, RNAi has been extended to silence specifically one allele of a given gene in a heterozygous context, thus allowing to compare the contribution of different alleles to a phenotype in the same genetic background.	Methods Mol Biol. 2013;923:161-76.	Abstract Article complet
High-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions Marois E, Scali C, Soichot J, Kappler C, Levashina EA, Catteruccia F. Abstract : BACKGROUND: Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures. METHODS: A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females. RESULTS: Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers. CONCLUSIONS: The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors.	Malar J 2012 Aug 28;11:302	Abstract Article complet
The major yolk protein vitellogenin interferes with the anti-plasmodium response in the malaria mosquito Anopheles gambiae Abstract : Abstract : When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.	PloS Biology Vol. 8(7):e1000434	Abstract Article complet
Focusing on complement in the antiparasitic defense of mosquitoes. Volohonsky G, Steinert S, Levashina EA Abstract : Malaria is an infectious disease caused by Plasmodium and transmitted to humans by the Anopheles mosquitoes. The mosquito immune system predominantly targets Plasmodium at the ookinete stage, and efficiently eliminates the majority of invading parasites. Identification of the components of the mosquito complement system now provides new focus for studies on the activation and control of this pathway, whose manipulation is expected to block malaria transmission at the vector level.	Trends Parasitol. 2009: 26(1):1-3	Abstract Article complet
Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae. Blandin SA, Wang-Sattler R, Lamacchia M, Gagneur J, Lycett G, Ning Y, Levashina EA, Steinmetz LM. Abstract : The ability of Anopheles gambiae mosquitoes to transmit Plasmodium parasites is highly variable between individuals. However, the genetic basis of this variability has remained unknown. We combined genome-wide mapping and reciprocal allele-specific RNA interference (rasRNAi) to identify the genomic locus that confers resistance to malaria parasites and demonstrated that polymorphisms in a single gene encoding the antiparasitic thioester-containing protein 1 (TEP1) explain a substantial part of the variability in parasite killing. The link between TEP1 alleles and resistance to malaria may offer new tools for controlling malaria transmission. The successful application of rasRNAi in Anopheles suggests that it could also be applied to other organisms where RNAi is feasible to dissect complex phenotypes to the level of individual quantitative trait alleles.	Science 2009 Oct 2;326(5949):147-50	Abstract Article complet
RNAi in the malaria vector, Anopheles gambiae. Catteruccia F, Levashina EA. Abstract : Malaria is a disease that kills more than a million people each year in tropical and subtropical countries. The disease is caused by Plasmodium parasites and is transmitted to humans exclusively by mosquitoes of the genus Anopheles. The lack of functional approaches has hampered study of the biological networks that determine parasite transmission by the insect vector. The recent discovery of RNA interference and its adaptation to mosquitoes is now providing crucial tools for the dissection of vector-parasite interactions and for the analysis of aspects of mosquito biology influencing the vectorial capacity. Two RNAi approaches have been established in mosquitoes: transient gene silencing by direct injection of double-stranded RNA, and stable expression of hairpin RNAs from transgenes integrated in the genome. Here we describe these methods in detail, providing information about their use and limitations.	Methods Mol Biol. 2009;555:63-75	Abstract Article complet
Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium. Fraiture M, Baxter RH, Steinert S, Chelliah Y, Frolet C, Quispe-Tintaya W, Hoffmann JA, Blandin SA, Levashina EA. Abstract : Plasmodium development within Anopheles mosquitoes is a vulnerable step in the parasite transmission cycle, and targeting this step represents a promising strategy for malaria control. The thioester-containing complement-like protein TEP1 and two leucine-rich repeat (LRR) proteins, LRIM1 and APL1, have been identified as major mosquito factors that regulate parasite loads. Here, we show that LRIM1 and APL1 are required for binding of TEP1 to parasites. RNAi silencing of the LRR-encoding genes results in deposition of TEP1 on Anopheles tissues, thereby depleting TEP1 from circulation in the hemolymph and impeding its binding to Plasmodium. LRIM1 and APL1 not only stabilize circulating TEP1, they also stabilize each other prior to their interaction with TEP1. Our results indicate that three major antiparasitic factors in mosquitoes jointly function as a complement-like system in parasite killing, and they reveal a role for LRR proteins as complement control factors.	Cell Host Microbe. 2009 Mar 19;5(3):273-84	Abstract Article complet
Molecular and cellular components of the mating machinery in Anopheles gambiae females. Rogers DW, Whitten MM, Thailayil J, Soichot J, Levashina EA, Catteruccia F. Abstract : Anopheles gambiae mosquitoes are the principal vectors of malaria. A major determinant of the capacity of these mosquitoes as disease vectors is their high reproductive rate. Reproduction depends on a single insemination, which profoundly changes the behavior and physiology of females. To identify factors and mechanisms relevant to the fertility of A. gambiae, we performed a comprehensive analysis of the molecular and cellular machinery associated with copulation in females. Initial whole-body microarray experiments comparing virgins with females at 2 h, 6 h, and 24 h after mating detected large transcriptional changes. Analysis of tissue localization identified a subset of genes whose expression was strikingly regulated by mating in the lower reproductive tract and, surprisingly, the gut. In the atrium of virgin females, where the male seminal fluid is received, our studies revealed a "mating machinery" consisting of molecular and structural components that are turned off or collapse after copulation, suggesting that this tissue loses its competence for further insemination. In the sperm storage organ, we detected a number of mating-responsive genes likely to have a role in the maintenance and function of stored sperm. These results identify genes and mechanisms regulating the reproductive biology of A. gambiae females, highlighting considerable differences with Drosophila melanogaster. Our data inform vector control strategies and reveal promising targets for the manipulation of fertility in field populations of these important disease vectors.	Proc Natl Acad Sci. 2008 Dec 9;105(49):19390-5	Abstract Article complet
Host-parasite interactions: the balance of trade Levashina EA Abstract : Living organisms are constantly exposed to microbes. Nevertheless a disease as an outcome of the pathogen attack remains an exception rather than a rule, and most species are remarkably resistant to infectious agents. In some cases, pathogens invade more than one host to complete their complex life cycle. To warrant a successful transmission from one host to another, parasites have developed a number of strategies and tricks, many of which rely on the passive or active participation of the host. This is especially true for parasitic protozoa such as trypanosomes, leishmania and Plasmodium, single cell organisms that are obligatorily transmitted to mammals by insect vectors. Inside their hosts, the parasites change looks and coats and invade a diverse set of cells and tissues, some for a transient passage, whereas others to establish a long-term infection. The past decade has shown an explosion in the field of host?pathogen interactions. The rapid progress was made possible by the availability of sequence information and bioinformatic tools and development of a series of functional assays, including RNAi screens and transgenic technologies relevant for both the host and the pathogen. These efforts, although in their very early days, firmly established an intricate crosstalk between the interacting organisms and have already identified a number of key molecules involved in it. Finally, development of in vivo imaging, high throughput methodologies, and computational techniques has gathered conceptual breakthroughs that infringe on some reputable dogmas. In this issue of the journal, selected contributions summarize new developments and current challenges related to three host?protozoa combinations. Two reviews outline recent progress in the understanding of interactions between flies and Euglenozoa parasites, whereas the third one focuses on the Plasmodium interactions with its mammalian host. Leishmania parasites are the causative agents of leishmaniasis, a debilitating disease transmitted by female phlebotomine sand flies. Complex interactions that occur between the haemoflagellate parasites and their vectors might be the basis of the narrow host specificity: out of the thousand sand fly species described to date, only 70 are proven or suspected vectors. The review by Bates discusses molecular events required for a successful parasite development within the sand fly midgut. Several molecules and structures have been identified that underlie leishmania motility (flagellum), midgut attachment (a leishmania surface lipophosphoglycan and a sand fly galectin), and regulation of metacyclogenesis, the differentiation process that leads to the accumulation of mammal-infective promastigotes. The review by Roditi and Lehane describes a fascinating crosstalk between tsetse flies and trypanosomes. African trypanosomes are insect-borne parasites that cause sleeping sickness in humans and nagana, the disease characterized by fever, lethargy, and edema, in domesticated animals. Although the development of transgenic tools to study biology of the trypanosome inside the insect vector is in its very beginning, it has already offered tools for in vivo imaging of parasites within the fly vector to elucidate regulation of gene expression and genetic exchange. On the sand fly side, a series of parameters that affect the infection rates has been established. The current challenge is to identify genes and molecules that underlie these factors and shape tsetse fly's capacity to transmit trypanosomes. Malaria remains today as one of the most devastating infectious diseases. The life cycle of its causative agent, Plasmodium, is split between the human host (the asexual stage) and the mosquito vector (the sexual stage). Review by Silvie et al. focuses on the asexual stages of the parasite development and highlights the advantages of the rodent in vivo models that continue to play crucial roles in the dissection of the genetic basis of the fine-tuned host?parasite interactions. Application of the high-end intravital imaging to follow fluorescently labeled P. berghei identified novel invasion routes and cryptic forms of parasites, demonstrating how new powerful technologies change our views on what was thought to be an established concept. The general message of this issue is that in spite of a long history, many more fascinating discoveries are awaiting us in the world of host?parasite interactions.	Curr Opin Microbiol. 2008 Aug;11(4):338-9	Abstract Article complet
Antimalarial responses in Anopheles gambiae: from a complement-like protein to a complement-like pathway. Blandin SA, Marois E, Levashina EA. Abstract : Malaria transmission between humans depends on the ability of Anopheles mosquitoes to support Plasmodium development. New perspectives in vector control are emerging from understanding the mosquito immune system, which plays critical roles in parasite recognition and killing. A number of factors controlling this process have been recently identified, and key among them is TEP1, a homolog of human complement factor C3 whose binding to the parasite surface targets it for subsequent killing. Here, we review our current knowledge of mosquito factors that respond to Plasmodium infection and elaborate on the activity and mode of action of the TEP1 complement-like pathway.	Cell Host Microbe 2008 Jun 12;3(6):364-74	Abstract Article complet
Reverse genetics analysis of antiparasitic responses in the malaria vector, Anopheles gambiae. Blandin SA, Levashina EA. Abstract : In Drosophila the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathways. The Toll signaling pathway responds mainly to Gram-positive bacterial and fungal infection while the Imd pathway mediates the response to Gram-negative bacteria. Microbial recognition upstream of Toll involves, at least in part, peptidoglycan recognition proteins (PGRPs). The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and Gram-negative binding protein 1 (GNBP1) that cooperate to detect the presence of lysine-type peptidoglycan in the host. Recently it has been shown that a loss-of-function mutation in peptidoglycan recognition protein SD (PGRP-SD) severely exacerbates the PGRP-SA and GNBP1 mutant phenotypes. Here we have solved the crystal structure of PGRP-SD at 1.5 ? resolution. Comparison with available structures of PGRPs in complex with their peptidoglycan (PGN) ligand strongly suggests a diaminopimelic acid (DAP) specificity for PGRP-SD. This result is supported by pull-down assays with insoluble PGNs. In addition we show that Toll pathway activation after infection by DAP-type PGN containing bacteria is clearly reduced in PGRP-SD mutant flies. Our hypothesis is that the role of PGRP-SD is the recognition of DAP-type PGNs responsible for the activation of the Toll pathway by Gram-negative bacteria.	Methods Mol Biol 2008;415:365-77	Abstract Article complet
Phagocytosis in mosquito immune responses Blandin SA, Levashina EA Abstract : Anopheles mosquitoes are the only vectors of human malaria parasites. Mosquito-parasite interactions are critical for disease transmission and therefore are a potential target for malaria control strategies. Mosquitoes mount potent immune responses that efficiently limit proliferation of a variety of infectious agents, including microbial pathogens and malaria parasites. The recent completion of the Anopheles gambiae genome sequencing project combined with the development of the powerful RNA interference-based gene silencing helped to identify major players of the immune defenses and uncovered evolutionarily conserved mechanisms in the anti-bacterial and anti-Plasmodium responses. The anti-bacterial responses are based on phagocytosis at early steps of infections, followed, several hours later, by the synthesis of anti-microbial peptides. The principal regulators of anti-parasitic responses are predominantly synthesized by the mosquito blood cells; however, the exact molecular mechanisms of parasite killing remain unclear. Several regulators of phagocytosis are also required for efficient parasite killing. Here, we summarize our current knowledge of the anti-bacterial and anti-parasitic responses, with the particular emphasis on the role of phagocytosis in mosquito immunity.	Immunological Reviews 2007 : Vol 219, 8-16	Abstract Article complet
Structural basis for conserved complement factor-like function in the antimalarial protein TEP1 (2007) Baxter RH, Chang CI, Chelliah Y, Blandin S, Levashina EA, Deisenhofer J Abstract : Thioester-containing proteins (TEPs) are a major component of the innate immune response of insects to invasion by bacteria and protozoa. TEPs form a distinct clade of a superfamily that includes the pan-protease inhibitors alpha2-macroglobulins and vertebrate complement factors. The essential feature of these proteins is a sequestered thioester bond that, after cleavage in a protease-sensitive region of the protein, is activated and covalently binds to its target. Recently, TEP1 from the malarial vector Anopheles gambiae was shown to mediate recognition and killing of ookinetes from the malarial parasite Plasmodium berghei, a model for the human malarial parasite Plasmodium falciparum. Here, we present the crystal structure of the TEP1 isoform TEP1r. Although the overall protein fold of TEP1r resembles that of complement factor C3, the TEP1r domains are repositioned to stabilize the inactive conformation of the molecule (containing an intact thioester) in the absence of the anaphylotoxin domain, a central component of complement factors. The structure of TEP1r provides a molecular basis for the differences between TEP1 alleles TEP1r and TEP1s, which correlate with resistance of A. gambiae to infection by P. berghei.	Proc. Natl. Acad. Sci. USA 2007 : Vol 104, 11615-11620	Abstract Article complet
Structural basis for conserved complement factor-like function in the antimalarial protein TEP1 Baxter RH, Chang CI, Chelliah Y, Blandin S, Levashina EA, Deisenhofer J. Abstract : Thioester-containing proteins (TEPs) are a major component of the innate immune response of insects to invasion by bacteria and protozoa. TEPs form a distinct clade of a superfamily that includes the pan-protease inhibitors α2-macroglobulins and vertebrate complement factors. The essential feature of these proteins is a sequestered thioester bond that, after cleavage in a protease-sensitive region of the protein, is activated and covalently binds to its target. Recently, TEP1 from the malarial vector Anopheles gambiae was shown to mediate recognition and killing of ookinetes from the malarial parasite Plasmodium berghei, a model for the human malarial parasite Plasmodium falciparum. Here, we present the crystal structure of the TEP1 isoform TEP1r. Although the overall protein fold of TEP1r resembles that of complement factor C3, the TEP1r domains are repositioned to stabilize the inactive conformation of the molecule (containing an intact thioester) in the absence of the anaphylotoxin domain, a central component of complement factors. The structure of TEP1r provides a molecular basis for the differences between TEP1 alleles TEP1r and TEP1s, which correlate with resistance of A. gambiae to infection by P. berghei.	Proc Natl Acad Sci U S A 2007 Jul 10;104(28):11615-20	Abstract Article complet
Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes Waterhouse RM, Kriventseva EV, Meister S, Xi Z, Alvarez KS, Bartholomay LC, Barillas-Mury C, Bian G, Blandin S, Christensen BM, Dong Y, Jiang H, Kanost MR, Koutsos AC, Levashina EA, Li J, Ligoxygakis P, Maccallum RM, Mayhew GF, Mendes A, Michel K, Osta MA, Paskewitz S, Shin SW, Vlachou D, Wang L, Wei W, Zheng L, Zou Z, Severson DW, Raikhel AS, Kafatos FC, Dimopoulos G, Zdobnov EM, Christophides GK Abstract : Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti (Aa), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa, the malaria vector Anopheles gambiae (Ag), and the fruit fly Drosophila melanogaster (Dm). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.	Science 2007: Vol 316, 1738-1743.	Abstract Article complet
Fz2 and cdc42 mediate melanization and actin polymerization but are dispensable for Plasmodium killing in the mosquito midgut Shiao SH, Whitten MM, Zachary D, Hoffmann JA, Levashina EA Abstract : The midgut epithelium of the mosquito malaria vector Anopheles is a hostile environment for Plasmodium, with most parasites succumbing to host defenses. This study addresses morphological and ultrastructural features associated with Plasmodium berghei ookinete invasion in Anopheles gambiae midguts to define the sites and possible mechanisms of parasite killing. We show by transmission electron microscopy and immunofluorescence that the majority of ookinetes are killed in the extracellular space. Dead or dying ookinetes are surrounded by a polymerized actin zone formed within the basal cytoplasm of adjacent host epithelial cells. In refractory strain mosquitoes, we found that formation of this zone is strongly linked to prophenoloxidase activation leading to melanization. Furthermore, we identify two factors controlling both phenomena: the transmembrane receptor frizzled-2 and the guanosine triphosphate-binding protein cell division cycle 42. However, the disruption of actin polymerization and melanization by double-stranded RNA inhibition did not affect ookinete survival. Our results separate the mechanisms of parasite killing from subsequent reactions manifested by actin polymerization and prophenoloxidase activation in the A. gambiae-P. berghei model. These latter processes are reminiscent of wound healing in other organisms, and we propose that they represent a form of wound-healing response directed towards a moribund ookinete, which is perceived as damaged tissue.	PLoS Pathog. 2006: Vol 2, 1152-1164	Abstract Article complet
Boosting NF-κB-Dependent Basal Immunity of Anopheles gambiae Aborts Development of Plasmodium berghei Frolet C, Thoma M, Blandin S, Hoffmann JA, Levashina EA Abstract : Anopheles gambiae, the major vector for the protozoan malaria parasite Plasmodium falciparum, mounts powerful antiparasitic responses that cause marked parasite loss during midgut invasion. Here, we showed that these antiparasitic defenses were composed of pre- and postinvasion phases and that the preinvasion phase was predominantly regulated by Rel1 and Rel2 members of the NF-kappaB transcription factors. Concurrent silencing of Rel1 and Rel2 decreased the basal expression of the major antiparasitic genes TEP1 and LRIM1 and abolished resistance of Anopheles to the rodent malaria parasite P. berghei. Conversely, depletion of a negative regulator of Rel1, Cactus, prior to infection, enhanced the basal expression of TEP1 and of other immune factors and completely prevented parasite development. Our findings uncover the crucial role of the preinvasion defense in the elimination of parasites, which is at least in part based on circulating blood molecules.	Immunity 2006: Vol 25, 677-85.	Abstract Article complet
Mosquito midguts and malaria: cell biology, compartmentalization and immunology Whitten MM, Shiao SH, Levashina EA Abstract : The malaria parasite Plasmodium has an absolute requirement for both a vertebrate and a mosquito host in order to complete its life cycle, and its interactions with the latter provide the focus for this review. The mosquito midgut represents one of the most challenging environments for the survival and development of Plasmodium, and is thus also one of the most attractive sites for novel targeted malaria control strategies. During their attempts to cross the midgut epithelium en route to the salivary glands, motile ookinetes are swiftly detected and labelled by mosquito recognition factors and targeted for destruction by a variety of immune responses that recruit killing factors both from the midgut and from other tissues in the surrounding body cavity. The exact interplay between these factors and the parasite is highly species- and strain-specific, as are the timing and the route of parasite invasion. These features are paramount to determining the success of the infection and the vector competence of the mosquito. Here we discuss recent advances in genomic analyses, coupled with detailed microscopical investigations, which are helping to unravel the identity and roles of the major players of these complex systems.	Parasite Immunol. 2006: Vol 4, 121-130	Abstract Article complet
Innate Immunity Aderem A, Levashina EA Abstract : No abstract.	Current Opinion in Immunology 2005: Vol 17, 1-3	Abstract Article complet
In vivo identification of novel regulators and conserved pathways of phagocytosis in A. gambiae Moita LF, Wang-Sattler R, Michel K, Zimmermann T, Blandin S, Levashina EA, Kafatos FC Abstract : Anopheles gambiae uses effective immune responses, including phagocytosis, to fight microbial infection. We have developed a semiquantitative phagocytosis test and used it in conjunction with dsRNA gene silencing to test the in vivo roles of 71 candidate genes in phagocytosis of Escherichia coli and Staphylococcus aureus. Here, we show that inactivation of 26 genes changes the phagocytic activity by more than 45% and that two pathways similar to those that mediate apoptotic cell removal in Caenorhabditis elegans are used in A. gambiae for phagocytosis of microorganisms. Simultaneous inactivation of the identified regulators of phagocytosis and conserved components defining each signaling pathway permitted provisional assignment of the novel regulators to one or the other pathway. Pathway inactivation enhances at least three times the ability of E. coli and S. aureus to proliferate in the mosquito. Interestingly, mosquito survival is not compromised even if both pathways are perturbed simultaneously.	Immunity 2005: Vol 23, 65-73	Abstract Article complet
Mosquito Immune Response Against Malaria Parasite Blandin S, Levashina EA Abstract : Anopheline mosquitoes are the major vectors oh human malaria. Mosquito-parasite interactions are a critical aspect of disease transmission and a potential target for malaria control. Mosquitoes vary in their innate ability to support development of the malaria parasite, but the molecular mechanisms that determine vector competence are poorly understood. This area research has been revolutionized by recent advances I nthe mosquito genome characterization and by the development of new tools for functional gene analysis.	Current Opinion in Immunology 2004: Vol 16, 1-5	Abstract Article complet
Thioester-containing proteins and insect immunity Blandin S, Levashina EA Abstract : Here, we discuss the role of thioester-containing proteins in innate immune responses of insects. TEPs are represented by multi-member families both in the fruitfly, Drosophila melanogaster, and in the mosquito, Anopheles gambiae. Phylogenetic analysis of the family suggests that in these two dipteran species evolution of TEPs followed independent scenarios as a result of specific adaptation to distinct ecological environments. Research on these two relatively simple model systems, which lack adaptive immunity, may provide new insights into the evolutionary origins and functions of this important protein family.	Mol. Immunol. 2004: Vol 40, 903-908	Abstract Article complet
Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA Abstract : Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.	Cell 2004: Vol 116, 661-670	Abstract Article complet
Bacterial a2-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome ? Budd A, Blandin S, Levashina EA, Gibson TJ Abstract : Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor a2-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion.	Genome Biology 2004: Vol 5	Abstract Article complet
Immune responses in Anopheles gambiae Levashina EA Abstract : Transmission of human malaria requires a successful development of Plasmodium parasites in anopheline mosquitoes. Insects have developed efficient immune responses to oppose microbial and eukaryotic invaders. The completion of the sequencing of the Anopheles genome provides a wealth of information on putative immune genes that are homologous to components of the Drosophila and mammalian immune systems. In this review, we will summarize our present knowledge of immune responses in the mosquito Anopheles gambiae and attempt a comparative analysis of insect immune systems.	Insect Biochem. Mol. Biol. 2004: Vol 34, 673-678	Abstract Article complet
Immune response and parasite transmission in blood-feeding insects Lehane MJ, Aksoy S, Levashina EA Abstract : The detailed model of insect immunity being built for Drosophila, allied to mass sequencing programs for blood-feeding insects, has led to advances in our understanding of the interaction between pathogens and insect vectors. An outline of insect immunity is given here based on the Drosophila studies, which is used as a framework to discuss recent work on Plasmodium–mosquito and Trypanosoma–tsetse interactions.	Trends in Parasitology 2004: Vol 20, 433-439	Abstract Article complet
Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila Goto A, Blanbin S, Royet J, Reichhart JM, Levashina EA Abstract : Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence-specific manner. Here we show that the direct injection of dsRNA can be used in adult Drosophila flies to disrupt function of endogenous genes in vivo. As a proof of principle, we habe used this method to silence components of a major signalling cascade, the Toll pathway, which controls fruit fly resistance to fungal and Gram-positive bac terial infections. We demonstrate that the knockout is efficient only if dsRNA is injected in 4- or more day-old flies and that it lasts for at least 1 week. Furthermore, we report dsRNA-based epistatic gene analysis via injection of a mixture of two dsRNAs and propose that injection of dsRNA represents a powerful method for rapid functional analysis of genes in Drosophila melanogaster adults, particularly of those whose mutations are lethal during development.	Nucleic Acids Research 2003: Vol 31, 6619-6623	Abstract Article complet
Immunity-related genes and gene families in Anopheles gambiae Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, Brey PT, Collins FH, Danielli A, Dimopoulos G, Hetru C, Hoa NT, Hoffmann JA, Kanzok SM, Letunic I, Levashina EA, Loukeris TG, Lycett G, Meister S, Michel K, Moita LF, Muller HM, Osta MA, Paskewitz SM, Reichhart JM, Rzhetsky A, Troxler L, Vernick KD, Vlachou D, Volz J, von Mering C, Xu J, Zheng L, Bork P, Kafatos FC Abstract : We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.	Science 2002: Vol 298, 159-165.	Abstract Article complet
Thioester-Containing Proteins of Protostomes. In "Infectious Disease: Innate Immunity" Levashina EA, Blandin S, Moita LF, Lagueux M, Kafatos FC Abstract : We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.	Ezekowitz R.A.B. and Hoffmann J.A., eds. Humana Press Inc 2002: Totowa, NJ, 155-173.	Abstract Article complet
Reverse genetics in the mosquito, Anopheles gambiae: targeted disruption of the Defensin gene Blandin S, Moita LF, Kocher T, Wilm M, Kafatos FC, Levashina EA Abstract :	EMBO Reports 2002: 3, 852-856.	Abstract Article complet
Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae Levashina EA, Moita LF, Blandin S, Vriend G, Lagueux M, Kafatos FC Abstract : We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.	Cell 2001: Vol 104, 709-718.	Abstract Article complet
Innate immune defence against malaria infection in the mosquito Dimopoulos G, Muller HM, Levashina EA, Kafatos FC Abstract :	Curr Opin Immunol. 2001: 13, 79-88 (Review)	Abstract Article complet
Constitutive expression of a complement-like protein in Toll and JAK gain-of-function mutants of Drosophila Lagueux M, Perrodou E, Levashina EA, Capovilla & Hoffmann JA Abstract : We show that Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of the complement C3/alpha(2)-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development, and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in the larval fat body, we observe a constitutive expression in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced inducibility in loss-of-function hop mutants. We also observe a constitutive expression in gain-of-function Toll mutants. We discuss the possible roles of these novel complement-like proteins in the Drosophila host defense.	Proc. Natl. Acad. Sci. USA 2000: 97, 11427-11432	Abstract Article complet
The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2 Green C, Levashina E, McKimmie C, Dafforn T, Reichhart JM, Gubb D Abstract : Mutants of the necrotic (nec) gene in Drosophila melanogaster die in the late pupal stage as pharate adults, or hatch as weak, but relatively normal-looking, flies. Adults develop black melanized spots on the body and leg joints, the abdomen swells with hemolymph, and flies die within 3 or 4 days of eclosion. The TOLL-mediated immune response to fungal infections is constitutively activated in nec mutants and pleiotropic phenotypes include melanization and cellular necrosis. These changes are consistent with activation of one or more proteolytic cascades. The nec gene corresponds to Spn43Ac, one of a cluster of three putative serine proteinase inhibitors at 43A1.2, on the right arm of chromosome 2. Although serpins have been implicated in the activation of many diverse pathways, lack of an individual serpin rarely causes a detectable phenotype. Absence of Spn43Ac, however, gives a clear phenotype, which will allow a mutational analysis of critical features of the molecular structure of serpins.	Genetics 2000: Vol 156, 1117-1127.	Abstract Article complet
Constitutive activation of Toll-mediated antifungal defense in serpin-deficient Drosophila Levashina E, Langley E, Green C, Gubb D, Ashburner M, Hoffmann JA & Reichhart JM Abstract : The antifungal defense of Drosophila is controlled by the spaetzle/Toll/cactus gene cassette. Here, a loss-of-function mutation in the gene encoding a blood serine protease inhibitor, Spn43Ac, was shown to lead to constitutive expression of the antifungal peptide drosomycin, and this effect was mediated by the spaetzle and Toll gene products. Spaetzle was cleaved by proteolytic enzymes to its active ligand form shortly after immune challenge, and cleaved Spaetzle was constitutively present in Spn43Ac-deficient flies. Hence, Spn43Ac negatively regulates the Toll signaling pathway, and Toll does not function as a pattern recognition receptor in the Drosophila host defense.	Science 1999: 285 1917-1919	Abstract Article complet
Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin Levashina E, Ohresser S, Lemaitre B & Imler JL Abstract : Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Here we present evidence that induction of metchnikowin gene expression can be mediated either by the TOLL pathway or by the imd gene product. We show that the gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient, imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. Our results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene.	J. Mol. Biol 1998: 278, 515-527	Abstract Article complet
Similarities between insect and plant host defenses Levashina EA Abstract :	Trends Cell Biol. 1997: 7, 316 (Review)	Abstract Article complet
Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties. LEVASHINA E, OHRESSER S, BULET P, REICHHART JM, HETRU C, HOFFMANN JA Abstract :	Eur. J. Biochem. 1995 : 233 694-700	Abstract Article complet
The Influence of Plant Phenotype on the Processes of Regeneration Lutova LA, Bondarenko LV, Buzovkina IS, Levashina EA, Tikhodeev ON, Khodzhaiova LT, Sharova NV, and Shishkova SO Abstract :	Russian Journal of Genetics 1994: 30: 1065-1074	Abstract Article complet
Sterol Biosynthesis Mutants of Higher Plants Lutova LA, Levashina EA, Bondarenko LV, Bayramova NL, Andronova EV, Inge-Vechtomov SG Abstract :	Genetika 1992: 28: 129-136	Abstract Article complet