NHGRI/NIAID-Approved White Paper: Genome Analysis Of Vectorial Capacity In Major Anopheles Vectors Of Malaria Parasites

The development of transgenic insect technology has the potential to combat a broad range of vector-borne diseases and insect pests. Current technologies involve transposable genetic elements such as piggyBac, Hermes, mariner-Mos1 and Minos. These have been used successfully to integrate transgenes into the genomes of numerous insect species, including Diptera, Lepidoptera, Coleoptera and Hymenoptera. However, such transformation vectors are inefficient, have limited carrying capacity and can give rise to position effects, insertional mutagenesis and instability. As an alternative approach, we previously reported a site-specific integration mechanism in Aedes aegypti using a viral integrase system from the Streptomyces phage phiC31. First, piggyBac transformation was used to introduce an attP target site into the genome, then strains were tested for site-specific integration of attB at the target site. The phiC31 system was highly successful at increasing integration efficiency, partly from the intrinsic efficiency of phiC31 integrase and partly from a doubling of survival when embryos and larvae were no longer exposed to piggyBac transposase. The phiC31 integration system has subsequently been developed in Anopheles gambiae. As before, we used piggyBac transformation to introduce the attP target site into the genome, generating multiple independent phase 1 ̳targeting‘ strains. These strains are now established, genetically characterised and in use for phase 2 integrations. One strain has been used for sitespecific integration of a putative anti-malarial peptide (Vida 3), under control of the Anopheles gambiae carboxypeptidase (AgCP) promoter, to drive post blood-meal expression. This phase 2 strain has been fully characterised and tested in infection studies. Since it is unlikely that a single anti-malarial peptide will be capable of blocking 100% of parasites, a major goal of this project is to target Plasmodium in different tissues of the same mosquito. We have characterised the Anopheles gambiae anti-platelet protein gene to use its control regions for expression of anti-malarial genes in the salivary glands. Our own experience from the generation of phase 2 transformants, plus information from the literature and other laboratories, would suggest that a major factor in limiting transformation efficiency in the phiC31 system is delivery of functional integrase mRNA together with the phase 2 plasmid. We are therefore planning to generate an integrase driver line to synthesise mRNA from the Anopheles gambiae nanos promoter. The ability to efficiently target transgenes to specific chromosomal locations and potentially to integrate very large transgenes has broad applicability, with interest to research on many medically and economically important species. Site-specific integration should circumvent many of the limitations associated with transposable elements and would facilitate true comparative transgene analyses, such as those required for promoter optimization. VectorBase: Driving Biological Projects Program F.H. Collins and Kitsos Louis for the VectorBase Consortium Eck Institute for Global Health University of Notre Dame Notre Dame, IN 46556, USA The VectorBase Bioinformatics Resource Center, a NIH-funded resource for management and analysis of genomes and genome-related data for arthropod vectors of human pathogens, is scheduled to begin its second 5 year funding cycle in September 2009. A major feature of this second cycle will be the establishment and implementation of a grant program for the vector research community called the VectorBase Driving Biological Projects program. This program will enable VectorBase to collaborate with its users by funding community research projects that will generate experimental evidence of value to the annotation of the genomes, proteomes or metabolomes of the targeted organisms managed by VectorBase. This talk will give a preliminary view of the nature and scope of the Driving Biological Projects Program and how it will likely be implemented. Leucine-rich repeat protein complex activates a mosquito complement-like pathway in defense against Plasmodium parasites Michael Povelones*, Robert M. Waterhouse, Fotis C. Kafatos & George K. Christophides Division of Cell and Molecular Biology, Department of Life Sciences, Imperial College, London, United Kingdom Leucine-rich repeat (LRR) containing proteins are central to host defense in plants and animals. One of the most striking examples of the versatility of the LRR motif in mediating recognition of diverse pathogen-associated molecules is the adaptive immune system of jawless vertebrates. In these animals variable lymphocyte receptor antibodies are generated via combinatorial assembly of LRR modules instead of immunoglobulin segments as in jawed vertebrates. Two Anopheles gambiae LRR proteins, LRIM1 and APL1C, have been previously reported as major antagonists of infections with Plasmodium berghei. We found that LRIM1 and APL1C circulate in the hemolymph as a 260 kDa disulfide-bonded multimeric complex and that the LRIM1/APL1C complex is the only form of the proteins in the mosquito hemolymph. The complex interacts with the complement C3-like protein, TEP1, promoting its cleavage or stabilization, and its subsequent localization on the surface of midgut-invading parasites, targeting them for destruction. Based on their shared structural features (signal peptide, LRRs, cysteine pattern and coiled-coils), we identify LRIM1 and APL1C as founding members of an A. gambiae protein family with over 20 members that can be divided into 4 distinct sub-families: long LRIMs, like LRIM1 and APL1C, with 10 or more LRRs, short LRIMs with 6 or 7 LRRs, transmembrane LRIMs exhibiting the defining features but with a predicted C-terminal transmembrane domain and coil-less LRIMs, which are missing only the coiled-coil domain. Similar numbers of orthologs are present in other disease-vector mosquitoes but not in any other species. This novel LRIM family of putative recognition receptors may be important in mosquito innate defense against human and animal pathogens. Ongoing experiments in the lab are focused on determining whether LRIMs play a role in specificity of mosquito immune responses to diverse pathogens. TWO C-TYPE LECTINS COOPERATE TO DEFEND ANOPHELES GAMBIAE AGAINST GRAM-NEGATIVE BACTERIA Anna K.D. Schnitger 1 , Hassan Yassine 2 , Fotis C. Kafatos 1 and Mike A. Osta 2 1 Imperial College London, Division of Cell and Molecular Biology, Exhibition Road, London, SW7 2AZ, United Kingdom. 2 Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Bliss Street, Beirut 11072020, Lebanon. Insect C-type lectins (CTLs) have been shown to mediate immune effector responses in vitro, however, the relative contribution of these CTLs to immune defenses in vivo is still poorly understood. Here, we report that two C-type lectin-like molecules, CTL4 and CTLMA2, which were shown previously to inhibit Plasmodium berghei ookinete melanization in the malaria vector Anopheles gambiae, are required for the clearance of Escherichia coli, but not Staphylococcus aureus, from adult female mosquitoes. Silencing either CTL by RNA interference dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria. Further, molecular characterization reveals that both CTLs are secreted into the mosquito hemolymph mainly in the form of a disulfide-linked heterodimer. This association explains the similar roles of these CTLs in bacterial defense as well as in the melanization response to P. berghei ookinetes. Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae. Immune responses triggered by bacterial isolates from the midgut of Anopheles mosquitoes Lotta Burström, Romanico B Arrighi, Yang Jiang, Jenny M Lindh, Ülo Langel, Ingrid Faye Department of Genetics, Microbiology and Toxicology, Stockholm University, Sweden Malaria is a parasitic disease transmitted by Anopheles mosquitoes. One novel approach to control malaria is paratransgenesis, in which a bacterium from the mosquito midgut is genetically modified to produce an anti-parasitic peptide. In this study we evaluate the immune response mounted by the Anopheles gambiae when three candidate bacteria, previously isolated from Anopheles mosquito midguts, were reintroduced with a blood meal. An ideal bacterium would elicit an immune response, to which it is resistant itself, in order to sustain in the gut and express anti-parasitic molecule(s). The immune response was monitored separately in midguts and carcasses for the expression of Cecropin A1, Gambicin and Defensin for 6hrs using quantative RT-PCR. Two Gram-negatives, Pantoea stewartii and Elizabethkingia sp (Lindh et al.2008), and one Gram-positive, Bacillus sp. (B2.1b) were tested (Lindh et al. 2005). The peptide genes were strongly up-regulated in the midguts, up to12 fold. The peak was seen between 3 -6 hrs post-feeding for all the bacteria. Interestingly, in the carcasses the peptides were peaking earlier, between 1-3 hrs post-feeding, reaching a lower level of expression. Viable counts revealed that P. stewartii and Elizabethkingia sp follow the same growth pattern in the gut of the mosquito as in blood kept at the same temperature. When the bacteria were tested for sensitivity to AgCecropin A, Elizabethkingia sp showed complete resistance, while the P. stewartii and Bacillus sp. isolates were sensitive. Thus, based on antimicrobial peptide resistance, the most suitable candidate for paratransgenics among the three tested would be Elizabethkingia sp. However, further criteria like transformability, expression of anti-parasitic peptides and long term sustainability in the mosquito need to be explored. References: Lindh JM, Borg-Karlson AK, Faye I (2008) Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteriacontaining water. Acta Trop 107(3):242-50. Lindh JM, Terenius O, Faye I. (2005) 16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts. Appl Environ Microbiol 71(11):7217-23 Title: Wolbachia-induced immune upregulation in mosquitoes and effects on vector competence Steven Sinkins Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford. [email protected] Abstract Infections of the maternally inherited bacterium Wolbachia pipientis are thought to be, in general, hidden from the host insect immune system. We designed and hybridized full genome microarrays against Wolbachia-infected and uninfected Aedes aegypti lines, and found that, in contrast to natural mosquito-Wolbachia combinations, an artificial transinfection induced a major upregulation of a number of immune genes. qRT-PCR experiments provided broad support for the array data and confirmed that the immune upregulation occurred throughout adult life. Based on the antimicrobial peptides encoded by some of the upregulated genes, we predicted a protective effect against bacterial pathogens of insects, and this was confirmed using Erwinia challenge. We also predicted an increased ability to kill the filarial nematodes that cause human lymphatic filariasis, and using Brugia pahangi as a model, showed significant reductions of filarial development to the infectious L3 stage in Wolbachia-infected females. These data have implications for the application of Wolbachia to tropical disease control, potentially providing a self-spreading non-GM method for population replacement.Infections of the maternally inherited bacterium Wolbachia pipientis are thought to be, in general, hidden from the host insect immune system. We designed and hybridized full genome microarrays against Wolbachia-infected and uninfected Aedes aegypti lines, and found that, in contrast to natural mosquito-Wolbachia combinations, an artificial transinfection induced a major upregulation of a number of immune genes. qRT-PCR experiments provided broad support for the array data and confirmed that the immune upregulation occurred throughout adult life. Based on the antimicrobial peptides encoded by some of the upregulated genes, we predicted a protective effect against bacterial pathogens of insects, and this was confirmed using Erwinia challenge. We also predicted an increased ability to kill the filarial nematodes that cause human lymphatic filariasis, and using Brugia pahangi as a model, showed significant reductions of filarial development to the infectious L3 stage in Wolbachia-infected females. These data have implications for the application of Wolbachia to tropical disease control, potentially providing a self-spreading non-GM method for population replacement. ANTIMICROBIAL PEPTIDES AS PART OF TICK IMMUNITY Daffre, S.*, ESTEVES, E., Cruz, C.E., Belmonte, R. Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo. CEP 05508-900, São Paulo, SP, Brazil. * e-mail: [email protected] The tick Rhipicephalus (Boophilus) microplus is the most important blood-sucking ectoparasite of cattle in the southern hemisphere and the biological vector of babesiosis and anaplasmosis. It has been already shown for mosquito the participation of immune system for control of parasites in several tissues (Cell Microbiol. 5: 3-14, 2003). We have been studying two aspects of the immune system of the cattle tick R. (B). microplus: i. phagocytosis and production of reactive oxygen species by hemocytes after microbial challenge (Exp Parasitol 99: 66-72, 2001); and ii. involvement of the antimicrobial peptides named microplusin, defensin (Dev Comp Immunol 28: 191-2000, 2004) and ixidodin (Peptides 27: 667674, 2006) in the Anaplasma infection. It has been observed by RTqPCR that the expression levels of the defensin and ixodidin genes in the tick embryonic cell line (BME 26) increase at 72 hours post-infection, by the other hand the levels of expression of the microplusin remaining unaltered. Experiments on RNA interference are underway to address the importance of AMPs genes for Anaplasma infection establishment. Another antimicrobial peptide (Hb33-61) derived from the alpha-chain of bovine hemoglobin has been purified from the gut contents of the tick R. (B). microplus (J. Biol. Chem. 274: 2533025334, 1999). Some animals have the strategy of utilizing hemoglobin as a source of endogenous biologically active peptides through it`s hydrolysis by specific proteases. These peptides have several properties such as antimicrobial, hormornal, opioid, antihibernatic and Ca+2 regulatory activities. We report the purification and biochemical characterization of the two proteinases involved on the cleavage of bovine hemoglobin to generate Hb33-61. One aspartic proteinase was purified from tick gut through ion exchange, hydrophobic interaction and gel filtration chromatography. Enzyme activity was determined with the fluorogenic substrate containing the aminoacid sequence 29-35 of the alpha-chain of bovine hemoglobin (SF 29-35). Cleavage of this substrate was observed between the residues M32 and F33. The cloned aspartic proteinase has a predicted molecular mass of 40 kDa and showed 82% identity with longepsin from Haemaphysalis longicornis. By the other hand, through ion exchange chromatography, we found a major cysteine proteinase that cleaves the fluorogenic substrate SF 57-67 between the residues A63 and A64. Interestingly, BmCL1, the cysteine endoproteinase immunolocalized to the tick gut (Insect Mol. Biol. 11(4): 325-328, 2002), showed the same cleavage specificity of SF 57-67. One study about the specificity of this protease was carried on using a combinatory library of synthetic peptides. Presently we are analyzing hemoglobin processing through in vitro digestion of the purified alpha-chain of hemoglobin by aspartic and cysteine proteinases. Other fragments originated from the hemoglobin digestion in the tick gut were purified by filtration and reversedphase chromatography showing antimicrobial activity. All of them are structurally correlated having alpha-helix as motif. Therefore, we hypothesize that the hemoglobin proteolysis might be important not only as nutrition source but also for microorganism control in tick gut. Supported by FAPESP and CNPq A set of ontologies to drive tools for the control of vector-borne diseases Pantelis Topalis 1 , Emmanuel Dialynas 1 , Elvira Mitraka 2 , Elena Deliyanni 1 , Inga Siden-Kiamos 1 and Christos Louis 1,2 1 Institute of Molecular Biology and Biotechnology, Foundation for Research and TechnologyHellas, 711 10 Heraklion, Crete, Greece; 2 Department of Biology, University of Crete, 711 10 Heraklion, Crete, Greece