RESUMEN
Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.
Asunto(s)
Aedes/efectos de los fármacos , Anopheles/efectos de los fármacos , Proteínas de Insectos/antagonistas & inhibidores , Repelentes de Insectos/farmacología , Receptores Odorantes/antagonistas & inhibidores , Compuestos Orgánicos Volátiles/farmacología , Aedes/fisiología , Aldehídos/química , Aldehídos/farmacología , Animales , Anopheles/fisiología , Monoterpenos Bicíclicos/química , Monoterpenos Bicíclicos/farmacología , Unión Competitiva , Cinamatos/química , Cinamatos/farmacología , Cimenos/química , Cimenos/farmacología , DEET/química , DEET/farmacología , Relación Dosis-Respuesta a Droga , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Repelentes de Insectos/química , Cinética , Monoterpenos/química , Monoterpenos/farmacología , Mosquitos Vectores/efectos de los fármacos , Mosquitos Vectores/fisiología , Odorantes/análisis , Unión Proteica , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Olfato/fisiología , Relación Estructura-Actividad , Compuestos Orgánicos Volátiles/químicaRESUMEN
In this work we report a fast and efficient virtual screening protocol for discovery of novel bioinspired synthetic mosquito repellents with lower volatility and, in all likelihood, increased protection time as compared with their plant-derived parental compounds. Our screening protocol comprises two filtering steps. The first filter is based on the shape and chemical similarity to known plant-derived repellents, whereas the second filter is based on the predicted similarity of the ligand's binding mode to the Anopheles gambiae odorant binding protein (AgamOBP1) relative to that of DEET and Icaridin to the same OBP. Using this protocol, a chemical library containing 42,755 synthetic molecules was screened in silico and sixteen selected compounds were tested for their affinity to AgamOBP1 in vitro and repellence against A. gambiae female mosquitoes using a warm-body repellent assay. One of them showed DEET-like repellence (91%) but with significantly lower volatility (2.84â¯×â¯10-6â¯mmHg) than either DEET (1.35â¯×â¯10-3â¯mmHg) or its parental cuminic acid (3.08â¯×â¯10-3â¯mmHg), and four other compounds were found to exhibit repellent indices between 69 and 79%. Overall, a correlation was not evident between repellence and OBP-binding strength. In contrast, a correlation between binding mode and repellence was found.
Asunto(s)
Descubrimiento de Drogas/métodos , Repelentes de Insectos/análisis , Receptores Odorantes/agonistas , Animales , Culicidae , Femenino , Cobayas , Ligandos , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas PequeñasRESUMEN
There is increasing interest in the development of effective mosquito repellents of natural origin to reduce transmission of diseases such as malaria and yellow fever. To achieve this we have employed an in vitro competition assay involving odorant-binding proteins (OBPs) of the malaria mosquito, Anopheles gambiae, with a predominantly female expression bias to identify plant essential oils (EOs) containing bioactive compounds that target mosquito olfactory function. EOs and their fractions capable of binding to such OBPs displayed repellence against female mosquitoes in a laboratory repellent assay. Repellent EOs were subjected to gas chromatographic analysis linked to antennogram (EAG) recordings from female A. gambiae to identify the biologically active constituents. Among these compounds cumin alcohol, carvacrol, ethyl cinnamate and butyl cinnamate proved as effective as DEET at an equivalent dose in the repellent assay, and combinations of carvacrol with either butyl cinnamate or cumin alcohol proved to be significantly more effective than DEET in the assay. When tested as spatial repellents in experimental shelters housing sleeping humans in northern Nigeria a binary mixture of carvacrol plus cumin alcohol caused mosquitoes to leave shelters in significantly higher numbers to those induced by DEET in female Anopheles spp. and in numbers equivalent to that of DEET in Culex spp. mosquitoes. These findings indicate an approach for the identification of biologically active molecules of natural origin serving as repellents for mosquitoes.
Asunto(s)
Anopheles , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Insectos , Repelentes de Insectos/farmacología , Receptores Odorantes , Caracteres Sexuales , Animales , Anopheles/genética , Anopheles/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Receptores Odorantes/biosíntesis , Receptores Odorantes/genéticaRESUMEN
Insect olfactory receptors (ORs) are heteromeric ligand-gated cation channels composed of a common olfactory receptor subunit (ORco) and a variable subunit (ORx) of as yet unknown structures and undetermined stoichiometries. In this study, we examined the allosteric modulation exerted on Anopheles gambiae heteromeric ORx/ORco olfactory receptors in vitro by a specific class of ORco agonists (OAs) comprising ORcoRAM2 and VUAA1. High OA concentrations produced stronger functional responses in cells expressing heteromeric receptor channels relative to cells expressing ORco alone. These OA-induced responses of ORx/ORco channels were also notably much stronger than those obtained upon administration of ORx-specific ligands to the same receptors. Most importantly, small concentrations of OAs were found to act as strong potentiators of ORx/ORco function, increasing dramatically both the efficacy and potency of ORx-specific odorants. These results suggest that insect heteromeric ORs are highly dynamic complexes adopting different conformations that change in a concerted fashion as a result of the interplay between the subunits of the oligomeric assemblies, and that allosteric modulation may constitute an important element in the modulation and fining tuning of olfactory reception function.
RESUMEN
In this study, we have deleted the lef8 gene of the baculovirus BmNPV, which encodes one of the viral RNA polymerase subunits, in order to create a knockout bacmid, Δlef8, directing cytopathology-free single-cell infections for gene transduction and recombinant protein production. However, while removal of the complete lef8 ORF produced the expected phenotype, it also affected the function of the closely linked essential gene orf40, thus hampering the mutant bacmid rescue in cultured Bombyx cells expressing recombinant LEF8. Subsequently, we determined that several diverse sequences can substitute for the orf40 5'-upstream sequences that were removed by the deletion of the lef8 gene and also showed that neither a physical linkage nor expression of the two relevant genes under native promoter control is a prerequisite for a fully functional virus. Based on these findings, we generated a rescue-competent lef8-null vector, which contained a heterologous promoter-driven orf40. This lef8-deficient vector, which produces productive infections and progeny virus lacking lef8 in deficiency-complementing cells expressing LEF8, could be used as the basis for an alternative to current silkmoth transduction systems.
Asunto(s)
Bombyx/virología , ARN Polimerasas Dirigidas por ADN/genética , Eliminación de Gen , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Transducción Genética , Proteínas Virales/genética , Animales , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Viral de la Expresión Génica , Proteínas Virales/metabolismoRESUMEN
Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig, and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as "middle", and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization, and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins.
Asunto(s)
Bombyx/genética , Corion , Sitios de Carácter Cuantitativo , Animales , Bombyx/metabolismo , Biología Computacional/métodos , Proteínas del Huevo , Cáscara de Huevo , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica , Biblioteca de Genes , Orden Génico , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Proteoma , Proteómica/métodos , Transcripción GenéticaRESUMEN
The silkmoth chorion was studied extensively by F.C. Kafatos' group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis.
Asunto(s)
Bombyx/genética , Corion , Genoma de los Insectos , Animales , Bombyx/embriología , Mapeo Cromosómico , Estructuras Cromosómicas , Cromosomas Artificiales Bacterianos , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Anotación de Secuencia MolecularRESUMEN
The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca(2+)-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET.
Asunto(s)
Anopheles/fisiología , Repelentes de Insectos , Receptores Odorantes/metabolismo , Animales , Anopheles/metabolismoRESUMEN
Differential regulation at the level of transcription provides a means for controlling gene expression in eukaryotes, especially during development. Insect model systems have been extensively used to decipher the molecular basis of such regulatory cascades, and one of the oldest such model systems is the regulation of chorion gene expression during ovarian follicle maturation. Recent experimental and technological advances have shed new light onto the system, allowing us to revisit it. Thus, in this review we try to summarize almost 40 years' worth of studies on chorion gene regulation while-by comparing Bombyx mori and Drosophila melanogaster models-attempting to present a comprehensive, unified model of the various regulatory aspects of choriogenesis that takes into account the evolutionary conservation and divergence of the underlying mechanisms.
Asunto(s)
Bombyx/genética , Drosophila melanogaster/genética , Proteínas del Huevo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/genética , Animales , Evolución Biológica , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Corion/crecimiento & desarrollo , Corion/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas del Huevo/metabolismo , Proteínas de Insectos/metabolismo , Pupa/crecimiento & desarrollo , Pupa/metabolismoRESUMEN
We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.
Asunto(s)
Autoanticuerpos/metabolismo , Músculo Esquelético/metabolismo , Miastenia Gravis/inmunología , Miastenia Gravis/terapia , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/uso terapéutico , Animales , Autoanticuerpos/sangre , Western Blotting , Bungarotoxinas/metabolismo , Línea Celular , Dicroismo Circular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos , Humanos , Técnicas de Inmunoadsorción , Radioisótopos de Yodo/metabolismo , Mariposas Nocturnas , Pichia , Plásmidos/genética , Estructura Terciaria de Proteína/fisiología , RadioinmunoensayoRESUMEN
Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases.
Asunto(s)
Anopheles/química , Proteínas de Insectos/química , Lipocalinas/química , Multimerización de Proteína , Animales , Anopheles/genética , Anopheles/metabolismo , Antenas de Artrópodos/química , Antenas de Artrópodos/metabolismo , Cristalografía por Rayos X , Femenino , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER) in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer's larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.
Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Proteínas de Insectos/genética , Mariposas Nocturnas/genética , Interferencia de ARN , Animales , Baculoviridae/genética , Hidrolasas de Éster Carboxílico/metabolismo , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hemolinfa/metabolismo , Proteínas de Insectos/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Microscopía Fluorescente , Mariposas Nocturnas/crecimiento & desarrollo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Genética Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A simple technique to enhance the humoral immune response to intracellular protein antigens in genetic immunizations is demonstrated in mice. In this approach, the intracellular protein is intentionally secreted from expressing cells as a chimeric protein, comprising an N-terminal secreted protein fused to the intracellular protein antigen. Using the Leishmania chagasi Ldccys1 cysteine protease (411CP) as an example of an intracellular protein antigen and both human and murine granulocyte colony stimulating factor (GMCSF) as examples of N-terminal secretion competent fusion partners in chimeric proteins, a humoral response in plasmid DNA immunized mice could only be detected by Western blotting when the expressed 411CP was secreted.
Asunto(s)
Antígenos/inmunología , Inmunidad Humoral/inmunología , Inmunogenética/métodos , Proteínas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Antígenos/genética , Antígenos/metabolismo , Western Blotting , Células CHO , Cricetinae , Cricetulus , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inmunización , Espacio Intracelular/inmunología , Espacio Intracelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Plásmidos/inmunología , Proteínas/genética , Proteínas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Vacunas de ADN/metabolismoRESUMEN
We present the characterization of BmVMP90, a vitelline membrane protein (VMP) of the silkmoth Bombyx mori bearing similarities with dipteran VMPs whose existence had recently been suggested by an in silico analysis of the silkmoth genome and follicular cell RNA expression analyses. Using a specific antibody, we determine the presence of BmVMP90 protein in ovarian follicular cell extracts at the end of vitellogenesis and in vitelline membrane extracts but not in the chorion of fractionated eggshells isolated from ovulated follicles. Whole mount follicle immunofluorescence studies reveal a pattern of BmVMP90 deposition matching the «imprinted¼ pattern of follicular cells on the vitelline membrane surface. Antisense DNA-directed inhibition BmVMP90 expression in ex vivo cultures of early vitellogenic follicles produced a phenotype of kidney- or bean-shaped follicles with detached follicular epithelia, suggestive of the importance of BmVMP90 for the integrity of developing follicles and normal deposition of the chorion structure that follows vitelline membrane formation but no adverse effects on the execution of the follicular cell-imprinted program of choriogenesis per se.
Asunto(s)
Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Proteínas del Huevo/metabolismo , Proteínas de Insectos/metabolismo , Animales , Bombyx/genética , Proteínas del Huevo/genética , Femenino , Expresión Génica , Proteínas de Insectos/genética , Masculino , Folículo Ovárico/crecimiento & desarrollo , Conejos , Análisis de Secuencia de ADN , VitelogénesisRESUMEN
A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses.
Asunto(s)
Bombyx/enzimología , Desoxirribonucleasas/genética , Proteínas de Insectos/genética , Ribonucleasas/genética , Secuencia de Aminoácidos , Animales , Bombyx/química , Bombyx/clasificación , Bombyx/genética , Células Cultivadas , Citoplasma/química , Citoplasma/enzimología , Citoplasma/genética , ADN/genética , ADN/metabolismo , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Filogenia , Transporte de Proteínas , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Ribonucleasas/química , Ribonucleasas/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
In the tripartite parasitization system of the lepidopteran host Manduca sexta, the endoparasitoid wasp Cotesia congregata and its endosymbiotic virus, C. congregata Bracovirus (CcBV), the expression of viral proteins is necessary for successful parasitization. Here we have examined the in vitro effects of six members of the ankyrin-repeat protein family (Ank) of CcBV, which are thought to interfere with the host's induced innate immune responses, on the transcriptional activity of a heterologous lepidopteran Rel/NFκB transcription factor, Relish1 of Bombyx mori. Using as transcriptional activator BmRelish1-d2 (R1d2), a constitutively active mutant of the major regulator of the Imd pathway, BmRelish1, in conjunction with a reporter gene controlled by a B. mori antimicrobial peptide gene promoter, we have found that 5 of the 6 examined Anks suppress R1d2-dependent transcriptional activity to various degrees. Immunofluorescence studies have also revealed that while some of the Ank proteins have a rather strict cytoplasmic localization, others are detected both in the cytoplasm and the nucleus of the expressing cells and that colocalization with R1d2 occurs exclusively in the nucleus. Thus, our results suggest that functional and spatial differences among the various CcBV Ank family members may be responsible for the observed differential inhibition of R1d2 activity.
Asunto(s)
Interacciones Huésped-Parásitos , Proteínas de Insectos/metabolismo , Manduca/inmunología , Proteínas Virales/metabolismo , Avispas/virología , Animales , Repetición de Anquirina , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica , Manduca/parasitología , Manduca/virologíaRESUMEN
To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.
Asunto(s)
Anopheles/metabolismo , Receptores Odorantes/metabolismo , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Dimerización , Colorantes Fluorescentes/farmacología , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptores Odorantes/química , Receptores Odorantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensilos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.
Asunto(s)
Regulación de la Expresión Génica , Lepidópteros/genética , Lepidópteros/inmunología , Interferencia de ARN , Animales , Bases de Datos Genéticas , Epidermis/crecimiento & desarrollo , Silenciador del Gen , Inmunidad Innata , Proteínas de Insectos/efectos de los fármacos , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Lepidópteros/efectos de los fármacos , Lepidópteros/crecimiento & desarrollo , ARN Bicatenario/efectos de los fármacos , Proyectos de InvestigaciónRESUMEN
A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel 'topology screen' assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties.
Asunto(s)
Anopheles/metabolismo , Membrana Celular/metabolismo , Proteínas de Insectos/metabolismo , Receptores Odorantes/metabolismo , Secuencia de Aminoácidos , Animales , Anopheles/genética , Western Blotting , Línea Celular , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Insectos/genética , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Odorantes/genética , TransfecciónRESUMEN
Odor-detection in the malaria mosquito Anopheles gambiae involves large families of diverse proteins, including multiple odorant binding proteins (AgOBPs) and olfactory receptors (AgORs). The receptors AgOR1 and AgOR2, as well as the binding protein AgOBP1, have been implicated in the recognition of human host odors. In this study, we have explored the expression of these olfactory proteins, as well as the ubiquitous odorant receptor heteromerization partner AgOR7, in the thirteen flagellomeres (segments) of female and male antenna. Expressing cells were visualized by adapting a whole mount fluorescence in situ hybridization method. In female mosquitoes, AgOR1-expressing olfactory receptor neurons (ORNs) were almost exclusively segregated in segments 3 to 9, whereas AgOR2-expressing ORNs were distributed over flagellomeres 2 to 13. Different individuals comprised a similar number of cells expressing a distinct AgOR type, although their antennal topography and number per flagellomere varied. AgOBP1-expressing support cells were present in segments 3 to 13 of the female antenna, with increasing numbers towards the distal end. In male mosquitoes, total numbers of AgOR- and AgOBP1-expressing cells were much lower. While AgOR2-expressing cells were found on both terminal flagellomeres, AgOR1 cells were restricted to the most distal segment. High densities of AgOBP1-expressing cells were identified in segment 13, whereas segment 12 comprised very few. Altogether, the results demonstrate that both sexes express the two olfactory receptor types as well as the binding protein AgOBP1 but there is a significant sexual dimorphism concerning the number and distribution of these cells. This may suggest gender-specific differences in the ability to detect distinct odorants, specifically human host-derived volatiles.