RESUMEN
Many herbivorous insects are mono- or oligophagous, having evolved to select a limited range of host plants. They specifically identify host-plant leaves using their keen sense of taste. Plant secondary metabolites and sugars are thought to be key chemical cues that enable insects to identify host plants and evaluate their quality as food. However, the neuronal and behavioral mechanisms of host-plant recognition are poorly understood. Here, we report a two-factor host acceptance system in larvae of the silkworm Bombyx mori, a specialist on several mulberry species. The first step is controlled by a chemosensory organ, the maxillary palp (MP). During palpation at the leaf edge, the MP detects trace amounts of leaf-surface compounds, which enables host-plant recognition without biting. Chemosensory neurons in the MP are tuned with ultrahigh sensitivity (thresholds of attomolar to femtomolar) to chlorogenic acid (CGA), quercetin glycosides, and ß-sitosterol (ßsito). Only if these 3 compounds are detected does the larva make a test bite, which is evaluated in the second step. Low-sensitivity neurons in another chemosensory organ, the maxillary galea (MG), mainly detect sucrose in the leaf sap exuded by test biting, allowing larvae to accept the leaf and proceed to persistent biting (feeding). The two-factor host acceptance system reported here may commonly underlie stereotyped feeding behavior in many phytophagous insects and determine their feeding habits.
Asunto(s)
Bombyx/fisiología , Conducta de Elección , Dieta , Conducta Alimentaria/fisiología , Larva/fisiología , Papilas Gustativas/fisiología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Conducta Animal/fisiología , Bombyx/anatomía & histología , Bombyx/crecimiento & desarrollo , Células Quimiorreceptoras/fisiología , Quimiotaxis/fisiología , Señales (Psicología) , Conducta Exploratoria/fisiología , Interacciones Huésped-Parásitos/fisiología , Larva/anatomía & histología , Larva/citología , Morus/química , Hojas de la Planta/química , Gusto/fisiología , Papilas Gustativas/anatomía & histologíaRESUMEN
Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Bombyx/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Insecticidas/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Bombyx/crecimiento & desarrollo , Endotoxinas/química , Células HEK293 , Proteínas Hemolisinas/química , Humanos , Insecticidas/química , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Conformación Proteica , Especificidad por SustratoRESUMEN
The taste sensing system is crucial for food recognition in insects and other animals. It is commonly believed that insect gustatory receptors (Grs) expressed in gustatory organs are indispensable for host plant selection. Many behavioral studies have shown that mono- or oligo-phagous lepidopteran insects use the balance between feeding attractants and feeding deterrents in host plants and that these are sensed by taste organs for host plant recognition. However, the molecular mechanism underlying taste recognition, especially of feeding deterrents, remains to be elucidated. To better understand this mechanism, we studied orphan Grs, including Bombyx mori Gr (BmGr) 16, BmGr18, and BmGr53, from the mono-phagous insect, Bombyx mori. Using Calcium imaging in mammalian cells, we first confirmed in lepidoptera insects that three of the putative bitter Grs widely responded to structurally different feeding deterrents. Although the phylogenetic distance of these Grs was considerable, they responded to partially overlapping deterrents of plant secondary metabolites. These findings suggest that not only these three Grs but also most of the Grs that have been assigned to putative bitter Grs are feeding-deterrent receptors that play a role in host plant recognition.
Asunto(s)
Bombyx/fisiología , Interacciones Huésped-Parásitos , Plantas/metabolismo , Plantas/parasitología , Receptores de Superficie Celular/metabolismo , Metabolismo Secundario , Gusto/fisiología , Animales , Bombyx/efectos de los fármacos , Bombyx/genética , Calcio/metabolismo , Cumarinas/farmacología , Conducta Alimentaria , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Proteínas de Insectos/metabolismo , Especificidad de Órganos , Metabolismo Secundario/efectos de los fármacos , Gusto/efectos de los fármacosRESUMEN
Information about the receptor-interaction region of Cry toxins, insecticidal proteins produced by Bacillus thuringiensis, is needed to elucidate the mode of action of Cry toxins and improve their toxicity through protein engineering. We analyzed the interaction sites on Cry1Aa with ABC transporter C2 (ABCC2), one of the most important Cry1A toxin receptors. A competitive binding assay revealed that the Bombyx mori ABCC2 (BmABCC2) Cry1A binding site was the same as the BtR175 binding site, suggesting that the loop region of Cry1Aa domain II is a binding site. Next, we constructed several domain II loop mutant toxins and tested their binding affinity in an SPR analysis, and also performed a cell swelling assay to evaluate receptor-mediated cytotoxicity. Our results indicate that the loop regions required for BtR175 and BmABCC2 binding and the regions important for cytotoxicity partially overlap. Our results also suggest that receptor binding is necessary but not sufficient for cytotoxicity. This is the first report showing the region of interaction between ABCC2 and Cry1Aa and the cytotoxicity-relevant properties of the Cry1Aa domain II loop region.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Bombyx/metabolismo , Cadherinas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Larva/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Toxinas Bacterianas/metabolismo , Sitios de Unión/fisiología , Proteínas de Insectos/metabolismo , Insecticidas/metabolismo , Unión Proteica/fisiología , Células Sf9RESUMEN
Herbivorous insects can identify their host plants by sensing plant secondary metabolites as chemical cues. We previously reported the two-factor host acceptance system of the silkworm Bombyx mori larvae. The chemosensory neurons in the maxillary palp (MP) of the larvae detect mulberry secondary metabolites, chlorogenic acid (CGA), and isoquercetin (ISQ), with ultrahigh sensitivity, for host plant recognition and feeding initiation. Nevertheless, the molecular basis for the ultrasensitive sensing of these compounds remains unknown. In this study, we demonstrated that two gustatory receptors (Grs), BmGr6 and BmGr9, are responsible for sensing the mulberry compounds with attomolar sensitivity for host plant recognition by silkworm larvae. Calcium imaging assay using cultured cells expressing the silkworm putative sugar receptors (BmGr4-10) revealed that BmGr6 and BmGr9 serve as receptors for CGA and ISQ with attomolar sensitivity in human embryonic kidney 293T cells. CRISPR/Cas9-mediated knockout (KO) of BmGr6 and BmGr9 resulted in a low probability of making a test bite of the mulberry leaves, suggesting that they lost the ability to recognize host leaves. Electrophysiological recordings showed that the loss of host recognition ability in the Gr-KO strains was due to a drastic decrease in MP sensitivity toward ISQ in BmGr6-KO larvae and toward CGA and ISQ in BmGr9-KO larvae. Our findings have revealed that the two Grs, previously considered to be sugar receptors, are molecules responsible for detecting plant phenolics in host plant recognition.
Asunto(s)
Bombyx , Humanos , Animales , Larva/fisiología , Bombyx/metabolismo , Plantas , Gusto/fisiología , Azúcares/metabolismo , Hojas de la Planta/metabolismoRESUMEN
Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae.
Asunto(s)
Bombyx , Proteínas de Drosophila , Animales , Gusto , Células Enteroendocrinas/metabolismo , Sistema Digestivo/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas de Drosophila/metabolismo , Insectos/metabolismo , Larva/metabolismo , Bombyx/metabolismo , Mamíferos/metabolismoRESUMEN
Cry proteins from Bacillus thuringiensis (Bt) and other bacteria are pesticidal pore-forming toxins. Since 2010, when the ABC transporter C2 (ABCC2) was identified as a Cry1Ac protein resistant gene, our understanding of the mode of action of Cry protein has progressed substantially. ABCC2 mediates high Cry1A toxicity because of its high activity for helping pore formation. With the discovery of ABCC2, the classical killing model based on pore formation and osmotic lysis became nearly conclusive. Nevertheless, we are still far from a complete understanding of how Cry proteins form pores in the cell membrane through interactions with their host gut membrane proteins, known as receptors. Why does ABCC2 mediate pore formation with high efficiency unlike other Cry1A-binding proteins? Is the "prepore" formation indispensable for pore formation? What is the mechanism underlying the synergism between ABCC2 and the 12-cadherin domain protein? We examine potential mechanisms of pore formation via receptor interactions in this paper by merging findings from prior studies on the Cry mode of action before and after the discovery of ABC transporters as Cry protein receptors. We also attempt to explain Cry toxicity using Cry-receptor binding affinities, which successfully predicts actual Cry toxicity toward cultured cells coexpressing ABC transporters and cadherin.
Asunto(s)
Bacillus thuringiensis , Transportadoras de Casetes de Unión a ATP/química , Animales , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/toxicidad , Cadherinas/metabolismo , Endotoxinas/toxicidad , Proteínas Hemolisinas/genética , Proteínas de Insectos/metabolismo , Larva/metabolismoRESUMEN
Ivermectin (IVM), an avermectin-derivative anthelmintic, specifically binds to glutamate-gated chloride ion channels (GluCls), causing paralysis in invertebrates. IVM also exhibits other biological activities such as Wnt/ß-catenin pathway inhibition in vertebrates that do not possess GluCls. This study showed that affinity purification using immobilized IVM B1a isolated TELO2, a cofactor of phosphatidylinositol 3-kinase-related kinases (PIKKs), as a specific IVM B1a-binding protein. TELO2 knockdown reduced cytoplasmic ß-catenin and the transcriptional activation of ß-catenin/TCF. IVM B1a bound to TELO2 through the C-terminal α-helix, in which mutations conferred IVM resistance. IVM reduced the TELO2 and PIKK protein levels and the AKT and S6 kinase phosphorylation levels. The inhibition of mTOR kinase reduced the cytoplasmic ß-catenin level. Therefore, IVM binds to TELO2, inhibiting PIKKs and reducing the cytoplasmic ß-catenin level. In conclusion, our data indicate TELO2 as a druggable target for human diseases involving abnormalities of the Wnt/ß-catenin pathway and PIKKs, including mTOR.
RESUMEN
Most lepidopteran insect larvae exhibit stepwise feeding behaviors, such as palpation using the maxillary palps (MPs) followed by test biting and persistent biting. However, the purpose of palpation has been unclear. In particular, nothing is known about the neurons in the MP and their mode of recognition of undesired plants, although such neurons have been suggested to exist. In this study, we used larvae of the stenophagous insect Bombyx mori and compared the roles of palpation and test biting in the selection of feeding behavior. When the larvae were given non-host plant leaves, they did not initiate test biting, indicating that non-host plant leaves were recognized via palpation without biting, and that this behavior resulted in a lack of persistent biting, as the leaves were judged non-suitable for consumption. Surface extracts of inedible leaves significantly suppressed test biting of mulberry leaves, a host plant of B. mori, suggesting that secondary metabolites on the leaf surface of inedible leaves function as test biting suppressors, even when another conditions are suitable for test biting. The allelochemical coumarin, which is found in the inedible leaves of cherry, Cerasus speciosa, significantly suppressed test biting of mulberry leaves, suggesting that coumarin is a possible deterrent to the eating of cherry leaves. Using the electrophysiological method of tip recording and a leaf-surface extract as the test material, leaf-surface compound-responsive neurons were identified in the MP. In addition, several neurons that respond to coumarin in the attomolar range were identified, suggesting that the larvae use ultrasensitive neurons in the MP to recognize inedible leaves. In the HEK293T cell heterologous expression system, the B. mori gustatory receptors BmGr53 and BmGr19, which were previously found to be expressed in the MP and to respond to coumarin in the attomolar range, responded to a leaf-surface extract of C. speciosa, suggesting that these receptors may be present on the inedible-leaf-recognizing neurons of the MP. These findings suggest that ultrasensitive plant secondary metabolite-recognizing neurons in the MP allow for the recognition of non-host plants via palpation without risking damage caused by ingesting harmful allelochemicals.
Asunto(s)
Bombyx , Conducta Alimentaria/fisiología , Feromonas , Percepción del Gusto/fisiología , Animales , Bombyx/metabolismo , Bombyx/fisiología , Células Quimiorreceptoras/metabolismo , Cumarinas/farmacología , Células HEK293 , Humanos , Larva/metabolismo , Larva/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Feromonas/farmacología , Extractos Vegetales/farmacología , Hojas de la Planta/metabolismo , Receptores de Superficie Celular/efectos de los fármacos , Gusto/fisiologíaRESUMEN
When ABC transporter family C2 (ABCC2) and ABC transporter family B1 (ABCB1) were heterologously expressed in non-susceptible cultured cells, the cells swelled in response to Cry1A and Cry3 toxins, respectively. Consistent with the notion that 3D-Cry toxins form cation-permeable pores, Bombyx mori ABCC2 (BmABCC2) facilitated cation-permeable pore formation by Cry1A when expressed in Xenopus oocytes. Furthermore, BmABCC2 had a high binding affinity (KD) to Cry1Aa of 3.1 × 10-10 M. These findings suggest that ABC transporters, including ABCC2 and ABCB1, are functional receptors for 3D-Cry toxins. In addition, the Cry2 toxins most distant from Cry1A toxins on the phylogenetic tree used ABC transporter A2 as a receptor. These data suggest that 3D-Cry toxins use ABC transporters as receptors. In terms of inducing cell swelling, ABCC2 has greater activity than cadherin-like receptor. The pore opening of ABC transporters was hypothesized to be linked to their receptor function, but this was repudiated by experiments using mutants deficient in export activity. The synergistic relationship between ABCC2 and cadherin-like receptor explains their ability to cause resistance in one species of insect.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/toxicidad , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Transportadoras de Casetes de Unión a ATP/química , Animales , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Antígenos CD13/metabolismo , Endotoxinas/química , Proteínas Hemolisinas/química , Humanos , Proteína 2 Asociada a Resistencia a Múltiples MedicamentosRESUMEN
The cadherin-like protein in lepidopteran insects, known as a receptor for Bacillus thuringiensis Cry1A toxins, is a single-pass membrane protein that can be divided into extracellular and intracellular regions. The extracellular region is important for toxin binding and oligomerization, whereas the role of the intracellular region during Cry1A intoxication is unclear. In the present study, we generated a deletion mutant of Bombyx mori cadherin-like protein (BtR175) that lacked the intracellular region to investigate its role in mediating Cry1A toxicity. Like wild-type BtR175, the mutant protein conferred susceptibility to Cry1Aa and Cry1Ab toxins in Sf9 cells, suggesting that the intracellular region is not required to mediate intoxication. The deletion mutant maintained another role of cadherin-like proteins; that it, synergistic activity with B. mori ABC transporter C2 (ABCC2) when mediating Cry1Aa and Cry1Ab toxicity. In addition, we evaluated the effects of reagents that have been reported to inhibit Cry1A toxicity (e.g., protein kinase A inhibitors, EDTA, and sucrose) on Cry1A toxicity in BtR175-expressing cells. Our results suggest that Cry1Aa-induced cell death in BtR175-expressing cells was not caused by signal transduction but by osmotic lysis. Overall, our data indicate that BtR175 mediates the toxicity of Cry1Aa and Cry1Ab toxins entirely via its extracellular region. They also indicate that the synergism between cadherin-like protein and ABCC2 occurs outside of cells or in the cell membrane.
Asunto(s)
Bacillus thuringiensis/fisiología , Proteínas Bacterianas/farmacología , Bombyx/genética , Cadherinas/genética , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Proteínas de Insectos/genética , Animales , Bacillus thuringiensis/crecimiento & desarrollo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Cadherinas/metabolismo , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas de Insectos/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Células Sf9RESUMEN
Cry proteins from Bacillus thuringiensis (Bt) have been used to control insect pests either as formulated sprays or as in Bt-crops. However, field-evolved resistance to Bt proteins is threatening the long-term use of Bt products. The SeABCC2 locus has been genetically linked to resistance to a Bt bioinsecticide (Xentari™) in Spodoptera exigua (a mutation producing a truncated form of the transporter lacking an ATP binding domain was found in the resistant insects). Here, we investigated the role of SeABCC2 in the mode of action of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and two Cry1A-1Ca hybrids by expressing the receptor in Sf21 and HEK293T cell lines. Cell toxicity assays showed that Sf21â¯cells expressing SeABCC2 become susceptible to Cry1A proteins. HEK293T cells expressing the transporter were found susceptible to Cry1A proteins but not to Cry1Ca. The results with the Cry1A-1Ca hybrids suggest that domain II from Cry1Ab/c is crucial for the toxicity to Sf21â¯cells, whereas domain III from Cry1Aa/b is crucial for the toxicity to HEK293T cells. Binding assays showed that the Cry1Ac binding is of high affinity and specific to cells expressing the SeABCC2 transporter. Heterologous competition experiments support a model in which domain II of Cry1Ab/c has a common binding site in the SeABCC2 protein, whereas domain III of Cry1Aa/b binds to a different binding site in the SeABCC2 protein.
Asunto(s)
Proteínas Bacterianas/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas de Insectos/genética , Larva/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Spodoptera/efectos de los fármacos , Animales , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Células Clonales , Endotoxinas/química , Endotoxinas/metabolismo , Endotoxinas/farmacología , Expresión Génica , Células HEK293 , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacología , Humanos , Proteínas de Insectos/metabolismo , Larva/citología , Larva/genética , Larva/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera/citología , Spodoptera/genética , Spodoptera/metabolismo , Relación Estructura-Actividad , TransfecciónRESUMEN
Enteroendocrine cells in the insect midgut are thought to secrete peptide hormones in response to the nutritional state. However, the role of dietary compounds in inducing peptide hormone secretion from enteroendocrine cells in insects remains unknown. In the present study, we demonstrated that several dietary compounds from mulberry leaves, including glucose, amino acids, and the secondary metabolite chlorogenic acid, induced significant secretion of tachykinin-related peptides from isolated silkworm midguts at the luminal concentrations measured in fed larvae. This study provides evidence that the insect midgut senses a non-nutritious secondary metabolite in addition to nutrient metabolites to monitor luminal food status and secretes a feeding regulatory hormone, suggesting that a unique dietary sensory system modulates insect feeding via enteroendocrine control.
Asunto(s)
Aminoácidos/farmacología , Bombyx/metabolismo , Ácido Clorogénico/farmacología , Glucosa/farmacología , Taquicininas/metabolismo , Animales , Bombyx/crecimiento & desarrollo , ADN Complementario/genética , Sistema Digestivo/metabolismo , Células Enteroendocrinas/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Morus/metabolismo , Hojas de la Planta/metabolismo , Metabolismo Secundario , Taquicininas/genéticaRESUMEN
The Bacillus thuringiensis Cry toxin causes swelling and necrosis in insect cells, but the route(s) and significance of the water influx involved in its cytotoxicity are unclear. Here, we assessed the role of aquaporins (AQPs), known as water channels, in Cry toxin intoxication. An AQP inhibitor did not interfere with any known process to form the toxin pore, but it diminished the cell swelling and loss of membrane integrity induced by the Cry toxin. Overexpression of AQPs facilitated water influx and cytotoxicity. Our results demonstrate that water influx via aquaporin directly determines necrotic cell death induced by the Cry toxin.
Asunto(s)
Acuaporinas/metabolismo , Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Agua/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Transporte Biológico/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Filogenia , Unión Proteica/efectos de los fármacos , Células Sf9 , Sodio/metabolismo , Ácidos Sulfónicos/farmacología , Factores de TiempoRESUMEN
In this study, we examined insect and human ABCC transporters closely related to the lepidopteran ABC transporter C2 (ABCC2), a powerful receptor for the Bacillus thuringiensis Cry toxin, for their responses to various Cry toxins. ABCC2 and the lepidopteran ABC transporter C3 (ABCC3) conferred cultured cells with susceptibility to a lepidopteran-specific Cry1Aa toxin but not to lepidopteran-specific Cry1Ca and Cry1Da. One coleopteran ABCC transporter specifically responded to a coleopteran-specific Cry8Ca toxin. ABCC transporters from a dipteran insect and humans did not respond to any of the tested Cry toxins that are active to lepidopteran and coleopteran insects. These results yield important information for our understanding of insect specificity of Cry toxins and provide the first demonstration of a coleopteran ABCC transporter that serves as a Cry toxin receptor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/metabolismo , Lepidópteros/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Escarabajos/efectos de los fármacos , Dípteros/efectos de los fármacos , Endotoxinas/química , Endotoxinas/toxicidad , Células HEK293 , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidad , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Filogenia , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genéticaRESUMEN
Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770DYWL773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770DYWL773 of ECL 4 in the ABCC2.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bombyx/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Bombyx/genética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Células Sf9 , SpodopteraRESUMEN
Bacillus thuringiensis produces Cry toxins, which are used as insecticides in sprays and in transgenic crops. However, little is known about the function of Cry toxin receptors and the mechanisms that determine their binding specificity and activity. In this study, the cRNAs of Bombyx mori ABC transporter C2 (BmABCC2), the toxin-binding region of cadherin-like receptor (BtR175-TBR), or aminopeptidase N1 (BmAPN1) were injected into Xenopus oocytes, and the Cry1Aa-dependent cation-selective pore formation activities of these receptors were analyzed using a two-electrode voltage clamp. Cation current passing through the pores was detected within 25 s, and increased in a linear fashion in BmABCC2-expressing oocytes treated with 88 nm Cry1Aa. This result suggested that Cry1Aa continuously made stable pores with the help of BmABCC2. In contrast, no cation current was observed until 60 min after incubation with 500 nm Cry1Aa in BtR175TBR-expressing oocytes even though oligomerization of Cry1Aa progressed. This result indicated that in the presence of BtR175-TBR most of the oligomerized toxin could not enter the cell membrane. However, oocytes that simultaneously expressed both receptors demonstrated that BtR175-TBR exerted a synergistic effect with BmABCC2 on pore formation in the presence of 22 nm Cry1Aa. These results confirm that the main reason for moderate-level resistance in insects lacking the cadherin-like receptor but expressing ABCC2 is the absence of a similar synergistic promotion of toxin oligomerization. Similar to results from our previous report evaluating ectopic expression in the Sf9/Baculovirus system, BmAPN1 could not by itself cause Cry1A-related pore formation, despite the fact that BmAPN1 gathered toxin on the oocytes as well as BmABCC2 did.
Asunto(s)
Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Proteínas de Insectos/metabolismo , Oocitos/fisiología , Receptores de Superficie Celular/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/fisiología , Proteínas Bacterianas/genética , Bombyx/genética , Bombyx/metabolismo , Antígenos CD13/genética , Antígenos CD13/metabolismo , Femenino , Proteínas de Insectos/genética , Insectos/microbiología , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Receptores de Superficie Celular/genética , XenopusRESUMEN
In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.
Asunto(s)
Proteínas Bacterianas/toxicidad , Bombyx/genética , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Proteínas de Insectos/química , Resistencia a los Insecticidas/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Secuencia de Aminoácidos , Animales , Bacillus thuringiensis/genética , Bacillus thuringiensis/patogenicidad , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/biosíntesis , Bombyx/inmunología , Bombyx/microbiología , Clonación Molecular , Endotoxinas/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas Hemolisinas/biosíntesis , Interacciones Huésped-Patógeno , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Resistencia a los Insecticidas/inmunología , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutagénesis Insercional , Dominios Proteicos , Estructura Secundaria de Proteína , Receptores de Superficie Celular/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/toxicidad , Células Sf9 , SpodopteraRESUMEN
Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca(2+) imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca(2+) and Na(+) influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior.
Asunto(s)
Bombyx/genética , Inositol/metabolismo , Proteínas de Insectos/genética , Receptores de Superficie Celular/genética , Animales , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Conducta Alimentaria , Células HEK293 , Humanos , Proteínas de Insectos/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Ligandos , Filogenia , Receptores de Superficie Celular/metabolismoRESUMEN
Insects taste nonvolatile chemicals through gustatory receptors (Grs) and make choices for feeding, mating, and oviposition. To date, genome projects have identified 69 Gr genes in the silkworm, Bombyx mori; however, the expression sites of these Grs remain to be explored. In this study, we used reverse transcription (RT)-PCR to investigate expression of the B. mori Gr-6 (BmGr6) gene, a member of the putative sugar clade gene family in various tissues. BmGr6 is expressed in the midgut, central nervous system (CNS), and oral sensory organs. Moreover, immunohistochemistry using an anti-BmGr6 antiserum demonstrated that BmGr6 is expressed in cells by oral sensory organs, midgut and nervous system. Furthermore, double-immunohistochemistry indicated that BmGr6 is expressed in midgut enteroendocrine cells, also in CNS neurosecretory cells. In particular, a portion of BmGr6-expressing cells, in both midgut and CNS, secretes FMRFamide-related peptides (FaRPs). These results suggest that BmGr6 functions not only as a taste receptor, but also as a chemical sensor such as for the regulation of gut movement, physiological conditions, and feeding behavior of larvae.