RESUMO
The white potato worm Premnotrypes vorax (Hustache) (Coleoptera: Curculionidae) is one of the most destructive insect pests of potato crops in South America. Like many coleopteran insects, P. vorax shows low susceptibility to Cry insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). However, the presence of Cry toxin receptors in the midgut of this this insect has never been studied. The main Cry-binding proteins described in other insect species are cadherin (CAD), aminopeptidase N (APN), alkaline phosphatase (ALP) and ATP-binding cassette (ABC) transporters. In this study, we analyzed and validated a de novo assembled transcriptome of Illumina sequencing data to identify and to characterize homologs of Cry toxin receptors. We identified the protein sequences in P. vorax that show high identity with their orthologous sequences of the Cry toxin binding proteins in other coleopteran larvae such as APN, ALP, CAD and ABC transporter. This study provides preliminary identification of putative receptor genes of Cry proteins that would be useful for future studies involving biocontrol of this important potato crop pest.
Assuntos
Besouros , Gorgulhos , Animais , Gorgulhos/genética , Transcriptoma , Proteínas de Insetos/genética , Transportadores de Cassetes de Ligação de ATP , Fosfatase Alcalina , Antígenos CD13/genética , Caderinas , CorantesRESUMO
Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.
RESUMO
BACKGROUND: Although much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion. RESULTS: In this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses. CONCLUSIONS: This study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae.
Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Trato Gastrointestinal/metabolismo , Proteínas Hemolisinas/farmacologia , Inseticidas/farmacologia , Larva/genética , Transcriptoma , Animais , Toxinas de Bacillus thuringiensis , Análise por Conglomerados , Biologia Computacional/métodos , Resistência a Medicamentos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Concentração Inibidora 50 , Anotação de Sequência Molecular , Reprodutibilidade dos TestesRESUMO
Bacillus thuringiensis produces insecticidal Cry and Cyt proteins that are toxic to different insect orders. In addition, Cyt toxins also display haemolytic activity. Both toxins are pore-forming proteins that form oligomeric structures that insert into the target membrane to lyse cells. Cyt toxins play an important role in mosquitocidal activity since they synergize Cry toxins and are able to overcome resistance to Cry toxins. Cry and Cyt toxins interact by specific epitopes, and this interaction is important to induce the synergistic activity observed. It was proposed that Cyt toxins do not interact with protein receptors but directly interacting with the specific midgut cell lipids. Here, we analysed if oligomerization and membrane insertion of Cyt1Aa are necessary steps to synergize Cry11Aa toxicity. We characterized Cyt1Aa helix α-C mutants that were affected in oligomerization, in membrane insertion and also in haemolytic and insecticidal activities. However, these mutants were still able to synergize Cry11Aa toxicity indicating these steps are independent events of Cyt1Aa synergistic activity. Furthermore, the data indicate that formation of stable Cyt1Aa-oligomeric structure is a key step for membrane insertion, haemolysis and insecticidal activity.
Assuntos
Aedes/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Hemolíticos/farmacologia , Inseticidas/farmacologia , Animais , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/química , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Hemólise , Hemolíticos/química , Inseticidas/química , Larva/efeitos dos fármacos , Estrutura Secundária de ProteínaRESUMO
Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/química , Membrana Celular/química , Endotoxinas/química , Proteínas Hemolisinas/química , Bicamadas Lipídicas/química , Modelos Químicos , Fosfolipídeos/química , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Larva/microbiologia , Bicamadas Lipídicas/metabolismo , Manduca/microbiologia , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Estrutura Terciária de ProteínaRESUMO
The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a ß-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the ß-sheet, allowing insertion of ß-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of ß-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.
Assuntos
Proteínas de Bactérias/química , Endotoxinas/química , Proteínas Hemolisinas/química , Inseticidas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aedes/efeitos dos fármacos , Animais , Toxinas de Bacillus thuringiensis , Larva/efeitos dos fármacos , Lipossomos/química , Membranas/química , Modelos Químicos , Conformação Proteica , Multimerização ProteicaRESUMO
Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop ß6-αE K198A, E204A and ß7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.