Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

A binary mosquitocidal toxin composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like toxin (Cry49Aa) was identified in Lysinibacillus sphaericus. Cry48Aa/Cry49Aa has action on Culex quinquefasciatus larvae, in particular, to those that are resistant to the Bin Binary toxin, which is the major insecticidal factor from L. sphaericus-based biolarvicides, indicating that Cry48Aa/Cry49Aa interacts with distinct target sites in the midgut and can overcome Bin toxin resistance. This study aimed to identify Cry48Aa/Cry49Aa ligands in C. quinquefasciatus midgut through binding assays and mass spectrometry. Several proteins, mostly from 50 to 120 kDa, bound to the Cry48Aa/Cry49Aa toxin were revealed by toxin overlay and pull-down assays. These proteins were identified against the C. quinquefasciatus genome and after analysis a set of 49 proteins were selected which includes midgut bound proteins such as aminopeptidases, amylases, alkaline phosphatases in addition to molecules from other classes that can be potentially involved in this toxin's mode of action. Among these, some proteins are orthologs of Cry receptors previously identified in mosquito larvae, as candidate receptors for Cry48Aa/Cry49Aa toxin. Further investigation is needed to evaluate the specificity of their interactions and their possible role as receptors

Molecular basis of interaction of the binary toxin of Bacillus biolarvicide shaericus with a-glucosidase from larvae culicids vectors

Made available in DSpace on 2015-05-27T13:35:00Z (GMT). No. of bitstreams: 2 579.pdf: 3990633 bytes, checksum: fe870db4eac6748eba2af6524ffbf216 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilO principal fator larvicida do Bacillus sphaericus (Bs) para culicídeos é a toxina binária (Bin), produzida sob a forma de um cristal, durante a esporulação. Esta toxina, ativada por meio da ação de proteases no lúmen intestinal, reconhece e liga-se a receptores específicos no intestino de larvas por meio de suasubunidade BinB, etapa crítica no seu modo de ação e essencial para a atividade larvicida. Os receptores em Culex pipiens, C. quinquefasciatus e Anopheles gambiae, denominados Cpm1, Cqm1, e Agm3, respectivamente, são alfa glicosidases de 66 kDa ligadas à membrana apical do epitélio intestinal por uma âncora de glicosilfosfatidilinositol (GPI). Larvas de Aedes aegypti expressam a alfaglicosidase Aam1, ortóloga ao receptor Cqm1 que, no entanto, não apresenta capacidade de ligação à toxina Bin. O principal objetivo deste trabalho foi caracterizar a base molecular da interação da toxina Bin do Bs com alfaglicosidases em larvas de culicídeos vetores. Para tal, a sequência protéica de Cqm1 foi alinhada a sequências ortólogas e parálogas de Ae. aegypti e An. gambiae, o que indicou trechos mais conservados na porção N-terminal e divergentes na porção C-terminal. A banálise funcional de fragmentos protéicos de 45 kDa da porção N-terminal de proteínas ortólogas e parálogas à Cqm1 sugeriu que o epitopo de ligação à toxina Bin está potencialmente localizado nesta região e que o estado conformacional nativo das proteínas é determinante para sua funcionalidade. Em seguida, foram avaliadas algumas características das alfa glicosidases Cqm1 e Aam1 de 66 kDa que podem estar envolvidas na sua capacidade de ligação à toxina Bin. Os resultados mostraram que as proteínas possuem diferenças estruturais e conformacionais que podem ter um papel determinante para a interação com a toxina Bin. Aanálise do padrão de glicosilação das proteínas revelou que a Aam1 posssui glicosilações em sua cadeia polipeptídica ao contrário da Cqm1 e que a ligação do receptor Cqm1 à toxina Bin não envolve N-glicanos. Na segunda parte do trabalho, foram investigados os motivos da subunidade BinB envolvidos na interação com o receptor Cqm1, através do estudo da funcionalidade de proteínas BinB mutantes com deleções nas regiões N- e C-terminal ou substituições de blocos de aminoácidos por alaninas. Os resultados obtidos indicam que o epitopo 147FQF149 da toxina é essencial a ligação ao receptor Cqm1 e que esta interação depende ainda do segmento N-terminal da BinB, compreendendo os primeiros 82 aminoácidos, uma região predita de segmentos não estruturados e de alfa hélice

Mecanismo molecular da resistência de uma colônia de Culex quinquefasciatus (Diptera : Culicidae) ao Bacillus sphaericus

A atividade larvicida do Bacillus sphaericus depende da ligação da toxina binária (Bin) com um receptor específico presente no epitélio intestinal de larvas de culicídeos. Este trabalho descreve a clonagem e sequenciamento do gene deste receptor em larvas de Culex quinquefasciatus de uma colônia susceptível (CqSF) e de uma resistente (CqRL1/2362) ao Bs, para elucidar o mecanismo molecular da resistência. Ensaios de afinidade in vitro mostraram que uma proteína de 60 kDa é o receptor da toxina na colônia CqSF e nenhuma molécula funcional foi observada em preparações da colônia CqRL1/2362. O sequenciamento do gene obtido da colônia CqSF mostrou uma seqüência de 1925 pb composta por regiões 5 e 3´não traduzidas, sequência codificadora e íntron. A respectiva sequência do gene para a colônia CqRL1/2362 mostrou uma deleção de 19 nucleotídeos, geradora de uma mudança na fase de leitura de 28 aminoácidos e de um códon de terminação da tradução prematuro. A presença deste códon prematuro pode desestabilizar o respectivo mRNA, e impedir sua tradução, bem como leva a síntese de uma proteína truncada sem a âncora GPI necessária a sua localização na membrana epitelial. Em ambos os casos a ausência do receptor no epitélio intestinal seria a causa da resistência. Neste trabalho foi produzida ainda uma proteína recombinante do receptor de 45 kDa que mostrou funcionalidade, demonstrando que o sítio de ligação está localizado na porção N-terminal da molécula. Anticorpos obtidos contra esta proteína recombinante demonstraram ser uma ótima ferramenta de detecção do receptor nativ

A Culex quinquefasciatus strain resistant to the binary toxin from Lysinibacillus sphaericus displays altered enzyme activities and energy reserves

Abstract Background The resistance of a Culex quinquefasciatus strain to the binary (Bin) larvicidal toxin from Lysinibacillus sphaericus is due to the lack of expression of the toxin’s receptors, the membrane-bound Cqm1 α-glucosidases. A previous transcriptomic profile of the resistant larvae showed differentially expressed genes coding Cqm1, lipases, proteases and other genes involved in lipid and carbohydrate metabolism. This study aimed to investigate the metabolic features of Bin-resistant individuals by comparing the activity of some enzymes, energy reserves, fertility and fecundity to a susceptible strain. Methods The activity of specific enzymes was recorded in midgut samples from resistant and susceptible larvae. The amount of lipids and reducing sugars was determined for larvae and adults from both strains. Additionally, the fecundity and fertility parameters of these strains under control and stress conditions were examined. Results Enzyme assays showed that the esterase activities in the midgut of resistant larvae were significantly lower than susceptible ones using acetyl-, butyryl- and heptanoyl-methylumbelliferyl esthers as substrates. The α-glucosidase activity was also reduced in resistant larvae using sucrose and a synthetic substrate. No difference in protease activities as trypsins, chymotrypsins and aminopeptidases was detected between resistant and susceptible larvae. In larval and adult stages, the resistant strain showed an altered profile of energy reserves characterized by significantly reduced levels of lipids and a greater amount of reducing sugars. The fertility and fecundity of females were similar for both strains, indicating that those changes in energy reserves did not affect these reproductive parameters. Conclusions Our dataset showed that Bin-resistant insects display differential metabolic features co-selected with the phenotype of resistance that can potentially have effects on mosquito fitness, in particular, due to the reduced lipid accumulation. Graphical Abstrac

A new allele conferring resistance to Lysinibacillus sphaericus is detected in low frequency in Culex quinquefasciatus field populations

Submitted by Adagilson Silva ([email protected]) on 2017-09-04T19:54:16Z No. of bitstreams: 1 26576515 2016 pai-ide.oa.pdf: 602511 bytes, checksum: a3bdfb10f98627983fa539648d310799 (MD5)Approved for entry into archive by Adagilson Silva ([email protected]) on 2017-09-05T18:53:50Z (GMT) No. of bitstreams: 1 26576515 2016 pai-ide.oa.pdf: 602511 bytes, checksum: a3bdfb10f98627983fa539648d310799 (MD5)Made available in DSpace on 2017-09-05T18:53:50Z (GMT). No. of bitstreams: 1 26576515 2016 pai-ide.oa.pdf: 602511 bytes, checksum: a3bdfb10f98627983fa539648d310799 (MD5) Previous issue date: 2016-02-04Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, BrasilThe Cqm1 α-glucosidase of Culex quinquefasciatus larvae acts as the midgut receptor for the binary toxin of the biolarvicide Lysinibacillus sphaericus. Mutations within the cqm1 gene can code for aberrant polypeptides that can no longer be properly expressed or bind to the toxin, leading to insect resistance. The cqm1 REC and cqm1 REC-2 alleles were identified in a laboratory selected colony and both displayed mutations that lead to equivalent phenotypes of refractoriness to L. sphaericus. cqm1 REC was first identified as the major resistance allele in this colony but it was subsequently replaced by cqm1 REC-2 , suggesting the better adaptive features of the second allele. The major aim of this study was to evaluate the occurrence of cqm1 REC-2 and track its origin in field populations where cqm1 REC was previously identified

Detection of an Allele Conferring Resistance to Bacillus sphaericus Binary Toxin in Culex quinquefasciatus Populations by Molecular Screening▿

The activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound α-glucosidase. A 19-nucleotide deletion in the cqm1 gene (cqm1REC) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus-treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1REC allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus, comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1REC allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus-treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through α-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1REC allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus. This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations

Novel mutations associated with resistance to Bacillus sphaericus in a polymorphic region of the Culex quinquefasciatus cqm1 gene

Submitted by Kamylla Nascimento ([email protected]) on 2018-04-10T11:52:47Z No. of bitstreams: 1 Novel Mutations Associated with Resistance to Bacillus sphaericus in a.pdf: 338176 bytes, checksum: 9e301f894bd190598a46f77cc5bdebc7 (MD5)Approved for entry into archive by Kamylla Nascimento ([email protected]) on 2018-04-10T12:06:15Z (GMT) No. of bitstreams: 1 Novel Mutations Associated with Resistance to Bacillus sphaericus in a.pdf: 338176 bytes, checksum: 9e301f894bd190598a46f77cc5bdebc7 (MD5)Made available in DSpace on 2018-04-10T12:06:16Z (GMT). No. of bitstreams: 1 Novel Mutations Associated with Resistance to Bacillus sphaericus in a.pdf: 338176 bytes, checksum: 9e301f894bd190598a46f77cc5bdebc7 (MD5) Previous issue date: 2012Este trabalho foi apoiado pelo Programa Estratégico de Apoio à Pesquisa em Saúde (PAPES) da Fundação Oswaldo Cruz - FIOCRUZ, Conselho Nacional de Pesquisa (CNPq Brasil, bolsa 403488 / 2008-7 ;), Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE Brasil, outorga APQ 0427-2.13 / 08 ).Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Microbiologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Bin toxin from Bacillus sphaericus acts on Culex quinquefasciatus larvae by binding to Cqm1 midgut-bound receptors, and disruption of the cqm1 gene is the major cause of resistance. The goal of this work was to screen for a laboratory-selected resistance cqm1(REC) allele in field populations in the city of Recife, Brazil, and to describe other resistance-associated polymorphisms in the cqm1 gene. The cqm1(REC) allele was detected in the four nontreated populations surveyed at frequencies from 0.001 to 0.017, and sequence analysis from these samples revealed a novel resistant allele (cqm1(REC-D16)) displaying a 16-nucletotide (nt) deletion which is distinct from the 19-nt deletion associated with cqm1(REC). Yet a third resistant allele (cqm1(REC-D25)), displaying a 25-nt deletion, was identified in samples from a treated area exposed to B. sphaericus. A comparison of the three deletion events revealed that all are located within the same 208-nt region amplified during the screening procedure. They also introduce equivalent frameshifts in the sequence and generate the same premature stop codon, leading to putative transcripts encoding truncated proteins which are unable to locate to the midgut epithelium. The populations analyzed in this study contained a variety of alleles with mutations disrupting the function of the corresponding Bin toxin receptor. Their locations reveal a hot spot that can be exploited to assess the resistance risk through DNA screening

Polymorphisms in GSTE2 is associated with temephos resistance in Aedes aegypti

The glutathione S-transferases (GSTs) are enzymes involved in several distinct biological processes. In insects, the GSTs, especially delta and epsilon classes, play a key role in the metabolism of xenobiotics used to control insect populations. Here, we investigated its potential role in temephos resistance, examining the GSTE2 gene from susceptible (RecL) and resistant (RecR) strains of the mosquito Aedes aegypti, vector for several pathogenic arboviruses. Total GST enzymatic activity and the GSTE2 gene expression profile were evaluated, with the GSTE2 cDNA and genomic loci sequenced from both strains. Recombinant GSTE2 and mutants were produced in a heterologous expression system and assayed for enzyme kinetic parameters. These proteins also had their 3D structure predicted through molecular modeling. Our results showed that RecR has a profile of total GST enzymatic activity higher than RecL, with the expression of the GSTE2 gene in resistant larvae increasing six folds. Four exclusive RecR mutations were observed (L111S, I150V, E178A and A198E), which were absent in the laboratory susceptible strains. The enzymatic activity of the recombinant GSTE2 showed different kinetic parameters, with the GSTE2 RecR showing an enhanced ability to metabolize its substrate. The I150V mutation was shown to induce significant changes in catalytic parameters and a 3D modeling of GSTE2 mapped two of the RecR changes (L111S and I150V) near the enzyme's catalytic pocket, also implying an impact on its catalytic activity. Our results reinforce a potential role for GSTE2 in the metabolic resistance phenotype while contributing to the understanding of the molecular basis for the resistance mechanism

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

A binary mosquitocidal toxin composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like toxin (Cry49Aa) was identified in Lysinibacillus sphaericus. Cry48Aa/Cry49Aa has action on Culex quinquefasciatus larvae, in particular, to those that are resistant to the Bin Binary toxin, which is the major insecticidal factor from L. sphaericus-based biolarvicides, indicating that Cry48Aa/Cry49Aa interacts with distinct target sites in the midgut and can overcome Bin toxin resistance. This study aimed to identify Cry48Aa/Cry49Aa ligands in C. quinquefasciatus midgut through binding assays and mass spectrometry. Several proteins, mostly from 50 to 120 kDa, bound to the Cry48Aa/Cry49Aa toxin were revealed by toxin overlay and pull-down assays. These proteins were identified against the C. quinquefasciatus genome and after analysis a set of 49 proteins were selected which includes midgut bound proteins such as aminopeptidases, amylases, alkaline phosphatases in addition to molecules from other classes that can be potentially involved in this toxin's mode of action. Among these, some proteins are orthologs of Cry receptors previously identified in mosquito larvae, as candidate receptors for Cry48Aa/Cry49Aa toxin. Further investigation is needed to evaluate the specificity of their interactions and their possible role as receptors