RESUMO
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.
Assuntos
Bacillus , Toxinas Bacterianas , Culex , Animais , Feminino , Toxinas Bacterianas/metabolismo , Culex/metabolismo , Lipídeos , Larva/genéticaRESUMO
Culex quinquefasciatus resistance to the binary (Bin) toxin, the major larvicidal component from Lysinibacillus sphaericus, is associated with mutations in the cqm1 gene, encoding the Bin-toxin receptor. Downregulation of the cqm1 transcript was found in the transcriptome of larvae resistant to the L. sphaericus IAB59 strain, which produces both the Bin toxin and a second binary toxin, Cry48Aa/Cry49Aa. Here, we investigated the transcription profiles of two other mosquito colonies having Bin resistance only. These confirmed the cqm1 downregulation and identified transcripts encoding the enzyme pantetheinase as the most downregulated mRNAs in both resistant colonies. Further quantification of these transcripts reinforced their strong downregulation in Bin-resistant larvae. Multiple genes were found encoding this enzyme in Cx. quinquefasciatus and a recombinant pantetheinase was then expressed in Escherichia coli and Sf9 cells, with its presence assessed in the midgut brush border membrane of susceptible larvae. The pantetheinase was expressed as a ~70 kDa protein, potentially membrane-bound, which does not seem to be significantly targeted by glycosylation. This is the first pantetheinase characterization in mosquitoes, and its remarkable downregulation might reflect features impacted by co-selection with the Bin-resistant phenotype or potential roles in the Bin-toxin mode of action that deserve to be investigated.
Assuntos
Amidoidrolases , Bacillaceae , Bacillus , Culex , Animais , Regulação para Baixo , Escherichia coli , Larva , Proteínas Ligadas por GPIRESUMO
Bacillus thuringiensis svar. israelensis (Bti) larvicides are effective in controlling Aedes aegypti; however, the effects of long-term exposure need to be properly evaluated. We established an Ae. aegypti strain that has been treated with Bti for 30 generations (RecBti) and is still susceptible to Bti, but females exhibited increased susceptibility to Zika virus (ZIKV). This study compared the RecBti strain to a reference strain regarding: first, the relative transcription of selected immune genes in ZIKV-challenged females (F30) with increased susceptibility detected in a previous study; then, the whole transcriptomic profile using unchallenged females (F35). Among the genes compared by RT-qPCR in the ZIKV-infected and uninfected females from RecBti (F30) and the reference strain, hop, domeless, relish 1, defensin A, cecropin D, and gambicin showed a trend of repression in RecBti infected females. The transcriptome of RecBti (F35) unchallenged females, compared with a reference strain by RNA-seq, showed a similar profile and only 59 differentially expressed genes were found among 9202 genes analyzed. Our dataset showed that the long-term Bti exposure of the RecBti strain was associated with an alteration of the expression of genes potentially involved in the response to ZIKV infection in challenged females, which is an important feature found under this condition.
Assuntos
Aedes , Bacillus thuringiensis , Infecção por Zika virus , Zika virus , Animais , Feminino , Bacillus thuringiensis/genética , LarvaRESUMO
BACKGROUND: Culex quinquefasciatus resistance to the binary toxin from Lysinibacillus sphaericus larvicides can occur because of mutations in the cqm1 gene that prevents the expression of the toxin receptor, Cqm1 α-glucosidase. In a resistant laboratory-selected colony maintained for more than 250 generations, cqm1REC and cqm1REC-2 resistance alleles were identified. The major allele initially found, cqm1REC , became minor and was replaced by cqm1REC-2 . This study aimed to investigate the features associated with homozygous larvae for each allele to understand the reasons for the allele replacement and to generate knowledge on resistance to microbial larvicides. RESULTS: Homozygous larvae for each allele were compared. Both larvae displayed the same level of resistance to the binary toxin (3500-fold); therefore, a change in phenotype was not the reason for the replacement observed. The lack of Cqm1 expression did not reduce the total specific α-glucosidase activity for homozygous cqm1REC and cqm1REC-2 larvae, which were statistically similar to the susceptible strain, using artificial or natural substrates. The expression of eight Cqm1 paralog α-glucosidases was demonstrated in resistant and susceptible larvae. Bioassays in which cqm1REC or cqm1REC-2 homozygous larvae were reared under stressful conditions showed that most adults produced were cqm1REC-2 homozygous (69%). Comparatively, in the offspring of a heterozygous sub-colony reared under optimal conditions for 20 generations, the cqm1REC allele assumed a higher frequency (0.72). CONCLUSION: Homozygous larvae for each allele exhibited a similar resistant phenotype. However, they presented specific advantages that might favor their selection and can be used in designing resistance management practices. © 2021 Society of Chemical Industry.
Assuntos
Toxinas Bacterianas , Culex , Proteínas de Insetos/genética , alfa-Glucosidases/genética , Alelos , Animais , Bacillaceae , Culex/enzimologia , Culex/genética , Larva/genéticaRESUMO
The mosquito Aedes aegypti is the vector of arboviruses such as Zika, Chikungunya, dengue and yellow fever. These infectious diseases have a major impact on public health. The unavailability of effective vaccines or drugs to prevent or treat most of these diseases makes vector control the main form of prevention. One strategy to promote mosquito population control is the use of synthetic insecticides to inhibit key enzymes in the metabolic pathway of these insects, particularly during larval stages. One of the main targets of the kynurenine detoxification pathway in mosquitoes is the enzyme 3-hydroxykynurenine transaminase (HKT), which catalyzes the conversion of 3-hydroxykynurenine (3-HK) into xanthurenic acid (XA). In this work, we report eleven newly synthesized oxadiazole derivatives and demonstrate that these compounds are potent noncompetitive inhibitors of HKT from Ae. aegypti. The present data provide direct evidence that HKT can be explored as a molecular target for the discovery of novel larvicides against Ae. aegypti. More importantly, it ensures that structural information derived from the HKT 3D-structure can be used to guide the development of more potent inhibitors.
Assuntos
Aedes/enzimologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Oxidiazóis/farmacologia , Transaminases/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Relação Estrutura-Atividade , Transaminases/metabolismoRESUMO
Maternal physical activity attenuates cardiorespiratory dysfunctions and transcriptional alterations presented by the carotid body (CB) of rats. Rats performed physical activity and were classified as inactive/active. During gestation and lactation, mothers received either normoprotein (NP-17% protein) or low-protein diet (LP-8% protein). In offspring, biochemical serum levels, respiratory parameters, cardiovascular parameters and the mRNA expression of hypoxia-inducible factor 1-alpha (HIF-1α), tyrosine hydroxylase (TH) and purinergic receptors were evaluate. LP-inactive pups presented lower RF from 1st to 14th days old, and higher RF at 30 days than did NP-inactive and NP-active pups. LP-inactive pups presented with reduced serum protein, albumin, cholesterol and triglycerides levels and an increased fasting glucose level compared to those of NP-inactive and NP-active groups. LP and LP-inactive animals showed an increase in the cardiac variability at the Low-Frequency bands, suggesting a major influence of sympathetic nervous activity. In mRNA analyses, LP-inactive animals showed increased HIF-1α expression and similar expression of TH and purinergic receptors in the CB compared to those of NP groups. All these changes observed in LP-inactive pups were reversed in the pups of active mothers (LP-active). Maternal physical activity is able to attenuate the metabolic, cardiorespiratory and HIF-1α transcription changes induced by protein malnutrition.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Desnutrição/prevenção & controle , Fenômenos Fisiológicos da Nutrição Materna/genética , Efeitos Tardios da Exposição Pré-Natal , Animais , Aptidão Cardiorrespiratória/fisiologia , Sistema Cardiovascular/fisiopatologia , Corpo Carotídeo/fisiopatologia , Dieta com Restrição de Proteínas , Feminino , Regulação da Expressão Gênica/genética , Humanos , Lactação/fisiologia , Desnutrição/genética , Desnutrição/fisiopatologia , Condicionamento Físico Animal , Gravidez , Ratos , Ratos Wistar , Sistema Nervoso Simpático/fisiopatologiaRESUMO
The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.
Assuntos
Leishmania infantum/metabolismo , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Protozoários/metabolismo , Motivos de Aminoácidos , Leishmania infantum/genética , Fosforilação/fisiologia , Proteínas de Ligação a Poli(A)/genética , Proteínas de Protozoários/genéticaRESUMO
The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Leishmania/genética , Leishmania/metabolismo , Iniciação Traducional da Cadeia Peptídica , Domínios e Motivos de Interação entre Proteínas , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Fosforilação , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica , Alinhamento de SequênciaRESUMO
The Cqm1 α-glucosidase, expressed within the midgut of Culex quinquefasciatus mosquito larvae, is the receptor for the Binary toxin (Bin) from the entomopathogen Lysinibacillus sphaericus. Mutations of the Cqm1 α-glucosidase gene cause high resistance levels to this bacterium in both field and laboratory populations, and a previously described allele, cqm1REC, was found to be associated with a laboratory-resistant colony (R2362). This study described the identification of a novel resistance allele, cqm1REC-2, that was co-selected with cqm1REC within the R2362 colony. The two alleles display distinct mutations but both generate premature stop codons that prevent the expression of midgut-bound Cqm1 proteins. Using a PCR-based assay to monitor the frequency of each allele during long-term maintenance of the resistant colony, cqm1REC was found to predominate early on but later was replaced by cqm1REC-2 as the most abundant resistance allele. Homozygous larvae for each allele were then generated that displayed similar high-resistance phenotypes with equivalent low levels of transcript and lack of protein expression for both cqm1REC and cqm1REC-2. In progeny from a cross of homozygous individuals for each allele at a 1 : 1 ratio, analyzed for ten subsequent generations, cqm1REC showed a higher frequency than cqm1REC-2. The replacement of cqm1REC by cqm1REC -2 observed in the R2362 colony, kept for 210 generations, indicates changes in fitness related to traits that are unknown but linked to these two alleles, and constitutes a unique example of evolution of resistance within a controlled laboratory environment.