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Development of novel insecticidal nanoformulations for plant protection
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Abstract(s)
Durante vários anos, as pragas foram responsáveis por perdas nos produtos hortícolas, o que, consequentemente, teve um impacto significativo na oferta e qualidade dos alimentos e na evolução da sociedade humana. Embora os recentes avanços na ciência e na tecnologia tenham reduzido consideravelmente a incidência e a severidade causadas por pragas, uma grande parte da produção real ainda é perdida todos os anos. Por esta razão, a proteção das culturas agrícolas exige o controle de vários insetos praga de forma segura, eficaz e de baixo custo. Esse desafio pode ser uma oportunidade para os agentes
de controle biológico uma vez que são alternativas mais seguras do que os pesticidas químicos usados atualmente. Desta forma, o objetivo desta tese foi produzir e formular moléculas biológicas conhecidas como fatores de virulência do nematode entomopatogénico da espécie Steinernema carpocapsae, para avaliar seu potencial uso como novos bioinseticidas contra insetos-praga.
Esta investigação iniciou-se com a clonagem e a expressão heteróloga de duas proteínas putativas tóxicas do nematode, conduzidas em Escherichia coli, sendo estas um domínio proteico, designado de péptido ScK1 e a proteína Sc-CAP. A primeira referida assemelha-se aos bloqueadores de canais de potássio das anémonas do mar e a segunda faz parte de uma extensa família de proteínas que recebe o nome de CAP (Proteínas
secretoras ricas em cisteína, antígeno 5 e proteínas 1 relacionadas à patogénese).
Devido à complexidade dessas proteínas ricas em cisteína, só foi possível a produção bem-sucedida das recombinantes em fusão com a proteína chaperone designada por isomerase de ligação dissulfeto (DsbC).
A estrutura secundária do péptido ScK1 que apresentou um tamanho molecular de 5.7-kDa, cujo a purificação indicou ser redox-sensível, exibiu um espectro de dicroísmo circular far-UV consistente com uma estrutura secundária alfa-helicoidal. A desnaturação térmica do péptido ScK1 permitiu estimar uma temperatura de desnaturação de 59,2 ± 0,1 ºC. A análise de interação proteína-proteína in silico revelou uma forte afinidade para com os canais de potássio dos insetos. Os resultados dos ensaios de toxicidade usando
Drosophila melanogaster como modelo mostraram que a injeção deste péptido é letal contra insetos de forma dose-dependente, com uma dose letal média (LD50) de 52,29 ng por adulto. A administração oral da proteína de fusão reduziu significativamente a atividade locomotora dos insetos tratados após 48 horas do ensaio. A estrutura terciária predita da proteína Sc-CAP revelou uma conformação de alfa-beta-alfa com grande homologia com proteínas de outros nematodes parasitas de vertebrados, conhecidos por terem capacidade de ligação a esteróis. A versão recombinante apresentou um tamanho molecular estimado de 50 kDa e apesar da proteína não apresentar toxicidade por injeção ou administração oral, os experimentos de interação
revelaram uma forte capacidade de ligação de esteróis. A análise de interação ligandoproteína mostrou uma alta afinidade do colesterol e do palmitato para diferentes motivos de ligação da proteína com uma energia de ligação de -6,3 e -4,5 kcal/mol, respetivamente. Estes resultados foram confirmados pelo ensaio in vitro, onde a proteína recombinante apresentou, de forma dose-dependente, grande afinidade de ligação ao colesterol.
A Nanoencapsulação destas duas proteínas toxicas foi conseguida através da preparação de nanopartículas de quitosano pelo método de gelificação iónica. A caracterização geral das nanopartículas revelou um tamanho nanométrico para as formulações NanoScK e NanoCAP, com um tamanho médio de 182,0 e 676,9 nm,
respetivamente, e uma liberação rápida inicial seguida por um perfil de liberação mais lento para ambas as formulações. Estas nanoformulações permitiram a libertação destas proteínas em insetos. Além disso, foi notável a acumulação dessas moléculas em diferentes tecidos do inseto, nomeadamente, no sistema digestivo e no sistema nervoso. A formulação NanoScK causou efeitos imediatos em insetos, induzindo uma evidente
redução da atividade locomotora, paralisia e mortalidade, ao contrário do NanoCAP, onde se observou baixa mortalidade. Por fim, foram avaliados os efeitos dessas formulações sobre a progenia do inseto. NanoScK desencadeou uma redução significativa no número de ovos depositados por insetos tratados, enquanto o NanoCAP causou uma redução significativa na taxa de emergência de adultos.
Os resultados desta tese conferem um estatuto de prova de conceito em relação ao uso de proteínas do secretado do nematode para o desenvolvimento de novas formulações inseticidas. Esta nova classe de biomoléculas pode dar origem à próxima geração de inseticidas biológicos visto que são específicos para as pragas alvo, biodegradáveis, não tóxicos, amigos do ambiente e de baixo custo de produção.
ABSTRACT: For several years, crop losses due to pests had a significant impact on the evolution of human society and food supply. Although recent advances in science and technology have considerably reduced the incidence and severity caused by plant pests, a big portion of actual production is still lost each year. For this reason, plant protection in agriculture demands the control of several insect pests in a safe, effective, and low-cost way. This challenge can be an opportunity for biological control agents since they are much safer alternatives than several chemical pesticides. Therefore, the purpose of this thesis was to produce and formulate biological molecules known as the virulence factors of entomopathogenic nematodes to evaluate their potential use as bioinsecticides against insect pests. This thesis started with the cloning and heterologous expression of two putative toxic proteins from Steinernema carpocapsae conducted in Escherichia coli. A small protein domain, named ScK1 peptide, which resembles a potassium channel blocker from a sea anemone, and a second larger protein, named Sc-CAP, from the cysteine-rich secretory protein/antigen 5/pathogenesis related-1 superfamily. Due to the complexity of these cysteine-rich proteins, the successful production of the recombinant proteins was only possible in fusion with the chaperone protein called disulfide bond isomerase. The secondary structure of the purified 5.7-kDa ScK1 peptide after proteolytic cleavage of the fusion protein appeared to be redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with alpha-helical secondary structure. ScK1 thermal denaturation allowed estimating a melting temperature of 59.2 ± 0.1 °C. In silico proteinprotein interaction analysis revealed a strong affinity towards insect potassium channels. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 52.29 ng per adult within 24 hours. Oral administration of the fusion protein significantly reduced the locomotor activity of the treated insects after 48 hours of the assay. The predicted tertiary structure of the Sc-CAP protein revealed a unique alphabeta-alpha sandwich with high structural homology to sterol-binding proteins from other parasitic nematodes. The recombinant version had an estimated molecular weight of 50 kDa and despite the protein did not show toxicity by injection or oral administration, the interaction experiments revealed a strong sterol binding capability. Docking analysis showed a high affinity of cholesterol and the palmitate to different binding motifs of the protein with an energy binding of -6.3 and -4.5 kcal/mol, respectively. These results were confirmed by the In vitro assay, where the recombinant protein showed increasing binding affinity to the cholesterol in a dose-response manner. The Nano-encapsulation of these two venom proteins was achieved by the preparation of chitosan nanoparticles via the ionic gelation method. The overall characterization of chitosan nanoparticles revealed a nanometric size for both NanoScK and NanoCAP formulations, with a mean size of 182.0 and 676.9 nm, respectively, and an initial rapid release followed by a slower release profile in both formulations. These nanoformulations allowed the delivery of the venom proteins into insects. Furthermore, it was notable the increase of these molecules in different insect tissues, such as the gut and nervous system. The NanoScK formulation caused immediate effects in treated insects with an evident reduced locomotor activity, paralysis, and mortality, contrary to the NanoCAP, where low mortality occurred later in the assay. Lastly, the effects of these formulations on the insect progeny were assessed. NanoScK triggered a significant reduction in the number of eggs laid by treated insects, while the NanoCAP caused a significant reduction in adult emergence. The outputs of this thesis confer a proof-of-concept status regarding the use of nematode venom proteins for the development of new insecticidal formulations. This new class of nanoinsecticides may soon become the next generation of insecticides because they are cost-effective, non-toxic, biodegradable, and beneficial to the environment.
ABSTRACT: For several years, crop losses due to pests had a significant impact on the evolution of human society and food supply. Although recent advances in science and technology have considerably reduced the incidence and severity caused by plant pests, a big portion of actual production is still lost each year. For this reason, plant protection in agriculture demands the control of several insect pests in a safe, effective, and low-cost way. This challenge can be an opportunity for biological control agents since they are much safer alternatives than several chemical pesticides. Therefore, the purpose of this thesis was to produce and formulate biological molecules known as the virulence factors of entomopathogenic nematodes to evaluate their potential use as bioinsecticides against insect pests. This thesis started with the cloning and heterologous expression of two putative toxic proteins from Steinernema carpocapsae conducted in Escherichia coli. A small protein domain, named ScK1 peptide, which resembles a potassium channel blocker from a sea anemone, and a second larger protein, named Sc-CAP, from the cysteine-rich secretory protein/antigen 5/pathogenesis related-1 superfamily. Due to the complexity of these cysteine-rich proteins, the successful production of the recombinant proteins was only possible in fusion with the chaperone protein called disulfide bond isomerase. The secondary structure of the purified 5.7-kDa ScK1 peptide after proteolytic cleavage of the fusion protein appeared to be redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with alpha-helical secondary structure. ScK1 thermal denaturation allowed estimating a melting temperature of 59.2 ± 0.1 °C. In silico proteinprotein interaction analysis revealed a strong affinity towards insect potassium channels. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 52.29 ng per adult within 24 hours. Oral administration of the fusion protein significantly reduced the locomotor activity of the treated insects after 48 hours of the assay. The predicted tertiary structure of the Sc-CAP protein revealed a unique alphabeta-alpha sandwich with high structural homology to sterol-binding proteins from other parasitic nematodes. The recombinant version had an estimated molecular weight of 50 kDa and despite the protein did not show toxicity by injection or oral administration, the interaction experiments revealed a strong sterol binding capability. Docking analysis showed a high affinity of cholesterol and the palmitate to different binding motifs of the protein with an energy binding of -6.3 and -4.5 kcal/mol, respectively. These results were confirmed by the In vitro assay, where the recombinant protein showed increasing binding affinity to the cholesterol in a dose-response manner. The Nano-encapsulation of these two venom proteins was achieved by the preparation of chitosan nanoparticles via the ionic gelation method. The overall characterization of chitosan nanoparticles revealed a nanometric size for both NanoScK and NanoCAP formulations, with a mean size of 182.0 and 676.9 nm, respectively, and an initial rapid release followed by a slower release profile in both formulations. These nanoformulations allowed the delivery of the venom proteins into insects. Furthermore, it was notable the increase of these molecules in different insect tissues, such as the gut and nervous system. The NanoScK formulation caused immediate effects in treated insects with an evident reduced locomotor activity, paralysis, and mortality, contrary to the NanoCAP, where low mortality occurred later in the assay. Lastly, the effects of these formulations on the insect progeny were assessed. NanoScK triggered a significant reduction in the number of eggs laid by treated insects, while the NanoCAP caused a significant reduction in adult emergence. The outputs of this thesis confer a proof-of-concept status regarding the use of nematode venom proteins for the development of new insecticidal formulations. This new class of nanoinsecticides may soon become the next generation of insecticides because they are cost-effective, non-toxic, biodegradable, and beneficial to the environment.
Description
Tese de Doutoramento, Biologia, 11 de dezembro de 2023, Universidade dos Açores.
Keywords
Proteção de Plantas Nematodes Fatores de Virulência Bioinsecticidas Nanoformulações
Pedagogical Context
Citation
Frias, Jorge Miguel Vieira de. (2022). "Development of novel insecticidal nanoformulations for plant protection". 154 p. (Tese de Doutoramento em Biologia). Ponta Delgada: Universidade dos Açores, 2022. Disponível em http://hdl.handle.net/10400.3/7152