Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate

Detalhes bibliográficos
Ano de defesa: 2020
Autor(a) principal: Camargo, Stella de Lima
Orientador(a): Não Informado pela instituição
Banca de defesa: Não Informado pela instituição
Tipo de documento: Tese
Tipo de acesso: Acesso aberto
Idioma: eng
Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
Programa de Pós-Graduação: Não Informado pela instituição
Departamento: Não Informado pela instituição
País: Não Informado pela instituição
Palavras-chave em Português:
Link de acesso: https://www.teses.usp.br/teses/disponiveis/64/64133/tde-19092022-103522/
Resumo: Sphaerospermopsis torques-reginae is a planktonic freshwater cyanobacteria species with broad distribution in several Brazilian aquatic environments. Some strains of this species are known to produce the neurotoxic organophosphate guanitoxin (GNT) as well as spumigin and anabaenopeptin, both protease inhibitors. GNT is a powerful irreversible inhibitor of the acetylcholinesterase enzyme which acts in the peripheral nervous cells of mammals. While GNT has been known for decades, the genes encoding its biosynthetic pathway are unknown. The sequencing and assembly of the S. torques-reginae ITEP-024 5.2 Mbp genome allowed us to identify a 12.5 kbp gene cluster potentially involved in GNT production. To rigorously validate our genomic finding, we over-expressed, purified and characterized many of the GNT biosynthetic enzymes in vitro, confirming our proposed biosynthetic cluster. Also, a vector was designed that assembled two of the biosynthetic genes with the goal to produce the intermediate enduracididine, a rare amino acid. The combination of in vivo and in vitro experiments successfully validated our proposed gene cluster. Using our results, we showed that this biosynthetic gene cluster was actively expressed in one of the largest cyanobacterial blooms hotspots, Lake Erie, Toledo, USA. This suggest that the potent neurotoxin guanitoxin could be in drinking water supplies around the world. Worryingly, guanitoxin is often overlooked and rarely tested for in reservoirs in spite of the danger it poses. This study contributed to the characterization of the GNT biosynthetic pathway, which may generate a patent enabling the development of rapid and sensitive methods for the detection and monitoring of this toxin in freshwaters used for public supply. Moreover, this work may offer potential for the development of new neurological medicines and tools
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spelling Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphateRevelando a biossíntese da guanitoxina, um organofosfato neurotóxico de ocorrência naturalAtividade enzimáticaCianobactériaCianotoxinaCyanobacteriaCyanotoxinEnzyme activityGenomaGenomePublic healthSaúde públicaSphaerospermopsis torques-reginae is a planktonic freshwater cyanobacteria species with broad distribution in several Brazilian aquatic environments. Some strains of this species are known to produce the neurotoxic organophosphate guanitoxin (GNT) as well as spumigin and anabaenopeptin, both protease inhibitors. GNT is a powerful irreversible inhibitor of the acetylcholinesterase enzyme which acts in the peripheral nervous cells of mammals. While GNT has been known for decades, the genes encoding its biosynthetic pathway are unknown. The sequencing and assembly of the S. torques-reginae ITEP-024 5.2 Mbp genome allowed us to identify a 12.5 kbp gene cluster potentially involved in GNT production. To rigorously validate our genomic finding, we over-expressed, purified and characterized many of the GNT biosynthetic enzymes in vitro, confirming our proposed biosynthetic cluster. Also, a vector was designed that assembled two of the biosynthetic genes with the goal to produce the intermediate enduracididine, a rare amino acid. The combination of in vivo and in vitro experiments successfully validated our proposed gene cluster. Using our results, we showed that this biosynthetic gene cluster was actively expressed in one of the largest cyanobacterial blooms hotspots, Lake Erie, Toledo, USA. This suggest that the potent neurotoxin guanitoxin could be in drinking water supplies around the world. Worryingly, guanitoxin is often overlooked and rarely tested for in reservoirs in spite of the danger it poses. This study contributed to the characterization of the GNT biosynthetic pathway, which may generate a patent enabling the development of rapid and sensitive methods for the detection and monitoring of this toxin in freshwaters used for public supply. Moreover, this work may offer potential for the development of new neurological medicines and toolsSphaerospermopsis torques-reginae é uma espécie planctônica de cianobactéria de água doce com ampla distribuição em ambientes aquáticos brasileiros. Sabe-se que algumas linhagens desta espécie produzem o organofosforado neurotóxico guanitoxina (GNT), bem como espumigina e a anabaenopeptina, ambos inibidores da protease. GNT é um potente inibidor irreversível da enzima acetilcolinesterase, atuando em células nervosas periféricas de mamíferos. Embora GNT seja conhecido há décadas, os genes que codificam sua via biossintética são desconhecidos. O sequenciamento e montagem do genoma da cianobactéria S. torques-reginae ITEP-024 de tamanho 5,2 Mbp, nos permitiu identificar um agrupamento gênico de 12,5 kbp potencialmente envolvido na produção de GNT. Para validar nosso achado genômico a maioria das enzimas biossintéticas foram superexpressadas, purificadas e caracterizadas in vitro, confirmando o agrupamento gênico proposto. Além disso, um vetor foi desenvolvido com os dois primeiros genes biossintéticos com o objetivo de produzir o intermediário enduracididina, um raro aminoácido. A combinação de experimentos in vivo e in vitro validou com sucesso nosso agrupamento gênico. Usando nossos resultados, mostramos que esse agrupamento gênico foi expresso ativamente em um dos maiores locais do mundo de florações de cianobactérias, o Lago Erie, Toledo, EUA. Isso sugere que essa potente neurotoxina, guanitoxina, pode estar em reservatórios de água potável em todo o mundo, usados para abastecimento público. O preocupante é que a guanitoxina é frequentemente negligenciada e raramente testada em reservatórios, apesar do perigo que representa. Este estudo contribuiu para a caracterização da via biossintética da GNT, podendo gerar uma patente que permita o desenvolvimento de métodos rápidos e sensíveis para a detecção e monitoramento dessa toxina em águas doces utilizadas para abastecimento público. Além disso, este trabalho pode oferecer potencial para o desenvolvimento de novos medicamentos neurológicosBiblioteca Digitais de Teses e Dissertações da USPFiore, Marli de FatimaCamargo, Stella de Lima2020-05-08info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttps://www.teses.usp.br/teses/disponiveis/64/64133/tde-19092022-103522/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPLiberar o conteúdo para acesso público.info:eu-repo/semantics/openAccesseng2022-10-24T18:23:28Zoai:teses.usp.br:tde-19092022-103522Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212022-10-24T18:23:28Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false
dc.title.none.fl_str_mv Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
Revelando a biossíntese da guanitoxina, um organofosfato neurotóxico de ocorrência natural
title Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
spellingShingle Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
Camargo, Stella de Lima
Atividade enzimática
Cianobactéria
Cianotoxina
Cyanobacteria
Cyanotoxin
Enzyme activity
Genoma
Genome
Public health
Saúde pública
title_short Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
title_full Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
title_fullStr Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
title_full_unstemmed Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
title_sort Revealing the biosynthesis of guanitoxin, a naturally occurring neurotoxic organophosphate
author Camargo, Stella de Lima
author_facet Camargo, Stella de Lima
author_role author
dc.contributor.none.fl_str_mv Fiore, Marli de Fatima
dc.contributor.author.fl_str_mv Camargo, Stella de Lima
dc.subject.por.fl_str_mv Atividade enzimática
Cianobactéria
Cianotoxina
Cyanobacteria
Cyanotoxin
Enzyme activity
Genoma
Genome
Public health
Saúde pública
topic Atividade enzimática
Cianobactéria
Cianotoxina
Cyanobacteria
Cyanotoxin
Enzyme activity
Genoma
Genome
Public health
Saúde pública
description Sphaerospermopsis torques-reginae is a planktonic freshwater cyanobacteria species with broad distribution in several Brazilian aquatic environments. Some strains of this species are known to produce the neurotoxic organophosphate guanitoxin (GNT) as well as spumigin and anabaenopeptin, both protease inhibitors. GNT is a powerful irreversible inhibitor of the acetylcholinesterase enzyme which acts in the peripheral nervous cells of mammals. While GNT has been known for decades, the genes encoding its biosynthetic pathway are unknown. The sequencing and assembly of the S. torques-reginae ITEP-024 5.2 Mbp genome allowed us to identify a 12.5 kbp gene cluster potentially involved in GNT production. To rigorously validate our genomic finding, we over-expressed, purified and characterized many of the GNT biosynthetic enzymes in vitro, confirming our proposed biosynthetic cluster. Also, a vector was designed that assembled two of the biosynthetic genes with the goal to produce the intermediate enduracididine, a rare amino acid. The combination of in vivo and in vitro experiments successfully validated our proposed gene cluster. Using our results, we showed that this biosynthetic gene cluster was actively expressed in one of the largest cyanobacterial blooms hotspots, Lake Erie, Toledo, USA. This suggest that the potent neurotoxin guanitoxin could be in drinking water supplies around the world. Worryingly, guanitoxin is often overlooked and rarely tested for in reservoirs in spite of the danger it poses. This study contributed to the characterization of the GNT biosynthetic pathway, which may generate a patent enabling the development of rapid and sensitive methods for the detection and monitoring of this toxin in freshwaters used for public supply. Moreover, this work may offer potential for the development of new neurological medicines and tools
publishDate 2020
dc.date.none.fl_str_mv 2020-05-08
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
format doctoralThesis
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dc.language.iso.fl_str_mv eng
language eng
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dc.rights.driver.fl_str_mv Liberar o conteúdo para acesso público.
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Liberar o conteúdo para acesso público.
eu_rights_str_mv openAccess
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dc.publisher.none.fl_str_mv Biblioteca Digitais de Teses e Dissertações da USP
publisher.none.fl_str_mv Biblioteca Digitais de Teses e Dissertações da USP
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