Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris

The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool...

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Autor principal: Lacerda, Ariane Ferreira
Outros Autores: Grossi, Maria Fátima
Formato: doctoralThesis
Idioma:por
Publicado em: Universidade Federal do Rio Grande do Norte
Assuntos:
Antifungal defensin
rDrr230a
rCD1
Pichia pastoris
Phakopsora pachyrhizi
Fusarium tucumaniae
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
Endereço do item:https://repositorio.ufrn.br/jspui/handle/123456789/20692
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id ri-123456789-20692
record_format dspace
institution Repositório Institucional
collection RI - UFRN
language por
topic Antifungal defensin
rDrr230a
rCD1
Pichia pastoris
Phakopsora pachyrhizi
Fusarium tucumaniae
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
spellingShingle Antifungal defensin
rDrr230a
rCD1
Pichia pastoris
Phakopsora pachyrhizi
Fusarium tucumaniae
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
Lacerda, Ariane Ferreira
Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris
description The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.
author2 Grossi, Maria Fátima
author_facet Grossi, Maria Fátima
Lacerda, Ariane Ferreira
format doctoralThesis
author Lacerda, Ariane Ferreira
author_sort Lacerda, Ariane Ferreira
title Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris
title_short Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris
title_full Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris
title_fullStr Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris
title_full_unstemmed Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris
title_sort avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em pichia pastoris
publisher Universidade Federal do Rio Grande do Norte
publishDate 2016
url https://repositorio.ufrn.br/jspui/handle/123456789/20692
work_keys_str_mv AT lacerdaarianeferreira avaliacaodaatividadeantimicrobianadasdefensinasrecombinantesdeervilhadrr230aecafecd1produzidasempichiapastoris
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spelling ri-123456789-206922019-01-29T23:47:41Z Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris Lacerda, Ariane Ferreira Grossi, Maria Fátima http://lattes.cnpq.br/0816835876517153 http://lattes.cnpq.br/3058512809761818 Scortecci, Kátia Castanho http://lattes.cnpq.br/4808910380593455 Santos, Elizeu Antunes dos http://lattes.cnpq.br/6762251930590306 Vasconcelos, Ilka Maria http://lattes.cnpq.br/0103197383336584 Pedrini, Márcia Regina da Silva http://lattes.cnpq.br/0099275569766870 Silva, Marília Santos http://lattes.cnpq.br/6395128945646125 Antifungal defensin rDrr230a rCD1 Pichia pastoris Phakopsora pachyrhizi Fusarium tucumaniae CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) A ineficácia de pesticidas químicos no controle de fungos fitopatogênicos na agricultura e a frequente incidência de doenças humanas causadas por bactérias resistentes a antibióticos levam à busca por compostos antimicrobianos alternativos. Neste contexto, defensinas vegetais constituem uma promissora ferramenta no controle de agentes patogênicos tanto de plantas quanto de humanos. Defensinas de plantas são peptídeos catiônicos com aproximadamente 50 resíduos de aminoácidos, ricas em cisteínas e cuja estrutura tridimensional é bastante conservada entre as diferentes espécies vegetais. Estas moléculas de ação antimicrobiana representam um importante componente inato da resposta de defesa vegetal contra patógenos e são expressas em diversos tecidos da planta, como folhas, tubérculos, flores, vagens e sementes. O presente trabalho teve por finalidade a avaliação da atividade antimicrobiana de duas defensinas vegetais contra diferentes espécies de fungos fitopatogênicos e bactérias patogênicas ao homem. A defensina Drr230a, cujo gene foi isolado de ervilha (Pisum sativum) e a defensina CD1, cujo gene foi identificado no transcriptoma de café (Coffea arabica) foram subclonadas em vetor de expressão de levedura e expressas em Pichia pastoris. O gene cd1 foi subclonado em duas formas recombinantes: CD1tC, contendo uma sequência codificante para seis histidinas (6xHis) na região C-terminal do peptídeo e CD1tN, contendo sequência codificante para 6xHis na região N-terminal. No caso da defensina Drr230a, a sequência codificante para 6xHis foi inserida apenas na região N-terminal da proteína. Ensaios de atividade antimicrobiana das proteínas recombinantes purificadas rDrr230a e rCD1 contra Phakopsora pachyrhizi, agente causal da ferrugem asiática da soja, foram realizados para analisar a inibição da germinação de esporos in vitro e a severidade da doença causada pelo fungo in planta. As duas defensinas recombinantes testadas foram capazes de inibir a germinação de uredosporos de P. pachyrhizi, não havendo diferença entre a ação antimicrobiana de CD1tC e CD1tN. Ademais, rDrr230a e rCD1 reduziram drasticamente a severidade da ferrugem asiática da soja, conforme demonstrado em ensaios in planta. Apesar de rCD1 não ter sido capaz de inibir a proliferação das bactérias patogênicas humanas Staplylococcus aureus e Klebsiella pneumoniae, rCD1 mostrou-se capaz de inibir o crescimento do fungo fitopatogênico Fusarium tucumaniae, causador da síndrome da morte súbita da soja. Os resultados obtidos mostram que tais defensinas vegetais são candidatas úteis para serem utilizadas em programas de engenharia genética de plantas para controlar doenças fúngicas impactantes na agricultura. 2016-06-13T20:26:34Z 2016-06-13T20:26:34Z 2015-06-30 doctoralThesis LACERDA, Ariane Ferreira. Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris. 2015. 125f. Tese (Doutorado em Bioquímica) - Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 2015. https://repositorio.ufrn.br/jspui/handle/123456789/20692 por Acesso Aberto application/pdf Universidade Federal do Rio Grande do Norte Brasil UFRN PROGRAMA DE PÓS-GRADUAÇÃO EM BIOQUÍMICA

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