Chalconas e curcuminoides como novas alternativas para combater o cancro cítrico e alguns patógenos de interesse clínico
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Data
2018-11-08
Autores
Morão, Luana Galvão [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O cancro cítrico é uma das doenças mais preocupantes no âmbito da citricultura. Causado pela bactéria Gram-negativa Xanthomonas citri subsp. citri está doença caracteriza-se pela aparição de lesões eruptivas e corticosas, castanho-amareladas em diversas partes aéreas das mais variadas espécies de Citros, sendo algumas mais susceptíveis que outras. Além disso, a infecção bacteriana na planta hospedeira desencadeia algumas alterações fisiológicas e metabólicas da planta dentre elas a produção demasiada de etileno, o que causa outro sintoma característico da doença, a queda prematura de frutos, o maior problema que o cancro cítrico acarreta, uma vez que os frutos não estão maduros e prontos o suficiente para serem colhidos e comercializados, podendo gerar prejuízo ao citricultor. Até o momento não há substâncias que sejam eficientes contra a bactéria Xanthomonas citri. Existem apenas medidas paliativas e, essencialmente, preventivas tais como a utilização de quebra ventos nos pomares, controle de mudas sadias, controle da larva minadora (Phyllocnistis citrella) e pulverização massiva de soluções de cobre fixo. A aplicação de soluções cúpricas é prejudicial ao meio ambiente, uma vez que o cobre tem efeito tóxico, cumulativo, carcinogênico e mutagênico, em pequenas quantidades. Sabe- se, também que há genes de resistência ao cobre, já existentes em X. citri. Paralelamente ao setor citrícola, tem-se a indústria farmacêutica e o extremo desafio em descobrir e produzir novas classes de antimicrobianos que apresentem capacidade de aniquilar diversas bactérias patogênicas de interesse clínico. O grande problema enfrentado, neste caso, é o uso incorreto de antimicrobianos existentes combinado com o aumento desenfreado de resistência bacteriana e ainda com a burocracia e alto custo nas pesquisas de ponta visando novas substâncias bioativas. Considerando a necessidade da obtenção de novas classes de substâncias potencialmente ativas e com menor taxa de toxicidade, tanto no setor citrícola quanto farmacêutico, nosso grupo de pesquisa, em colaboração com o Instituto de Química (Laboratório de Química Verde Medicinal) da UNESP de São José do Rio Preto e Universidade de Groningen, na Holanda, objetivaram a síntese de classes de compostos derivados de substâncias naturais, a fim de avaliar sua capacidade antimicrobiana frente ao fitopatógeno Xanthomonas citri subsp. citri bem como a alguns microrganismos de interesse clínico e modelos microbiológicos, tais como B. subtilis, E. coli, P. aeruginosa, S. enterica Typhimurium, E. faecalis e S. aureus. Ao todo foram testadas três classes de compostos sintéticos derivadas de curcumina e chalconas pelo método de microdiluição REMA. Também foi investigado o modo de ação dos compostos que foram bioativos, através de diversas técnicas: ensaio da integridade de membrana citoplasmática, ensaio de disrupção de septo de divisão celular, análise com FT-IR, análise da atividade GTPásica de FtsZ (associado ao septo de divisão celular), avaliação de síntese de macromoléculas através de radiomarcadores (para DNA, RNA, proteínas e parede celular) e dosagem de ATP intracelular para avaliar perturbação na via energética. Nove compostos foram ativos contra B. subtilis, seis contra X. citri e dois contra S. aureus, atingindo um percentual de inibição de crescimento bacteriano maior que 90%. Testes realizados em plantas infectadas com X. citri, tratada com os compostos, mostraram a eliminação da capacidade de causar cancro comparado aos controles negativos. Os testes do modo de ação destes compostos sugeriram que alguns podem ter ação multi target, outro composto mostrou ação específica na desestruturação da integridade da membrana citoplasmática e outro ainda demonstrou agir na disrupção do septo de divisão celular bacteriana. No entanto análises futuras e novos testes do modo de ação podem confirmar e completar esses resultados.
Citrus canker is one of the most worrying diseases in the citrus industry. Caused by Gramnegative bacteria Xanthomonas citri subsp. Citri this disease is characterized by the appearance of eruptive, yellow-brown lesions in several aerial parts of the most varied Citrus species, some being more susceptible than others. In addition, the bacterial infection in the host plant triggers some physiological and metabolic changes of the plant, among them the production of too much ethylene, which causes another characteristic symptom of the disease, premature fruit drop, the biggest problem that citrus canker causes, since the fruits are not ripe and ready enough to be harvested and marketed, may cause harm to the farmer. To date there are no substances that are effective against the bacterium Xanthomonas citri. There are only palliative and, essentially, preventive measures such as the use of windbreaks in orchards, control of healthy seedlings, control of the larvae (Phyllocnistis citrella) and massive spraying of fixed copper solutions. The application of copper solutions is harmful to the environment, since copper has a toxic, cumulative, carcinogenic and mutagenic effect, in small amounts. It is also known that copper resistance genes already exist in X. citri. Parallel to the citrus industry, there is the pharmaceutical industry and the extreme challenge of discovering and producing new classes of antimicrobials that have the capacity to kill several pathogenic bacteria of clinical interest. The major problem faced in this case is the misuse of existing antimicrobials combined with the unbridled increase in bacterial resistance and still with the bureaucracy and high cost in cutting-edge research aiming new bioactive substances. Considering the need to obtain new classes of potentially active substances with a lower toxicity rate, both in the citrus and pharmaceutical sectors, our research group, in collaboration with the Institute of Chemistry (Laboratório de Química Verde Medicinal) of UNESP of São José do Rio Preto and the University of Groningen, in the Netherlands, aimed to synthesize classes of compounds derived from natural substances in order to evaluate their antimicrobial capacity against the phytopathogen Xanthomonas citri subsp. Citri as well as to some microorganisms of clinical interest and microbiological models such as B. subtilis, E. coli, P. aeruginosa, S. enterica Typhimurium, E. faecalis and S. aureus. In all, three classes of synthetic compounds derived from curcumin and chalcones were tested by the REMA microdilution method. It was also investigated the mode of action of the compounds that were bioactive, through several techniques: cytoplasmic membrane integrity assay, cell division septum disruption assay, FT-IR analysis, analysis of GTPase activity of the FtsZ (associated with the cell division septum), evaluation of macromolecular synthesis through radiolabels (for DNA, RNA, proteins and cell wall) and dosage of intracellular ATP to evaluate disturbance in the energy pathway. Nine compounds were active against B. subtilis, six against X. citri and two against S. aureus, reaching a percentage of inhibition of bacterial growth greater than 90%. Tests performed on plants infected with X. citri treated with the compounds showed the elimination of the ability to cause canker compared to the negative controls. The mode of action tests of these compounds suggested that some may have multi target action, another compound showed specific action on cytoplasmic membrane integrity disturbance and another still showed to act on bacterial cell division septum disruption. However, future analyzes and new mode of action tests can confirm and complete these results.
Citrus canker is one of the most worrying diseases in the citrus industry. Caused by Gramnegative bacteria Xanthomonas citri subsp. Citri this disease is characterized by the appearance of eruptive, yellow-brown lesions in several aerial parts of the most varied Citrus species, some being more susceptible than others. In addition, the bacterial infection in the host plant triggers some physiological and metabolic changes of the plant, among them the production of too much ethylene, which causes another characteristic symptom of the disease, premature fruit drop, the biggest problem that citrus canker causes, since the fruits are not ripe and ready enough to be harvested and marketed, may cause harm to the farmer. To date there are no substances that are effective against the bacterium Xanthomonas citri. There are only palliative and, essentially, preventive measures such as the use of windbreaks in orchards, control of healthy seedlings, control of the larvae (Phyllocnistis citrella) and massive spraying of fixed copper solutions. The application of copper solutions is harmful to the environment, since copper has a toxic, cumulative, carcinogenic and mutagenic effect, in small amounts. It is also known that copper resistance genes already exist in X. citri. Parallel to the citrus industry, there is the pharmaceutical industry and the extreme challenge of discovering and producing new classes of antimicrobials that have the capacity to kill several pathogenic bacteria of clinical interest. The major problem faced in this case is the misuse of existing antimicrobials combined with the unbridled increase in bacterial resistance and still with the bureaucracy and high cost in cutting-edge research aiming new bioactive substances. Considering the need to obtain new classes of potentially active substances with a lower toxicity rate, both in the citrus and pharmaceutical sectors, our research group, in collaboration with the Institute of Chemistry (Laboratório de Química Verde Medicinal) of UNESP of São José do Rio Preto and the University of Groningen, in the Netherlands, aimed to synthesize classes of compounds derived from natural substances in order to evaluate their antimicrobial capacity against the phytopathogen Xanthomonas citri subsp. Citri as well as to some microorganisms of clinical interest and microbiological models such as B. subtilis, E. coli, P. aeruginosa, S. enterica Typhimurium, E. faecalis and S. aureus. In all, three classes of synthetic compounds derived from curcumin and chalcones were tested by the REMA microdilution method. It was also investigated the mode of action of the compounds that were bioactive, through several techniques: cytoplasmic membrane integrity assay, cell division septum disruption assay, FT-IR analysis, analysis of GTPase activity of the FtsZ (associated with the cell division septum), evaluation of macromolecular synthesis through radiolabels (for DNA, RNA, proteins and cell wall) and dosage of intracellular ATP to evaluate disturbance in the energy pathway. Nine compounds were active against B. subtilis, six against X. citri and two against S. aureus, reaching a percentage of inhibition of bacterial growth greater than 90%. Tests performed on plants infected with X. citri treated with the compounds showed the elimination of the ability to cause canker compared to the negative controls. The mode of action tests of these compounds suggested that some may have multi target action, another compound showed specific action on cytoplasmic membrane integrity disturbance and another still showed to act on bacterial cell division septum disruption. However, future analyzes and new mode of action tests can confirm and complete these results.
Descrição
Palavras-chave
Xanthomonas citri subsp. citri, Cancro cítrico, Patógenos, Compostos sintéticos, Antimicrobianos, Citrus canker, Pathogens, Synthetic compounds, Antimicrobials