Estudo metabolômico de fungos do gênero Colletotrichum e de organismos marinhos produtores de tambjaminas
Data
Autores
Título da Revista
ISSN da Revista
Título de Volume
Editor
Resumo
<span style="font-weight: 400;">O presente trabalho descreve o estudo metabolômico de fungos do gênero <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> e de organismos marinhos produtores de tambjaminas <span style="font-weight: 400;">Virididentula dentata<span style="font-weight: 400;">, <span style="font-weight: 400;">Tambja stegosauriformis<span style="font-weight: 400;">, <span style="font-weight: 400;">Tambja brasiliensis<span style="font-weight: 400;"> e <span style="font-weight: 400;">Roboastra ernsti<span style="font-weight: 400;">. Fungos do gênero <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> causam doenças em uma grande variedade de plantas de diversas famílias. Metabólitos secundários produzidos por estes micro-organismos podem constituir parte do processo de virulência associada à sua fitopatogenicidade. Apesar de muitos metabólitos de linhagens de <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> já terem sido isolados e identificados, a fitopatogenicidade associada a fungos deste gênero ainda não está bem estabelecida. Com o intuito de se investigar a diferença no metabolismo de linhagens endofíticas e fitopatogênica de <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> spp., 25 isolados de <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> foram investigados, dentro os quais 16 eram endofíticos e 9 fitopatogênicos. Os extratos dos meios de cultivo foram fracionados e analisados por UPLC-IMS-QToF-HRMS. Os dados brutos foram processados empregando-se ferramentas <span style="font-weight: 400;">in-house<span style="font-weight: 400;"> e <span style="font-weight: 400;">molecular networking<span style="font-weight: 400;">. A avaliação dos resultados permitiu concluir que linhagens endofíticas e fitopatogênicas de <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> spp. possuem metabolismo consideravelmente diferente. Linhagens endofíticas produzem compostos que são comuns à varias linhagens do grupo; já linhagens fitopatogênicas produzem compostos mais específicos a cada um dos isolados. O processo de desreplicação dos resultados de<span style="font-weight: 400;"> molecular networking <span style="font-weight: 400;">utilizando-se uma biblioteca <span style="font-weight: 400;">in-house<span style="font-weight: 400;"> de compostos isolados de <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> spp. permitiu a identificação de 5 compostos que apresentam atividade fitotóxica nas amostras relacionadas às linhagens fitopatogênicas. Tais resultados são forte indicativo de que a fitopatogenicidade de linhagens de <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> podem estar associada à produção de compostos fitotóxicos. Tambjaminas são um grupo de alcalóides quimicamente relacionados à prodigiosina e prodigionina, isoladas de bactérias e organismos marinhos como ascídias, briozoários e nudibrânquios. Além de apresentarem atividades biológicas diversificadas, as tambjaminas estão possivelmente relacionadas à mecanismos de defesa dos organismos a partir dos quais são isoladas. Diversos estudos de organismos marinhos produtores de tambjaminas envolvidos em relações presa-predador demonstraram que estes alcalóides foram detectados nos extratos tanto das presas quanto dos predadores. Nesse sentido, foram obtidos os extratos de <span style="font-weight: 400;">V. dentata, T. stegosauriformis, T. brasiliensis e R. ernsti<span style="font-weight: 400;">. A partir da análise dos extratos orgânicos e aquosos dos organismos marinhos por HPLC-UV-ELSD-MS e UPLC-QToF-MS/MS, foi possível confirmar a presença de 6 tambjaminas conhecidas: tambjaminas A, C, D e K e os aldeídos das tambjaminas A e B. A quantificação do conteúdo de tambjaminas nas amostras de diferentes invertebrados por HPLC com detector de fluorescência demonstrou que <span style="font-weight: 400;">T. stegosauriformis<span style="font-weight: 400;"> e <span style="font-weight: 400;">T. brasiliensis<span style="font-weight: 400;"> possuem conteúdo de tambjaminas de 8 e 14 vezes superiores à sua presa <span style="font-weight: 400;">V. dentata<span style="font-weight: 400;">, enquanto que <span style="font-weight: 400;">R. ernsti<span style="font-weight: 400;"> possui concentração de tambjaminas entre 3 e 5 vezes superiores à suas presas <span style="font-weight: 400;">T. brasiliensis<span style="font-weight: 400;"> e <span style="font-weight: 400;">T. stegosauriformis<span style="font-weight: 400;">. Adicionalmente, análises metabolômicas não-direcionadas do extrato do manto de <span style="font-weight: 400;">R. ernsti <span style="font-weight: 400;">permitiram verificar a presença de 6 novas tambjaminas. A síntese, purificação e caracterização das novas tambjaminas permitiu confirmar a estrutura de 1 das tambjaminas inéditas.
<span style="font-weight: 400;">The present study describes the metabolomic investigation of fungi of the genus <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> and tambjamine-producing marine organisms <span style="font-weight: 400;">Virididentula dentata<span style="font-weight: 400;">, <span style="font-weight: 400;">Tambja stegosauriformis<span style="font-weight: 400;">, <span style="font-weight: 400;">Tambja brasiliensis<span style="font-weight: 400;"> and <span style="font-weight: 400;">Roboastra ernsti<span style="font-weight: 400;">. Fungi belonging to the genus <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> cause disease in a wide variety of plants from different families. Secondary metabolites produced by these microorganisms can be part of the virulence process associated with their phytopathogenicity. Although many metabolites of <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> spp. have already been isolated and identified, the phytopathogenicity associated with fungi of this genus is not yet well established. In order to investigate the metabolic differences between endophytic and phytopathogenic strains of <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> spp., culture media produced by twenty-five isolates of <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> spp. were investigated, 16 of which were endophytic and 9 were phytopathogenic. The culture media were fractionated and analyzed by UPLC-IMS-QToF-HRMS. The raw data were processed using in-house tools and Molecular Networking. The evaluation of the results allowed us to conclude that endophytic and phytopathogenic strains of <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> spp. have considerably different metabolism. Endophytic strains produce compounds that are common to several strains in the group; phytopathogenic strains produce compounds more specific to each isolate. Dereplication of the GNPS exported results using an in-house library of compounds isolated from <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> fungi combined with <span style="font-weight: 400;">in-silico<span style="font-weight: 400;"> fragmentation tools allowed the identification of 5 compounds in samples related to phytopathogenic strains. Those compounds display phytotoxic activity. Such results are a strong indication that the phytopathogenicity of <span style="font-weight: 400;">Colletotrichum<span style="font-weight: 400;"> fungi may be associated with the production of phytotoxic compounds. Tambjamines are a group of alkaloids chemically related to prodigiosin and prodiginine, isolated from bacteria and marine organisms such as ascidians, bryozoans and nudibranchs. In addition to presenting diversified biological activities, tambjaminas are possibly related to the defense mechanisms of the organisms from which they are isolated. Several studies of tambjamine-producing marine organisms involved in prey-predator relationships have shown that these alkaloids were detected in extracts from both prey and predators. In this sense, extracts of <span style="font-weight: 400;">V. dentata<span style="font-weight: 400;">, <span style="font-weight: 400;">T. stegosauriformis<span style="font-weight: 400;">, <span style="font-weight: 400;">T<span style="font-weight: 400;">. <span style="font-weight: 400;">brasiliensis<span style="font-weight: 400;"> and <span style="font-weight: 400;">R. ernsti <span style="font-weight: 400;">were obtained. The analysis of organic and aqueous extracts from marine organisms by HPLC-UV-ELSD-MS and UPLC-QTOF-MS/MS allowed us to confirm the presence of 6 known tambjamines: tambjamines A, C, D and K, and the aldehydes of the tambjamines A and B. The quantification of tambjamine content in samples from different invertebrates performed by HPLC with a fluorescence detector demonstrated that <span style="font-weight: 400;">T. stegosauriformis<span style="font-weight: 400;"> and <span style="font-weight: 400;">T. brasiliensis<span style="font-weight: 400;"> have tambjamines content 8 and 14 times higher than their prey <span style="font-weight: 400;">V. dentata<span style="font-weight: 400;">, while <span style="font-weight: 400;">R. ernsti<span style="font-weight: 400;"> has tambjamines concentration between 3 and 5 times higher than their prey <span style="font-weight: 400;">T. brasiliensis<span style="font-weight: 400;"> and <span style="font-weight: 400;">T. stegosauriformis<span style="font-weight: 400;">. Additionally, non-targeted metabolomic analyzes of the extract from the mantle of <span style="font-weight: 400;">R. ernsti<span style="font-weight: 400;"> allowed to verify the presence of 6 new tambjamines. The synthesis, purification and characterization of the new tambjamines allowed to confirm the structure of 1 of the unprecedented tambjamines.