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Previous issue date: 2016-03-29 / FACEPE / Lectinas, proteínas que interagem com carboidratos, isoladas da entrecasca (MuBL), cerne (MuHL) e folha (MuLL) de Myracrodruon urundeuva apresentaram atividade termiticida contra cupins operários da espécie Nasutitermes corniger. O presente trabalho teve como objetivo investigar mecanismos da ação termiticida dessas lectinas. Inicialmente, foi realizado um estudo de caracterização das atividades enzimáticas encontradas no trato digestivo de operários, de forma comparativa com os soldados da mesma espécie. Extratos de intestinos dos cupins foram avaliados quanto à presença de celulases (endoglucanases, exoglucanase e β-glicosidades), hemicelulases (β-xilosidases, α-L-arabinofuranosidase e β-D-xilanase), amilases e proteases (proteases totais, tripsina, quimotripsina e queratinase). A primeira avaliação dos mecanismos de ação correspondeu à determinação do efeito de MuBL e MuLL sobre as atividades enzimáticas detectadas no intestino dos operários. Na segunda etapa, foram investigadas possíveis proteínas que sejam alvos de ligação das lectinas no intestino dos operários. Para tanto, extratos de intestino foram submetidos à cromatografia em matrizes MuBL-Sepharose e MuLL-Sepharose. As proteínas que adsorveram à matriz foram submetidas à eletroforese e espectrometria de massas para separação e identificação. Na última etapa, operários foram submetidos por 48 h a dietas artificiais compostas por matriz de celulose e suplementadas ou não com MuBL, MuHL ou MuLL (em suas respectivas CL50); em seguida, os insetos tiveram seu intestino médio dissecados, fixados e submetidos à análise histológica, contagem de células digestivas e regenerativas e visualização de células em proliferação, células enteroendócrinas, células em apoptose e da matriz peritrófica. Todas as atividades enzimáticas avaliadas foram detectadas nos extratos de operários e soldados, sendo endoglucanase e β-D-xilanase as principais atividades detectadas. As atividades enzimáticas de operários e soldados foram detectadas em níveis diferentes e apresentaram diferentes respostas a variações de temperatura e pH indicando que as castas possuem aparatos digestivos distintos. MuBL e MuLL foram capazes de modular diferentemente as atividades enzimáticas do trato digestivo dos operários. A atividade de exoglucanase foi neutralizada por MuBL enquanto MuLL promoveu aumento dessa atividade. Atividade de α-L-arabinofuranosidase foi inibida por MuLL e não afetada por MuBL. Ambas as lectinas estimularam a atividade de α-amilase e inibiram as atividades de proteases. As matrizes MuBL-Sepharose e MuLL-Sepharose ligaram a proteína apolipoforina. MuBL-Sepharose também ligou proteína com homologia a transportadores do tipo ABC (ATP-binding cassette) e tripsina. MuBL, MuHL e MuLL causaram forte desorganização no epitélio do intestino médio dos operários, reduzindo o número de células digestivas, regenerativas e enteroendócrinas. A ingestão das lectinas resultou em morte das células intestinais por apoptose. A matriz peritrófica foi visualizada nos tratamentos controle e com as lectinas, contudo a marcação foi menos intensa em intestinos de cupins que ingeriram MuBL. Os resultados mostram que as lectinas foram capazes de cruzar essa matriz peritrófica e causar danos às células do epitélio intestinal. Em conclusão, podemos afirmar que: 1) operários e soldados de N. corniger apresentam aparatos digestivos diferenciados, com elevada eficiência para digerir biomassa celulósica; 2) a ação termiticida das lectinas MuBL e MuLL pode estar relacionada com a modulação de processos digestivos e absortivos no trato intestinal de operários, tendo como alvos enzimas digestivas, a proteína transportadora apolipoforina e transportadores ABC; 3) MuBL, MuHL e MuLL atuam promovendo desestruturação do epitélio intestinal de operários, causando morte de células digestivas, regenerativas e enteroendócrinas. Esses resultados constituem os primeiros relatos sobre alvos moleculares e celulares de lectinas termiticidas. / Lectins, proteins that interact with carbohydrates, isolated from the bark (MuBL), heartwood (MuHL) and leaf (MuLL) of Myracrodruon urundeuva showed termiticidal activity against workers of Nasutitermes corniger termite. This study aimed to investigate mechanisms of the termiticidal action of these lectins. Initially, a study was conducted in order to characterize the enzyme activities found in the digestive tract of workers, in comparison with the soldiers of the same species. Termite gut extracts were assessed for the presence of cellulases (endoglucanase, exoglucanase, and β-glucosidase), hemicellulases (β-xylosidase, α-L-arabinofuranosidase and β-D-xylanase), amylases and proteases (total protease, trypsin, chymotrypsin and keratinase). The first evaluation of the action mechanisms corresponded to the determination of MuBL and MuLL effects on the enzyme activities detected in the gut of the workers. In the second step, we investigated possible proteins that would be lectin binding targets in the gut of workers. Therefore, worker gut extracts were subjected to chromatographies on MuBL-Sepharose and MuLL-Sepharose matrices. Proteins that adsorbed to the matrices were submitted to electrophoresis and mass spectrometry for separation and identification. In the last step, workers were submitted for 48 h to artificial diets composed of cellulose matrix and supplemented or not with MuBL, MuHL or MuLL (at their respective LC50); then, the insects had their midguts dissected, fixed and evaluated by histological analysis, counting of digestive and regenerative cells, and visualization of proliferative, enteroendocrine and apoptotic cells, as well as of peritrophic matrix. All the enzyme activities evaluated were detected in worker and soldier extracts, being endoglucanase and β-D-xylanase the main activities detected. The enzyme activities of workers and soldiers were detected at different levels and showed different responses to temperature and pH, indicating that these castes have different digestive apparatuses. MuBL and MuLL were able to modulate differently the enzyme activities from the digestive tract of workers. MuBL neutralized the exoglucanase activity while MuLL promoted increase in this activity. α-L-arabinofuranosidase activity was inhibited by MuLL and was not affected by MuBL. Both lectins stimulated the α-amylase activity and inhibit the activity of proteases. The MuBL-Sepharose and MuLL-Sepharose matrices bound the protein apolipophorin. MuBL-Sepharose also bound proteins with homology to ABC (ATP-binding cassette) transporters and trypsin. MuBL, MuHL and MuLL caused strong disorganization in the midgut epithelium of workers, reducing the number of digestive, regenerative and enteroendocrine cells. Ingestion of lectins resulted in death of intestinal cells by apoptosis. The peritrophic matrix was visualized in the control and lectin treatments but the labeling was less intense in gut of termites that ingested MuBL. These results show that the lectins were able to cross this peritrophic matrix and cause damage to the cells of the midgut epithelium. In conclusion, we can state that: 1) N. corniger workers and soldiers have different digestive apparatus, with high efficiency to digest cellulosic biomass; 2) the termiticidal action of MuBL and MuLL can be linked to modulation of digestive and absorptive processes in the intestinal tract of workers, targeting digestive enzymes, the transporter protein apolipophorin and ABC transporters; 3) MuBL, MuHL and MuLL promoted disruption of the midgut epithelium of workers, causing death of digestive, regenerative and enteroendocrine cells. These results are the first reports on molecular and cellular targets of termiticidal lectins.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufpe.br:123456789/24323 |
| Date | 29 March 2016 |
| Creators | LIMA, Thâmarah de Albuquerque |
| Contributors | http://lattes.cnpq.br/2885145995086459, PAIVA, Patrícia Maria Guedes, NAPOLEÃO, Thiago Henrique |
| Publisher | Universidade Federal de Pernambuco, Programa de Pos Graduacao em Bioquimica e Fisiologia, UFPE, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFPE, instname:Universidade Federal de Pernambuco, instacron:UFPE |
| Rights | Attribution-NonCommercial-NoDerivs 3.0 Brazil, http://creativecommons.org/licenses/by-nc-nd/3.0/br/, info:eu-repo/semantics/openAccess |
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