Avaliação quantitativa do proteoma do fungo Paracoccidioides brasiliensis, isolado Pb18, com distintos graus de virulência
Arquivos
Data
2014
Autores
Castilho, Daniele Goncalves 
Orientadores
Batista, Wagner Luiz
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Este trabalho avaliou de forma quantitativa a composicao proteica do fungo patogenico Paracoccidioides brasiliensis na fase de levedura. Esta analise foi realizada no isolado Pb18 com distintos perfis de infeccao em camundongo B10.A (Pb18 virulento e Pb18 atenuado). Alem disso, foi realizada a analise comparativa dos dados do proteoma de ambos os isolados para caracterizar possiveis fatores de virulencia em P. brasiliensis. Para tanto, o extrato proteico de celulas leveduriformes do isolado Pb18 atenuado (mantido por anos em cultura) e Pb18 virulento (recem-isolado de animal) foram digeridos com tripsina e, os peptideos resultantes foram marcados com formaldeido e D2-formaldeido e, apos a marcacao, as amostras foram analisadas por LC-MS/MS. Utilizando esta estrategia foi possivel identificar e quantificar 256 proteinas, sendo que 101 proteinas apresentaram expressao diferencial aumentada no isolado Pb18 virulento, e 129 proteinas no Pb18 atenuado. Entre as proteinas com expressao aumentada (> 6 vezes) no isolado Pb18 virulento estao a catalase peroxissomal (PADG_00324), a superoxido dismutase (PADG_07418), Cdc42 (PADG_05369), e a protease A vacuolar (PADG_00634). Todas as proteinas identificadas e quantificadas foram agrupadas de acordo com as suas categorias funcionais e, os resultados obtidos mostraram que o isolado Pb18 virulento apresenta uma reorganizacao global do metabolismo e aumento na expressao de proteinas que participam da resposta ao estresse. As diferencas quantitativas observadas foram confirmadas em analises de PCR em tempo real e atividades enzimaticas. Dessa forma, podemos sugerir que as proteinas identificadas neste trabalho contribuem para a virulencia de P. brasiliensis. Este e o primeiro trabalho que utiliza a estrategia de espectrometria de massas para identificar fatores de virulencia utilizando um mesmo isolado fungico com distintos graus de virulencia. Os achados apresentados neste trabalho poderao iniciar estudos para elucidar a relacao parasito-hospedeiro nesta importante micose sistemica
This study evaluated quantitatively the protein composition of the pathogenic fungus Paracoccidioides brasiliensis in yeast phase. This analysis was performed on isolate Pb18 with distinct profiles of infection in mice B10.A (virulent Pb18 and attenuated Pb18). Furthermore, comparative analysis was conducted of data from proteome of both isolates to characterize potential virulence factors of P. brasiliensis. For this, protein extracts of yeast cells attenuated Pb18 isolate (maintained for years in culture) and virulent Pb18 (freshly isolated animal) were digested with trypsin, the resulting peptides were labeled with formaldehyde and D2-formaldehyde and analyzed by LCMS/MS. We identified and quantified 256 proteins, of these 101 proteins were overexpressed in virulent Pb18 isolate, and 129 proteins in attenuated Pb18 isolate. Among the proteins with increased expression (> 6 times) in virulent Pb18 isolate are peroxisomal catalase (PADG_00324), superoxide dismutase (PADG_07418), Cdc42 (PADG_05369), and the vacuolar protease A (PADG_00634). All identified and quantified proteins were grouped according to their functional categories, and the results showed that the isolate Pb18 virulent presents comprehensive reorganization of metabolism and increase in expression of proteins involved in stress response. The differences observed, through proteomic analyzes, were confirmed by analyzes of realtime PCR and enzymatic activity. Thus, we suggest that the proteins identified in this study contribute to the virulence in P. brasiliensis. This is the first work that uses the strategy of mass spectrometry to explore virulence factors using the same fungal isolate with different degrees of virulence. The findings presented in this paper will initiate studies to elucidate the host-parasite relationship in this important systemic mycosis.
This study evaluated quantitatively the protein composition of the pathogenic fungus Paracoccidioides brasiliensis in yeast phase. This analysis was performed on isolate Pb18 with distinct profiles of infection in mice B10.A (virulent Pb18 and attenuated Pb18). Furthermore, comparative analysis was conducted of data from proteome of both isolates to characterize potential virulence factors of P. brasiliensis. For this, protein extracts of yeast cells attenuated Pb18 isolate (maintained for years in culture) and virulent Pb18 (freshly isolated animal) were digested with trypsin, the resulting peptides were labeled with formaldehyde and D2-formaldehyde and analyzed by LCMS/MS. We identified and quantified 256 proteins, of these 101 proteins were overexpressed in virulent Pb18 isolate, and 129 proteins in attenuated Pb18 isolate. Among the proteins with increased expression (> 6 times) in virulent Pb18 isolate are peroxisomal catalase (PADG_00324), superoxide dismutase (PADG_07418), Cdc42 (PADG_05369), and the vacuolar protease A (PADG_00634). All identified and quantified proteins were grouped according to their functional categories, and the results showed that the isolate Pb18 virulent presents comprehensive reorganization of metabolism and increase in expression of proteins involved in stress response. The differences observed, through proteomic analyzes, were confirmed by analyzes of realtime PCR and enzymatic activity. Thus, we suggest that the proteins identified in this study contribute to the virulence in P. brasiliensis. This is the first work that uses the strategy of mass spectrometry to explore virulence factors using the same fungal isolate with different degrees of virulence. The findings presented in this paper will initiate studies to elucidate the host-parasite relationship in this important systemic mycosis.
Descrição
Citação
CASTILHO, Daniele Gonçalves. Avaliação quantitativa do proteoma do fungo Paracoccidioides brasiliensis, isolado Pb18, com distintos graus de virulência. 2014. 146 f. Dissertação (Mestrado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2014.