RESUMO
Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4-7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host-parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.
Assuntos
Angiostrongylus cantonensis/metabolismo , Proteínas de Helminto/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/fisiologia , Animais , Eletroforese em Gel Bidimensional , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos/genética , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Estágios do Ciclo de Vida , Masculino , Proteoma/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caramujos/parasitologia , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções por Strongylida/parasitologiaRESUMO
It has been hypothesized that blood-brain barrier (BBB) dysfunction in Angiostrongylus cantonensis infection might be due to the apoptosis of the hosts' BBB cells. Here, we evaluated this hypothesis through several methods, all based on an in vitro mouse BBB model consisting of primary culture brain microvascular endothelial cells (BMECs) and brain astrocytic cells (BACs). In the present study, a four-hour percolation and HRP permeability experiment showed that A. cantonensis larvae extracts can increase the permeability of the BBB. Apoptosis among BMECs and BACs after exposure to larvae extracts was monitored by TUNEL and annexin-V-FITC/PI double staining. A. cantonensis larvae extracts were found to induce apoptosis in both BMECs and BACs. For this reason, we concluded that the induction of apoptosis might participate in the BBB dysfunction observed during angiostrongyliasis. Improved fundamental understanding of how A. cantonensis induces apoptosis may lead to new approaches to the treatment or prevention of this parasitic disease.