Unravelling the Neospora caninum secretome through the secreted fraction (ESA) and quantification of the discharged tachyzoite using high-resolution mass spectrometry-based proteomics

Particle and Fibre Toxicology

Ana Patrícia Yatsuda⁴  AF Maarten Altelaar³  Albert JR Heck³  Vinicius Tragante¹  Henk van den Toorn³  Ney Lemke²  Marcio Luis Acencio²  Harm Post³  Letícia Pollo-Oliveira⁴ 

[1] Division of Biomedical Genetics, Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands;Botucatu Institute of Biosciences, UNESP - Univ Estadual Paulista, Distrito de Rubião Jr, s/n, Botucatu, São Paulo 18918-970, Brazil;Netherlands Proteomics Centre, Padualaan 8, Utrecht 3884 CH, The Netherlands;Faculdade de Ciências Farmacêuticas de Ribeirão Preto e Núcleo de Apoio à Pesquisa em Produtos Naturais e Sintéticos (NPPNS), Universidade de São Paulo, Av do Café , s/n, Ribeirão Preto, SP 14040-903, Brazil

关键词: Relative quantification;    Shotgun;    Secretome;    Neospora caninum;    Mass spectrometry;   

Others  :  823752
DOI  :  10.1186/1756-3305-6-335

 received in 2013-08-30, accepted in 2013-11-15,  发布年份 2013

【 摘 要 】

Background

The apicomplexan parasite Neospora caninum causes neosporosis, a disease that leads to abortion or stillbirth in cattle, generating an economic impact on the dairy and beef cattle trade. As an obligatory intracellular parasite, N. caninum needs to invade the host cell in an active manner to survive. The increase in parasite cytosolic Ca²⁺ upon contact with the host cell mediates critical events, including the exocytosis of phylum-specific secretory organelles and the activation of the parasite invasion motor. Because invasion is considered a requirement for pathogen survival and replication within the host, the identification of secreted proteins (secretome) involved in invasion may be useful to reveal interesting targets for therapeutic intervention.

Methods

To chart the currently missing N. caninum secretome, we employed mass spectrometry-based proteomics to identify proteins present in the N. caninum tachyzoite using two different approaches. The first approach was identifying the proteins present in the tachyzoite-secreted fraction (ESA). The second approach was determining the relative quantification through peptide stable isotope labelling of the tachyzoites submitted to an ethanol secretion stimulus (discharged tachyzoite), expecting to identify the secreted proteins among the down-regulated group.

Results

As a result, 615 proteins were identified at ESA and 2,011 proteins quantified at the discharged tachyzoite. We have analysed the connection between the secreted and the down-regulated proteins and searched for putative regulators of the secretion process among the up-regulated proteins. An interaction network was built by computational prediction involving the up- and down-regulated proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD000424.

Conclusions

The comparison between the protein abundances in ESA and their measure in the discharged tachyzoite allowed for a more precise identification of the most likely secreted proteins. Information from the network interaction and up-regulated proteins was important to recognise key proteins potentially involved in the metabolic regulation of secretion. Our results may be helpful to guide the selection of targets to be investigated against Neospora caninum and other Apicomplexan organisms.

【 授权许可】

   
2013 Pollo-Oliveira et al.; licensee BioMed Central Ltd.

【 图 表 】

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