title: Unravelling the Neospora caninum secretome through the secreted fraction (ESA) and quantification of the discharged tachyzoite using high-resolution mass spectrometry-based proteomics.
abstract: 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.
authors.forename: Letícia
authors.initials: L
authors.lastname: Pollo-Oliveira
authors.forename: Harm
authors.initials: H
authors.lastname: Post
authors.forename: Marcio Luis
authors.initials: ML
authors.lastname: Acencio
authors.forename: Ney
authors.initials: N
authors.lastname: Lemke
authors.forename: Henk
authors.initials: H
authors.lastname: van den Toorn
authors.forename: Vinicius
authors.initials: V
authors.lastname: Tragante
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: A F Maarten
authors.initials: AF
authors.lastname: Altelaar
authors.affiliation: 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. ayatsuda@fcfrp.usp.br.
authors.forename: Ana Patrícia
authors.initials: AP
authors.lastname: Yatsuda
mesh_terms: D007553:Isotope Labeling; D013058:Mass Spectrometry; D018688:Neospora / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D015800:Protozoan Proteins / Q000032:analysis*
chemical_list: D020543:Proteome; D015800:Protozoan Proteins