Abstract
BackgroundThe 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.MethodsTo 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.ResultsAs 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.ConclusionsThe 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.
Highlights
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
Our study to comprehend the N. caninum secretome was based on the secretory stimulus via increased intracellular Ca2+, in vitro triggered by ethanol
Proteins identified from the N. caninum secreted fraction (ESA) The collected Tachyzoite secreted fraction (ESA) proteins were separated into 13 bands by One dimension (1D)-SDS-PAGE (Figure 1) and were in-gel digested
Summary
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. 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. As with all apicomplexan species, N. caninum is an obligate intracellular parasite that invades the host cell in a conserved active manner, which involves the release of proteins from phylum-specific secretory organelles and the activation of the parasite invasion motor. These organelles, known as micronemes, rhoptries and dense granules, secrete proteins crucial for apical attachment, moving junction formation, gliding motility, and parasitophorous vacuole formation and establishment [4,5,6]. Ethanol is a known trigger of apical organelle secretion by means of Ca2+ mobilisation [10]
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