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

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.

Dynamic secretome of Trichomonas vaginalis: Case study of β-amylases

“…However, our filter for distinguishing bona fide secreted proteins from contaminants resulted in only 89 proteins that fulfilled our strict criteria. Most experimental designs for studying the secretome of parasitic protists are based on a single time period of incubation under optimal conditions to minimize cell lysis, followed by MS analysis of the proteins released to the medium (conditioned medium) (31,34,(61)(62)(63)(64). Although this approach has identified important secreted proteins, eliminating contaminant proteins is problematic.…”

Identification of a TNF-α inducer MIC3 originating from the microneme of non-cystogenic, virulent Toxoplasma gondii

“…In contrast, only 1 ROP, 1 GRA, and 1 SRS showed elevated expression in TgCtwh3 ESAs compared with RH strain ESAs. SRSs appeared in ESAs, likely due to the proteolytic shedding from the parasite2030. Toxolysin 4 (TLN4) is a putative metalloprotease that is processed into multiple proteolytic fragments within the T. gondii secretory system31.…”

Proteomic data on Thrombospondin-related proteins (TRAP) from Neospora caninum (NcMIC2-like1 and NcMIC2)

“…1A). Among all the 615 proteins identified in ESA [10], NcMIC2-like1 was the 21 st most frequently identified and NcMIC2 ranked 93 rd . Since NcMIC2-like1 and NcMIC2 are predicted to be acidic (pI 4.5 for both), 2D gels were run carrying tachyzoite extracts using narrow acidic strips of 3-5.6 NL (nonlinear).…”

“…A shotgun (LC-MS/MS) proteomic approach on N. caninum, both from N. caninum tachyzoite lysate and secreted fraction (ESA), enabled the identification of a massive number of proteins, including NcMIC2-like1 and NcMIC2 [10]. In total, 490 peptide spectrum matches (PSMs) from NcMIC2-like1 and 168 from NcMIC2 were obtained, with a considerably higher presence of NcMIC2-like1 than NcMIC2 in the ESA (448 and 126 PSMs, respectively) and the same number of PSMs (42) in the tachyzoite extract (Fig.…”

“…The N. caninum data from high-resolution mass spectrometry-based proteomics was obtained as previously described [10]. Proteins present in the ESA and in the discharged tachyzoites (parasites submitted to an ethanol secretion stimulus) were identified using shotgun LC-MS/MS.…”