Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Abstract

S U M M A R YRecent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

Neospora caninum Recruits Host Cell Structures to Its Parasitophorous Vacuole and Salvages Lipids from Organelles

“…This would substantiate our observations illustrating that the Nc-Liv PV display a more important coverage with host mitochondria than the PV of T. gondii (Prugniaud), a parasite strain lacking TgMAF1 (56). A ROP2 gene is also present in N. caninum and is expressed by the parasite (83). NcROP2 shares 47% identity with TgROP2 and possesses features of ROP2 family proteins, including the R-rich amphipathic helix (RAH) domain at the N terminus that mediates the anchorage of ROP2 to the PV membrane.…”

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

“…Nuclear localization suggests that the TgCDPK1 may be responsible for phosphorylating nuclear proteins, potentially relaying a shift in transcription associated with invasion and/or other yet to be defined functions associated with cytokinesis of the daughter cells. We also show that intracellular Neospora and Toxoplasma exposed to BKI-1294 treatment exhibited markedly elevated BAG1 transcript levels and increased labeling by antibodies directed against BAG1 and by the mAbCC2, another marker for bradyzoite stage differentiation (18).…”

“…After 24 h, cultures were supplemented with 2.5 M BKI-1294; infected cultures treated with DMSO served as controls. After 2, 4, and 6 days of treatment, the monolayers were washed with 100 mM sodium cacodylate buffer (pH 7.3) and fixed with cacodylate buffer containing 2.5% glutaraldehyde for 10 min (18). Cells were collected using a rubber cell scraper and centrifuged for 10 min at 1,200 rpm and room temperature.…”

“…The supernatant was removed, and infected cells were fixed further in glutaraldehyde-cacodylate at 4°C overnight. Postfixation in 2% OsO 4 , dehydration, embedding in Epon 820 epoxy resin, and cutting of ultrathin sections was done as previously described (18,19). Specimens were viewed on a Phillips 400 transmission electron microscope (TEM) operating at 80 kV.…”

“…After initial growth for 24 h, specimens were exposed to 2.5 M BKI-1294 at periods of 2, 4, or 6 days. They were then washed once in PBS and fixed and processed for immunofluorescence as previously described (18). The following antibodies (Abs) were used: (i) a whole N. caninum extract rabbit antiserum (16), diluted 1:2,500; (ii) mAbCC2, a rat monoclonal Ab reacting with a yet uncharacterized cyst wall antigen (20), diluted 1:300; (iii) ␣-BAG1, a polyclonal rabbit Ab directed against BAG1, a classical bradyzoite-specific antigen (21-23), diluted 1:300; (iv) ␣-SAG1, a mouse monoclonal Ab directed against the major immunodominant tachyzoite surface antigen (24), diluted 1:300; and (v) a whole T. gondii extract rabbit antiserum, diluted 1:2,000.…”