GRA12, a novel dense granule protein from Neospora caninum.

Neospora caninum, an obligate intracellular parasite of the phylum Apicomplexa, is a major cause of abortion in cattle. After invasion, tachyzoites can reside in the parasitophorous vacuole (PV) and ingest nutrition through the intravacuolar network (IVN). Secreted dense granule proteins of N. caninum (NcGRAs) may play important roles in maintaining the structures of the PV and IVN. In this study, we predicted a NcGRA12 gene; aligned it with Toxoplasma gondii GRA12 for homology analysis; and analyzed the ORF, signal peptide and transmembrane domain. Then, we cloned the NcGRA12 gene, expressed the NcGRA12 protein, prepared polyclonal antibodies, and carried out colocalization analysis of NcGRA12 with NcGRA6 in extracellular tachyzoites and intracellular PVs using an immunofluorescence assay (IFA). Finally, we determined the solubility of the NcGRA12 protein. The results showed that NcGRA12 shared 59.13% nucleotide homology and 44.9% amino acid homology with TgGRA12. There was no predicted signal peptide or transmembrane domain. IFA data of extracellular tachyzoites showed that the NcGRA12 protein was secreted by the apical organ and located at the posterior end of tachyzoites, which was consistent with TgGRA12. IFA data of intracellular PVs identified NcGRA12 in the IVN membranes. Moreover, NcGRA12 could colocalize with NcGRA6 in intracellular PVs but not extracellular tachyzoites. Solubility analysis showed that NcGRA12 existed in soluble and membrane-related forms in the PV. Overall, we provide the first report of the novel NcGRA12 protein and verify that it is associated with the IVN membranes of PVs in N. caninum. These data lay a foundation for further research into the function of NcGRA12.

Neospora caninum: a new class of biopharmaceuticals in the therapeutic arsenal against cancer.

BACKGROUND: Microorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies. Viruses, bacteria and even protozoa have been largely explored with success as effective human antitumor agents. To date, only one oncolytic virus-T-VEC-has been approved by the US Food and Drug Administration for use in biological cancer therapy in clinical trials. The goal of our study is to evaluate the potential of a livestock pathogen, the protozoan Neospora caninum, non-pathogenic in humans, as an effective and safe antitumorous agent. METHODS/RESULTS: We demonstrated that the treatment of murine thymoma EG7 by subcutaneous injection of N. caninum tachyzoites either in or remotely from the tumor strongly inhibits tumor development, and often causes their complete eradication. Analysis of immune responses showed that N. caninum had the ability to 1) lyze infected cancer cells, 2) reactivate the immunosuppressed immune cells and 3) activate the systemic immune system by generating a protective antitumor response dependent on natural killer cells, CD8-T cells and associated with a strong interferon (IFN)-γ secretion in the tumor microenvironment. Most importantly, we observed a total clearance of the injected agent in the treated animals: N. caninum exhibited strong anticancer effects without persisting in the organism of treated mice. We also established in vitro and an in vivo non-obese diabetic/severe combined immunodeficiency mouse model that N. caninum infected and induced a strong regression of human Merkel cell carcinoma. Finally, we engineered a N. caninum strain to secrete human interleukin (IL)-15, associated with the alpha-subunit of the IL-15 receptor thus strengthening the immuno-stimulatory properties of N. caninum. Indeed, this NC1-IL15hRec strain induced both proliferation of and IFN-γ secretion by human peripheral blood mononuclear cells, as well as improved efficacy in vivo in the EG7 tumor model. CONCLUSION: These results highlight N. caninum as a potential, extremely effective and non-toxic anticancer agent, capable of being engineered to either express at its surface or to secrete biodrugs. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine.

Identification of secretion domain of Neospora caninum profilin.

Profilin (PROF) is a small actin-binding protein presented in apicomplexan protozoa. It was previously reported that Neospora caninum profilin (NcPROF) is secreted into the hemolymph of silkworm larvae regardless of the lack of an identified regular secretion signal peptide. To date, which domain is required for its secretion still remains unknown. To this end, we express a fluorescent protein (mCherry) fused with NcPROF at its N-terminus or C-terminus. Both fusion proteins were expressed and secreted into the culture supernatant from Bm5 cells or hemolymph from silkworm larvae, respectively. To further narrow down the C-terminal minimal domain required for its secretion, we constructed three truncated C-terminal domain constructions, ΔN (aa41-163), ΔN1 (aa50-163), and ΔN2 (aa144-163) respectively. All three fusion proteins were detected in the culture supernatant of Bm5 cells and silkworm hemolymph. Surprisingly, a 20-aa C-terminal α-helix domain facilitates the secretion of mCherry, allowing purification of ΔN2-mCherry from silkworm larval hemolymph by affinity chromatography. Taken together, the secretion domain from NcPROF was identified, indicating that can be utilized for the secretory expression of recombinant proteins in the future.

Neospora caninum antigens displaying virus-like particles as a bivalent vaccine candidate against neosporosis.

Neospora caninum is a causative and transmissible agent of dog and bovine neosporosis. The resulting reproductive failures in infected cattle lead to significant economic losses worldwide. However, there is no satisfactory treatment or vaccine currently available to combat this pathogen. Thus, the development of appropriate vaccines to manage its infection and transmission is urgently needed. In this study, we expressed Rous sarcoma virus-like particles (RSV-LP) that displayed dual N. caninum antigens in silkworms. The antigen candidates are modified by adding a transmembrane domain of GP64 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) to the C-terminus of surface antigen 1 (NcSAG1) and SAG1-related sequence 2 (NcSRS2). The NcSRS2 alone or the NcSAG1/NcSRS2 bivalent form displaying RSV-LPs were purified using sucrose density gradient centrifugation. These purified VLPs were then used for immunizations in gerbils, Meriones unguiculatus, to evaluate the anti-N. caninum effects in vivo. The results demonstrated that antigens displaying RSV-LPs in immunized gerbils produced the antigen-specific antibody, leading to a relatively lower parasite load after infections of N. caninum. To the best of our knowledge, this is the first study to present an RSV-LP vaccine displaying bivalent antigens from neosporosis. Taken together, our strategy suggests that silkworm-expressed virus-like particles (VLPs) are promising bivalent vaccine candidates against N. caninum infections.

The soluble fraction of Neospora caninum treated with PI-PLC is dominated by NcSRS29B and NcSRS29C.

Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.

Actin from the apicomplexan Neospora caninum (NcACT) has different isoforms in 2D electrophoresis.

Apicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum - a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique. The mass spectrometry data indicated that N. caninum has at least nine different actin isoforms, possibly caused by post-translational modifications. In addition, the C4 pan-actin antibody detected specifically actin in N. caninum cellular extract. Extracellular N. caninum tachyzoites were treated with toxins that act on actin, jasplakinolide and cytochalasin D. Both substances altered the peripheric cytoplasmic localization of actin on tachyzoites. Our findings add complexity to the study of the apicomplexan actin in cellular processes, since the multiple functions of this important protein might be regulated by mechanisms involving post-translational modifications.

First Characterization of the Neospora caninum Dense Granule Protein GRA9.

The obligate intracellular apicomplexan parasite Neospora caninum (N. caninum) is closely related to Toxoplasma gondii (T. gondii). The dense granules, which are present in all apicomplexan parasites, are important secretory organelles. Dense granule (GRA) proteins are released into the parasitophorous vacuole (PV) following host cell invasion and are known to play important roles in the maintenance of the host-parasite relationship and in the acquisition of nutrients. Here, we provide a detailed characterization of the N. caninum dense granule protein NcGRA9. The in silico genomic organization and key protein characteristics are described. Immunofluorescence-based localization studies revealed that NcGRA9 is located in the dense granules and is released into the interior of the PV following host cell invasion. Immunogold-electron microscopy confirmed the dense granule localization and showed that NcGRA9 is associated with the intravacuolar network. In addition, NcGRA9 is found in the "excreted secreted antigen" (ESA) fraction of N. caninum. Furthermore, by analysing the distribution of truncated versions of NcGRA9, we provide evidence that the C-terminal region of this protein is essential for the targeting of NcGRA9 into the dense granules of N. caninum, and the truncated proteins show reduced secretion.

Constitutive expression and characterization of a surface SRS (NcSRS67) protein of Neospora caninum with no orthologue in Toxoplasma gondii.

Neospora caninum is a parasite of the Apicomplexa phylum responsible for abortion and losses of fertility in cattle. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed with the surface proteins known as the SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SAG related or SRS proteins have been a target of intense research due to its immunodominant pattern, exhibiting potential as diagnostic and/or vaccine candidates. The aim of this study was the cloning, expression and characterization of the gene NcSRS67 of N. caninum using a novel designed plasmid. The coding sequence of NcSRS67 (without the signal peptide and the GPI anchor) was cloned and expressed constitutively instead of the ccdB system of pCR-Blunt II-TOPO. The protein was purified in a nickel sepharose column and identified by mass spectrometry (MS/MS). The constitutive expression did not affect the final bacterial growth, with a similar OD 600nm compared to the non-transformed strains. The recombinant NcSRS67 was over expressed and the native form was detected by the anti-rNcSRS67 serum on 1D western blot as a single band of approximately 38kDa as predicted. On an in vitro assay, the inhibitory effect of the polyclonal antiserum anti-rNcSRS67 was nearly 20% on adhesion/invasion of host cells. The NcSRS67 native protein was localised on part of the surface of N. caninum tachyzoite when compared to the nucleus by confocal immunofluorescence.

A new microneme protein of Neospora caninum, NcMIC8 is involved in host cell invasion.

Microneme proteins play an important role in the invasion process of Apicomplexan parasites through adhesion to host cells. We discovered a new N. caninum protein, NcMIC8, which is highly identical to TgMIC8. The NcMIC8 sequence has 2049 bp and no intron in the open reading fragment. It has a molecular weight of 73.8 kDa and contains a signal peptide, a transmembrane region, a low complexity region and 10 epidermal growth factor (EGF) domains. Immuno-fluorescence assay showed that NcMIC8 is located in the microneme. NcMIC8 was secreted to culture medium under stimulation of 1% ethanol, and cleaved to form the mature body of 40 kDa before transporting to microneme or during secretion. Blocking NcMIC8 using anti-NcMIC8 serum effectively inhibited host cell invasion by tachyzoites in vitro. NcMIC8 in the form of mature body interacts with NcMIC3, and the two microneme proteins form a complex probably during transportation. NcMIC8 is a new microneme protein of N. caninum and could be an attractive target for the control of neosporosis.

Evaluation of Neospora caninum truncated dense granule protein 2 for serodiagnosis by enzyme-linked immunosorbent assay in dogs.

Neosporosis is an infectious disease caused by Neospora caninum, and it primarily affects cattle and dogs. An infection by N. caninum causes fetal abortion and neonatal mortality. Previous proteomics and immunoscreening analyses revealed that N. caninum dense granule antigen 2 (NcGRA2) has potential for serodiagnosis of N. caninum. Consequently, we expressed the truncated NcGRA2 (NcGRA2t), which lacks a signal peptide. We compared the serodiagnostic performances of recombinant NcGRA2t with that of truncated surface antigen 1 of N. caninum (NcSAG1t). Specificity testing using sera from mice infected with Toxoplasma gondii indicated that the NcGRA2t recombinant protein does not cross-react with T. gondii. In addition, we detected anti-NcGRA2t antibody at the acute stage in experimentally infected dogs, while detecting anti-NcSAG1t antibody during both the acute and chronic stages. Our results suggest that the levels of anti-NcGRA2 antibody reflect parasite activation in dogs. In conclusion, antibodies against NcGRA2t and NcSAG1t are suitable indicators to distinguish the acute and chronic stages of N. caninum infection.

Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice.

Three antigens (NcSAG1, NcSRS2 and NcMIC3) from Neospora caninum were expressed using the BmNPV bacmid system in silkworm larvae and purified from the hemolymph. From 20 silkworm larvae, 1.5, 1.2 and 1.4 mg of purified recombinant NcSAG1, NcSRS2 and NcMIC3 were obtained, respectively. When each purified recombinant antigen was immunized with Freund's incomplete adjuvant (FIA) to mice, recombinant NcSAG1 induced a Th2 immune response in immunized mice and produced a SAG1-specific antibody. In the experiment where NcSAG1-immunized mice were challenged with N. caninum, the cerebral N. caninum burden was significantly reduced compared with that of either the FIA- or PBS-immunized mice. Recombinant NcSRS2 or NcMIC3 induced both Th1 and Th2 immune responses, but NcMIC3-immunization did not induce significant production of NcMIC3-specific antibodies. These results suggest that the silkworm can produce recombinant antigens of N. caninum, which can be used as a recombinant vaccine against N. caninum.

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

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.

Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion.

Host cell invasion by the obligate intracellular apicomplexan parasites, including Plasmodium (malaria) and Toxoplasma (toxoplasmosis), requires a step-wise mechanism unique among known host-pathogen interactions. A key step is the formation of the moving junction (MJ) complex, a circumferential constriction between the apical tip of the parasite and the host cell membrane that traverses in a posterior direction to enclose the parasite in a protective vacuole essential for intracellular survival. The leading model of MJ assembly proposes that Rhoptry Neck Protein 2 (RON2) is secreted into the host cell and integrated into the membrane where it serves as the receptor for apical membrane antigen 1 (AMA1) on the parasite surface. We have previously demonstrated that the AMA1-RON2 interaction is an effective target for inhibiting apicomplexan invasion. To better understand the AMA1-dependant molecular recognition events that promote invasion, including the significant AMA1-RON2 interaction, we present the structural characterization of AMA1 from the apicomplexan parasites Babesia divergens (BdAMA1) and Neospora caninum (NcAMA1) by X-ray crystallography. These studies offer intriguing structural insight into the RON2-binding surface groove in the AMA1 apical domain, which shows clear evidence for receptor-ligand co-evolution, and the hyper variability of the membrane proximal domain, which in Plasmodium is responsible for direct binding to erythrocytes. By incorporating the structural analysis of BdAMA1 and NcAMA1 with existing AMA1 structures and complexes we were able to define conserved pockets in the AMA1 apical groove that could be targeted for the design of broadly reactive therapeutics.

Label-free molecular analysis of live Neospora caninum tachyzoites in host cells by selective scanning Raman micro-spectroscopy.

A selective scanning method was used to measure spatially resolved Raman spectra of live Neospora caninum tachyzoites colonizing human brain microvascular-endothelial cells. The technique allowed the detection of nucleic acids, lipids and proteins linked to the parasites and their cellular micro-environment at ∼10× shorter acquisition time compared to raster scanning.

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE.

Identification of differentially expressed proteins during Neospora caninum tachyzoite-bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5-fold in relative abundance, p<0.05, t-test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde-3-phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.

The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured Neospora caninum.

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.

The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum.

A Neospora caninum 17-19 kDa antigenic protein fraction (p17) in one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) is the immunodominant antigen recognized by sera from bovines naturally infected by N. caninum. To identify the proteins making up the p17 fraction, we screened a new N. caninum tachyzoite cDNA library with an affinity-purified antibody against p17 (APA17). We isolated several cDNA clones with 100% sequence identity to the NcGRA7 gene. This previously described gene encodes a dense granule protein with an apparent molecular mass of 33 kDa. A second line of evidence emerged through a combined proteomic approach associating two-dimensional PAGE (2D-PAGE) to Western blotting and to mass spectrometry to characterize the p17 fraction. Two acidic immunodominant but minority protein spots were recognized by APA17 and by bovine sera. These antigens of 17 and 33 kDa are respectively composed of 4 and 2 isoforms. Furthermore, p17 isolation by 2D-PAGE and peptide sequencing by tandem mass spectrometry yielded a partial sequence of 17 amino acids, which allowed the putative amino terminal region of the NcGRA7 protein to be identified unambiguously. The NcGRA7 protein, without the putative signal peptide at the NH2-terminus, was cloned and expressed in Escherichia coli and when the purified recombinant protein (rNcGRA7) was analysed by SDS-PAGE and mass spectrometry, 2 bands of 24 and 33 kDa were resolved and identified as NcGRA7. These results demonstrate that the immunodominant 17 kDa antigen of N. caninum is encoded by the NcGRA7 gene.

Subcellular fractionation and molecular characterization of the pellicle and plasmalemma of Neospora caninum.

A characteristic structural feature of Toxoplasma gondii and Neospora caninum is the presence of a triple-membrane pellicle, on the zoite stages of their complex life-cycle. Here we report the results of electron microscopic studies which show that the pellicle is made of a typical plasmalemma covered on its cytoplasmic side by a system of flattened vesicles named the inner membrane complex. Using methods described previously for the purification of pellicle and plasmalemma fractions from T. gondii, we have evaluated the same methodology for the preparation of pellicles and plasmalemma from N. caninum. The approach used involved subcellular fractionation and sucrose gradient centrifugation to prepare fractions containing pellicles. Plasmalemma was prepared by extraction of this fraction with a high salt glycerol treatment. Fractions containing membrane structures were identified by electron microscopy, and the proteins and antigens present in them were subsequently studied by SDS-PAGE and Western blotting. Electron microscopy of the pellicle fractions of N. caninum demonstrated preservation of the triple-membrane structure which is identical to that found in T. gondii. SDS-PAGE of the pellicle fractions revealed it contained several major proteins. Analyses revealed that the plasmalemma of N. caninum contained 2 abundant proteins in addition to other much lower abundance antigens detectable by monoclonal antibodies. These studies therefore report, for the first time, a detailed molecular characterization of the pellicle and plasmalemma of N. caninum.

Toxoplasma gondii dense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif.

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory-secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine 'KKXX' endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

Comparison of proteome and antigenic proteome between two Neospora caninum isolates.

This study was conducted to explore the relationship between two isolates of Neospora caninum (N. caninum) (KBA-2 and VMDL-1) using proteomics. To achieve the goal, proteins of N. caninum tachyzoite lysates of KBA-2 and VMDL-1 were separated by two-dimensional gel electrophoresis (2-DE), stained with silver-nitrate and analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to compare protein profiles. In addition, proteins separated by 2-DE were transferred to membranes, probed with bovine anti-N. caninum KBA-2 immunoglobulin G, and reactive proteins were visualized and compared between the two isolates. Most spots on 2-DE profiles and antigenic spots on 2-DE immunoblot profiles were located at similar locations in terms of isoelectric point and molecular weight. Proteins common to both isolates included the following: heat shock protein 70, subtilisin-like serine protease, nucleoside triphosphatase, heat shock protein 60, pyruvate kinase, tubulin alpha, tubulin beta, enolase, putative protein disulfide isomerase, actin, fructase-1,6-bisphosphatase, putative ribosomal protein S2, microneme protein Nc-P38, lactate dihydrogenase, fructose-1,6-bisphosphatase aldolase, serine threonine phosphatase 2C, 14-3-3 protein homologue, N. caninum dense granule-1 and NcGRA2. As a consequence, even though N. caninum KBA-2 and VMDL-1 isolates were isolated from geographically distinct locations there were significant homology in the proteome and antigenic proteome profiles. In addition, proteomic approach was verified as a useful tool for understanding of host immune response against different isolates of protozoa.