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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites.

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.

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.

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.

Molecular characterization of a thrombospondin-related anonymous protein homologue in Neospora caninum.

Thrombospondin-related anonymous protein (TRAP) family members participate in attachment and invasion of host cells by apicomplexan parasites. A TRAP homologue in Neospora caninum strain Nc-1 (NcMIC2) was cloned, sequenced and found to be 61% identical (75% similar) at the amino acid level to Toxoplasma gondii MIC2 (TgMIC2). Similar to TgMIC2, the predicted amino acid sequence of NcMIC2 contains one integrin-like domain (I or A domain), five thrombospondin (TSP) repeats, a putative transmembrane spanning region and intracellular C-terminus, and was localized to micronemes by cryo-immunoelectron microscopy. The secretion of NcMIC2 was temperature dependent and was induced at or above 25 degrees C. The secreted form of NcMIC2 released into the medium was found to be proteolytically processed such that it lacked the C-terminal domain. Secretion of NcMIC2 was regulated by calcium, since several agents which raise intracellular calcium levels were shown to promote NcMIC2 secretion and chelation of [Ca(2+)](i) abrogated release. As a member of the growing family of apicomplexan TRAP proteins, NcMIC2 may play an important role in attachment and invasion by N. caninum into host cells.

Detection of surface-associated and intracellular glycoconjugates and glycoproteins in Neospora caninum tachyzoites.

The surface-associated molecules of the invasive stages of apicomplexan parasites such as Neospora caninum and Toxoplasma gondii are most likely crucially involved in mediating the interaction between the parasite and its host cell. In N. caninum, several antigens have recently been identified which could participate in host cell adhesion and/or invasion. These are antigens which are either constitutively expressed on the outer plasma membrane, or antigens which are only transiently localised on the surface as they are expulsed from the secretory vesicles either prior, or after host cell invasion. Some of these proteins have been characterised at the molecular level, and it has been shown that they are, with respect to protein sequences, closely related to homologous counterparts in T. gondii. Nevertheless, there is only a low degree of cross-antigenicity between the two species. In microbial interactions it has been shown that carbohydrates could also play a crucial role in host cell recognition and immunological host parasite interactions. In this study we present data which strongly suggest that the surface of N. caninum tachyzoites is glycosylated. In SDS-PAGE, glycoproteins comigrated largely with glycosylphosphatidylinositol-anchored proteins which were identified using in vivo [3H]ethanolamine labelling followed by autoradiography. The lectin Con A reacted strongly with the surface of these parasites, binding of which is indicative for the presence of N-glycans. Additional surface binding was observed, although only in a subpopulation of all tachyzoites, for wheat germ agglutinin and Jacalin. Intracellular binding sites for Con A were mainly associated with the parasite dense granules. By lectin labelling of Western blots of N. caninum protein extracts, glycoproteins were identified which reacted specifically with the lectins Con A, wheat germ agglutinin, Jacalin and soy bean agglutinin.

Identification and characterisation of a dense granule-associated protein in Neospora caninum tachyzoites.

Neospora caninum is an apicomplexan parasite which is morphologically and ultrastructurally very similar to Toxoplasma gondii. In order to identify molecules involved in host cell entry and subsequent modification of the parasitophorous vacuole, a polyclonal antiserum directed against N. caninum tachyzoites was raised in a rabbit. Subcellular fractionation of tachyzoites was performed using the non-ionic detergent Triton-X-114. Membrane fractions were analysed by immunoblotting using the polyclonal antiserum. One of the immunoreactive protein bands had a mol. wt of 33,000 and was subsequently named Nc-p33. Affinity-purified anti-Nc-p33 antibodies were used to characterise this polypeptide using SDS-PAGE, isoelectric focusing, Western blot analysis and immuno-EM. Nc-p33 was found in two isolates of N. caninum (NC-1 and Liverpool), but could not be detected in T. gondii tachyzoites. Immunogold EM revealed that Nc-p33 constituted a dense granule-associated protein, and Western blotting demonstrated that Nc-p33 was most likely identical to the recently described antigen NCDG1. Shortly after invasion, this dense granule protein was targeted to the parasitophorous vacuole membrane, and, at later timepoints after infection, was also found on the parasitophorous vacuolar network. This suggested that Nc-p33 could play a functional role in the modification of the parasitophorous vacuole and its membrane.

Immunization of dogs with a canine herpesvirus vector expressing Neospora caninum surface protein, NcSRS2.

In order to develop a vaccine against Neospora caninum in dogs, we constructed recombinant canine herpesvirus (CHV) expressing N. caninum surface protein, NcSRS2. Indirect immunofluorescence indicated that the antigenic structure of the recombinant NcSRS2 was similar to the authentic parasite protein. The dogs immunised with recombinant virus produced IgG antibody to N. caninum, and their sera recognised the parasite protein on Western blot. The dogs inoculated with recombinant virus showed no clinical symptoms and infectious CHV was not recovered from the dogs, suggesting that recombinant CHV expressing N. caninum proteins may lead to a vaccine against neosporosis in dogs.

Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells.

We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2.