Apical membrane antigen 1 is a cross-reactive antigen between Neospora caninum and Toxoplasma gondii, and the anti-NcAMA1 antibody inhibits host cell invasion by both parasites

The cross-reactive antigens of Neospora caninum and Toxoplasma gondii are important in the exploration to determine the common mechanisms of parasite-host interaction. In this study, a gene encoding N. caninum apical membrane antigen 1 (NcAMA1) was identified by immunoscreening of a N. caninum tachyzoite cDNA expression library with antisera from mice immunized with recombinant T. gondii apical membrane antigen 1 (TgAMA1). NcAMA1 was encoded by an open reading frame of 1695 bp, which encoded a protein of 564 amino acids. The single-copy NcAMA1 gene was interrupted by seven introns. NcAMA1 showed 73.6% amino acid identity to TgAMA1. Mouse polyclonal antibodies raised against the recombinant NcAMA1 (rNcAMA1) recognized a 69-kDa native parasite protein by Western blotting. Immunofluorescence analysis showed that NcAMA1 was localized to the apical end of tachyzoites. Two-dimensional electrophoresis and Western blotting indicated that an approximately 57-kDa cleavage product was released into the excretory/secretory products of N. caninum. Preincubation of free tachyzoites with anti-rNcAMA1 IgG antibodies inhibited the invasion into host cells by N. caninum and T. gondii. These results indicated that AMA1 is a cross-reactive antigen between N. caninum and T. gondii and a potential common vaccine candidate to control two parasites.     

Identification of ribosomal phosphoprotein P0 of Neospora caninum as a potential common vaccine candidate for the control of both neosporosis and toxoplasmosis

The characterization of the cross-reactive antigens of two closely related apicomplexan parasites, Neospora caninum and Toxoplasma gondii, is important to elucidate the common mechanisms of parasite-host interactions. In this context, a gene encoding N. caninum ribosomal phosphoprotein P0 (NcP0) was identified by immunoscreening of a N. caninum tachyzoite cDNA expression library with antisera from mice immunized with T. gondii tachyzoites. The NcP0 was encoded by a gene with open reading frame of 936 bp, which encoded a protein of 311 amino acids. The NcP0 gene existed as a single copy in the genome and was interrupted by a 432 bp intron. The NcP0 showed 94.5% amino acid identity to T. gondii P0 (TgP0). Anti-recombinant NcP0 (rNcP0) sera recognized a native parasite protein with a molecular mass of 34 kDa in Western blot analysis. Immunofluorescence analysis showed that the NcP0 was localized to the surface of N. caninum tachyzoites. A purified anti-rNcP0 IgG antibody inhibited the growth of N. caninum and T. gondii in vitro in a concentration-dependent manner. These results indicate that P0 is a cross-reactive antigen between N. caninum and T. gondii and a potential common vaccine candidate to control both parasites.     

Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.     

Identification and characterization of Neospora caninum cyclophilin that elicits gamma interferon production

Gamma interferon (IFN-gamma) response is essential to the development of a host protective immunity in response to infections by intracellular parasites. Neosporosis, an infection caused by the intracellular protozoan parasite Neospora caninum, is fatal when there is a complete lack of IFN-gamma in the infected host. However, the mechanism by which IFN-gamma is elicited by the invading parasite is unclear. This study has identified a microbial protein in the N. caninum tachyzoite N. caninum cyclophilin (NcCyP) as a major component of the parasite responsible for the induction of IFN-gamma production by bovine peripheral blood mononuclear cells (PBMC) and antigen-specific CD4(+) T cells. NcCyP has high sequence homology (86%) with Toxoplasma gondii 18-kDa CyP with a calculated molecular mass of 19.4 kDa. NcCyP is a secretory protein with a predicted signal peptide of 17 amino acids. Abundant NcCyP was detected in whole-cell N. caninum tachyzoite lysate antigen (NcAg) and N. caninum tachyzoite culture supernatant. In N. caninum tachyzoite culture supernatant, three NcCyP bands of 19, 22, and 24 kDa were identified. NcAg stimulated high levels of IFN-gamma production by PBMC and CD4(+) T cells. The IFN-gamma-inducing effect of NcAg was blocked by cyclosporine, a specific ligand for CyP, in a dose-dependent manner. Furthermore, cyclosporine abolished IFN-gamma production by PBMC from na?ve cows as well as PBMC and CD4(+) T cells from infected/immunized cows. These results indicate that the N. caninum tachyzoite naturally produces a potent IFN-gamma-inducing protein, NcCyP, which may be important for parasite survival as well as host protection.     

Identification of a major surface protein on Neospora caninum tachyzoites

Neospora caninum is a recently identified coccidian parasite that is closely related to Toxoplasma gondii. Molecules associated with the surface of N. caninum tachyzoites are likely to be involved in the process of adhesion and invasion of host cells. They probably also participate in the interaction of the parasite with the immune system, and they could play an important role in the pathogenesis of the parasite. To identify such surface molecules, we performed subcellular fractionation studies of isolated N. caninum tachyzoites. Employing the nonionic detergent Triton-X-114, we prepared a membrane fraction. Immunoblot analysis of this fraction using polyclonal antisera directed against tachyzoites of N. caninum and T. gondii resulted in the identification of a protein of approximately 43 kDa (Nc-p43). This molecule was present in two isolates of Neospora (Nc-1 and Liverpool) but was absent in Toxoplasma (RH-strain) tachyzoites. Further immunofluorescence and immunogold transmission electron microscopy (TEM) studies using affinity-purified anti-Nc-p43 antibodies demonstrated the presence of this molecule on the surface of N. caninum tachyzoites.     

Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii

Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monoclonal antibodies to screen a cDNA expression library in lambda gt11, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18,959. As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate.     

Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells

Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.     

Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.     

Subcellular localization and functional characterization of Nc-p43, a major Neospora caninum tachyzoite surface protein.

Neospora caninum is a recently identified coccidian parasite which shares many features with, but is clearly distinct from, Toxoplasma gondii. N. caninum tachyzoites infect a wide range of mammalian cells both in vivo and in vitro. The mechanisms by which infection is achieved are largely unknown. Recent evidence has suggested that a receptor-ligand system in which one or several host cell receptors bind to one or several parasite ligands is involved. Parasite cell surface-associated molecules such as the recently identified Nc-p43 antigen are prime suspects for being implicated in this physical interaction. In this study it is shown that invasion of Vero cell monolayers by N. caninum tachyzoites in vitro is impaired on incubation of parasites with subagglutinating amounts of affinity-purified antibodies directed against Nc-p43. Postembedding immunogold labeling with anti-Nc-p43 antibodies demonstrated that Nc-p43 is localized not only on the parasite cell surface but also within dense granules and rhoptries. The fate of Nc-p43 during intracellular proliferation of N. caninum tachyzoites and subsequent maturation of the parasitophorous vacuole was also studied.     

Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta.     

Characterization of TgROP9 (p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified by T cell clone.

T cell clone 3Tx19 detects a Toxoplasma gondii tachyzoite protein which, in high resolution 2D gel electrophoresis, runs at 36 kDa apparent MW with two spots of pI 5.9 and 6.5, thus exhibiting a migration pattern distinct from those of other known Toxoplasma antigens. The sequences of peptide fragments from tryptic digestion of the more prominent protein spot allowed the design of oligonucleotide primers to obtain the coding cDNA sequence. Sequence analysis of cDNA from strain BK revealed a 363 amino acid open reading frame, defined by all nine peptide sequences determined. The deduced protein sequence contains two hydrophobic segments, one near the N-terminus including a predicted signal peptide and a shorter second at the carboxy terminus, but homology to any other known protein is lacking. With synthetic peptides covering the complete primary structure, the epitope for clone 3Tx19 was mapped within the deduced partial sequence, which had remained unconfirmed by tryptic peptides. Antibodies raised against another, putative B cell epitope peptide detected the same two protein spots in 2D gel, indicating that they are antigenically related isoforms. The protein p36 is expressed by T. gondii isolates of all three intraspecies subgroups, but not in the bradyzoite stage. In intracellular tachyzoites, p36 colocalizes with rhoptry proteins and has a distribution pattern disparate from that of dense granule and microneme proteins. Subcellular fractionation indicated that p36 is a soluble constituent of tachyzoites. We suggest that this T cell-stimulatory novel rhoptry protein of T. gondii be named ROP9. It represents a marker of the tachyzoite stage.     

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.     

Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii

A full-length dihydroorotase (DHOase) sequence was cloned from a Toxoplasma gondii tachyzoite cDNA library. The sequence had a calculated molecular mass of 44.2 kDa and a pI of 5.72, and was most similar to type IIa DHOases. A recombinant protein was expressed and purified with a yield of approximately 20 mg L(-1) of cell culture. Polyclonal antibodies raised against purified recombinant protein reacted with a band of the expected molecular mass in tachyzoite extracts. Specific activities of 18.3 micromol/min/mg in the biosynthetic direction and 18.4 micromol/min/mg in the degradative direction, with K(m, carbamyl aspartate) = 323 microM and K(m, dihydroorotate) = 64.3 microM, were measured for purified recombinant protein. Size exclusion chromatography/laser light scattering showed a single, monodisperse peak with a molecular mass of 45.6 kDa, suggesting that the native protein is a monomer.     

Identification and characterization of Neospora caninum tachyzoite antigens useful for diagnosis of neosporosis.

The purpose of the present study was to identify antigens of the protozoan Neospora caninum that could be useful for the diagnosis of neosporosis in domestic animals. As revealed by immunoblotting, immune sera from a wide range of animal species exhibited a similar recognition pattern of four major and several minor N. caninum antigens. In contrast to preinoculation sera, all tested immune sera recognized nonreduced immunodominant 17-, 29-, 30-, and 27-kDa antigens. A 46-kDa protein which showed faint recognition by preimmune sera also exhibited a strong response by immune sera. Immunolocalization of the four immunodominant N. caninum antigens was investigated by immunogold electron microscopy using monospecific polyclonal antisera. The 17-kDa antigen appears to be associated with the body part of the rhoptries, while the 29- and 30-kDa antigens were associated with the dense granules, network, and limiting membrane of the parasitophorous vacuole. Studies were also conducted to compare antibody responses to N. caninum and the related protozoan Toxoplasma gondii. Although N. caninum and T. gondii (RH strain) tachyzoites shared a few cross-reacting antigens, the immunodominant antigens of both parasites were not recognized by heterologous sera. Also, immunogold staining with rabbit anti-Neospora hyperimmune serum exhibited almost no labeling of external membranes of Neospora tachyzoites compared with the very marked labeling seen when Toxoplasma tachyzoites (RH strain) were incubated with rabbit anti-Toxoplasma hyperimmune serum. These unique antigenic differences should be useful in developing a diagnostic assay for N. caninum.     

The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels

A monoclonal antibody (MAb) has been generated against a novel 63 kDa surface/apical antigen of Toxoplasma gondii tachyzoites which is identified here as TgAMA-1, the Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1). Sequence analysis, phase partitioning in Triton X-114, and labeling of TgAMA-1 with iodonaphthalene azide all suggest that TgAMA-1 is a type I transmembrane protein. There is a high degree of sequence similarity between TgAMA-1 and Plasmodium AMA-1, most notably in the position of conserved cysteine residues within the protein's predicted extracellular domain. In contrast to full length Plasmodium AMA-1, which has previously been localized to the rhoptries, it is shown here by immunofluorescence and immunoelectron microscopy that intracellular TgAMA-1 is found in the micronemes. A 53 kDa N-terminal proteolytic fragment of TgAMA-1 is constitutively secreted from the parasite at 37 degrees C. As is the case with other microneme proteins, the proteolytic processing and secretion of TgAMA-1 is dramatically enhanced in response to treatments which increase intracellular calcium levels.     

Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.