The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii

Abstract

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii and has been found to be associated with neurological disorders in dogs and congenital infections and abortions in cattle. We have identified two surface proteins of 29 and 35 kDa (designated Ncp29 and Ncp35, respectively) from N. caninum tachyzoites that are the predominant antigens recognized by antisera from Neospora-infected animals. Monoclonal antibodies against Ncp29 and Ncp35 were used to analyze several independent and diverse N. caninum isolates; both antigens were recognized in all isolates, suggesting that they are well conserved. Localization studies and surface labeling with biotin demonstrated that Ncp29 and Ncp35 are membrane associated and displayed on the surface of the parasite. After treatment with phosphatidylinositol-specific phospholipase C, parasite lysates were analyzed with antibodies against the cross-reacting determinant of glycosylphosphatidylinositol anchors. Approximately six glycolipid-anchored surface proteins were identified, with the two most prominent corresponding to Ncp29 and Ncp35. Sequence comparisons of Ncp29 and Ncp35 with GenBank indicated that they are most similar to the T. gondii surface antigen 1 (SAG1) and surface antigen 1-related sequence 2 (SRS2), respectively. Consequently, Ncp29 has been designated NcSAG1 and Ncp35 has been designated NcSRS2. Both NcSAG1 and NcSRS2 contain a tandemly duplicated motif and 12 absolutely conserved cysteines which are also found in all of the SAG and SRS proteins of T. gondii. Maintenance of these motifs and the 12 cysteine residues suggests that these surface antigens share a similar secondary and tertiary structure that is presumably important for a conserved function that these antigens serve during infection.

FIG. 1

The Ncp29 and Ncp35 proteins were recognized as immunodominant antigens by antisera from a wide range of infected hosts. Sera from three experimentally infected mice, three experimentally infected dogs, or three naturally infected cows were tested by Western blotting against the total parasite antigen of the Nc-1 strain of N. caninum. The two most immunogenic antigens comigrated with the Ncp29 antigen, recognized by MAb 6C11 (α-p29), and the Ncp35 antigen, recognized by MAb 5H5 (α-p35). Sera from noninfected animals did not react with parasite antigen.

FIG. 2

The Ncp29 and Ncp35 surface antigens were recognized in different strains of N. caninum. The anti-Ncp29 (α-p29) MAb 6C11 (A) and the MAb 5H5 (α-p35) (B) reacted by Western blotting to parasite lysates of three independent N. caninum isolates from dogs (Nc-1, Nc-2, and Nc-LIV) and two independent N. caninum isolates from cattle (BPA-1 and BPA-3). These MAbs did not react to parasite lysates of T. gondii or S. neurona.

FIG. 3

Triton X-114 partitioning assays indicated that Ncp29 and Ncp35 are membrane associated. Parasite lysates were separated with Triton X-114 into an aqueous phase (lanes A) that contains soluble proteins and a detergent phase (lanes D) that contains membrane-associated proteins. Western blot analysis with MAb 6C11 (α-p29) indicated that Ncp29 partitioned exclusively with the detergent phase. Western blot analysis with MAb 5H5 (α-p35) indicated that Ncp35 partitioned primarily with the detergent phase, although a minor portion was found in the aqueous phase.

FIG. 4

Immuno-EM localization of Ncp29 and Ncp35 in extracellular tachyzoites of N. caninum. Both Ncp29 (MAb 6C11 (A) and Ncp35 (MAb 5H5) (B) were localized to the parasite cell membrane but were not found in micronemes, dense granules, or rhoptries. Occasional gold particles were found in the cytoplasm within small vesicles. No labeling of the parasites was observed with MAb DG52 against T. gondii SAG1 (C). Bars, approximately 0.2 μm.

FIG. 5

Approximately six dominant surface proteins, including Ncp29 and Ncp35, were revealed by surface biotinylation of N. caninum tachyzoites and Western blot detection with avidin-HRP conjugate. (A) Ncp29 (6C11) and Ncp35 (5H5) that were immunoprecipitated (IP) from labeled parasites were biotinylated, as determined by Western blotting with avidin-HRP. (B) Similarly, MAb 6C11 (α-p29) detected Ncp29 and MAb 5H5 (α-p35) detected Ncp35 in the biotinylated protein fraction precipitated with streptavidin-agarose (StAv) but not in the protein fraction from nonbiotinylated parasites, thus indicating that the streptavidin precipitation was specific for biotin-labeled proteins. Avidin-HRP detection was specific for biotinylated proteins, as revealed by Western blot analysis of nonbiotinylated parasites.

FIG. 6

Ncp29 and Ncp35 were detected by anti-CRD (α-CRD) after phosphatidylinositol-specific phospholipase (PI-PLC) treatment, indicating they contain a glycolipid anchor. Nc-1 strain N. caninum tachyzoites (N.c.) or RH strain T. gondii tachyzoites (T.g.) were lysed, treated with PI-PLC, and analyzed by Western blotting with antibodies against the CRD of GPI anchors. Approximately six GPI-anchored proteins were revealed in PI-PLC-treated N. caninum, two of which correspond to Ncp29 and Ncp35 (arrows). Anti-CRD antibodies did not react with parasites that had not been PI-PLC treated.

FIG. 7

Amino acid sequence alignment of Ncp35 with the SRS2 protein from T. gondii, showing the duplicated motif (shaded) and the 12 conserved cysteine residues (boxed). The putative signal peptide is underlined.