Identification Of Neospora Caninum MIC6and Preliminary Study Of Its Funtional Mechanism

Neospora caninum is an obligate intracellular parasite which has a world-wide distribution and causes neosporosis in many species. During the cell invasion by N. caninum, secretory organelles secrete a large number of proteins when initial contact is made between the parasite and the host cell. Microneme proteins (MICs) act as major cellular adhesion factors for host cells and participate in parasite recognition and adhesion. Although numerous reports have described the MICs (TgMICs) in phylum Apicomplexa, less information is available for N. caninum MICs. In the present study, we identified N. caninum microneme protein6(NcMIC6) and investigated its biological characteristics, function, protein-protein interactions (PPI) and action mechanism of PPI.Bioinformatics analysis showed that nucleotide sequence of NcMIC6gene is984bp, which had69%nucleotide similarity with TgMIC6. It encoded a precursor protein of327amino acids (aa).The overall amino acid sequence similarity was high, with47%identity shared between NcMIC6and TgMIC6. Using SignalP4.1, we predicted that the N-terminal portion of NcMIC6contained a signal peptide. TMHMM showed that there was only putative transmembrane domain in the NcMlC6Using SMART, we identified three EGF domains and two low complexity regions in the NcMIC6.To obtain proteins for antibody generation, we constructed prokaryotic expression vector pET28a-NcMIC1, pET28a-NcMIC4and pET28a-NcMIC6and then transformed into Escherichia coli for rNcM1C1, rNcMIC4and rNcMIC6expression and purification. Mouse polyclonal antibodies against rNcMIC1, rNcMIC4and rNcMIC6were prepared. Immunofluorescence analysis showed that NcMIC6had a polar labeling pattern, which was consistent with localization of micronemes in the apical region. Pulse invasion assays showed that NcMIC6translocated from the apical tip to the posterior end of the parasites. Host cell invasion assay demonstrated that anti-rNcMIC6could effectively inhabit N. caninum invasion. Secrtion assay showed that NcMIC6can secret into the supernatants and its secretion can be induced with different temperatures and reagents.To confirm the hypothesis that NcMIC1, NcMIC4and NcMIC6could interact physically in N. caninum, we performed the coimmunoprecipitation. The results indicated that NcMIC1, NcMIC4and NcMIC6form a complex in N. caninum tachyzoites. GST-pull down showed that NcMIC1interact with NcMIC4and NcMIC6respectively. In addition, GST-pull down demonstrated that the C-terminal cytoplasmic domain of NcMIC6interacted with aldolase. The key acid amino (from tryptophan to alanine) mutation of NcMIC6C-terminal could decrease the ability of NcMIC6and NcALD interaction. Immunofluorescence analysis showed that NcMIC6was found predominately at the apical tip of the intracellular tachyzoites and co-localized with NcALD.To obtain the proteins for immunization, we constructed a chimeric recombinant antigen NcMICl-4-6. After immunization of rNcMIC6and rNcMIC1-4-6, the immune response was evaluated using antibodies detection, cytokine assays and cerebral parasite load. The groups immunized with rNcMIC6or rNcMIC1-4-6developed a high level of IgG and significantly elevated IL-4production compared with the control groups. Moreover, rNcMIC6and rNcMIC1-4-6significantly lowered the cerebral parasite load. These results suggest that rNcMIC6and rNcMIC1-4-6are potential vaccine candidates against neosporosis.