Identification and characterization of a microneme protein (NcMIC6) in Neospora caninum

Neospora caninum, an Apicomplexa parasite, is the causative agent of neosporosis. As described for other members of Apicomplexa, microneme proteins (MICs) play a key role in attachment and invasion of host cells by N. caninum. Herein we identified N. caninum microneme protein 6 (NcMIC6) that is orthologous to Toxoplasma gondii microneme protein 6 (TgMIC6). The open reading frame of the NcMIC6 gene is 984 bp and encodes a 327 amino acid peptide. Sequence analysis showed that NcMIC6 included a signal peptide, a transmembrane region, three epidermal growth factor-like (EGF) domains, and two low complexity regions. Antibodies raised against recombinant NcMIC6 recognized an approximately 35-kDa native MIC6 protein in Western blots of N. caninum tachyzoites. Immunofluorescence analysis showed that NcMIC6 had a polar labeling pattern, which was consistent with localization of micronemes in the apical region. Pulse invasion assays showed that NcMIC6 translocated from the apical tip to the posterior end of the parasites. Secretion assays demonstrated that NcMIC6 was released into the supernatants. Importantly, it was clearly revealed by co-immunoprecipitation that NcMIC6 formed a complex with other two soluble microneme proteins (NcMIC1 and NcMIC4). In conclusion, identification and characterization of the novel microneme protein NcMIC6 may contribute to understanding how this protein functions during the parasite motility and host cell invasion.