Identification and function characterization of NcAP2XII-4 in Neospora caninum

Neospora caninum is a protozoan parasite in the Apicomplexa controlled by complex signaling pathways. Transcriptional control, an important way to regulate gene expression, has been almost absent in the N. caninum life process. However, to date, research on the transcriptional regulation of the AP2 family factors in N. caninum has been extremely limited. A prior study demonstrated that removing rhoptry protein 5 (ROP5), a significant virulence factor, resulted in abnormal expression levels of predicted NcAP2XII-4 in N. caninum, suggesting that the factor may regulate the function of ROP5. This study aimed to identify NcAP2XII-4 and its function in transcriptional regulation. The NcAP2XII-4 gene was identified by analyzing the N. caninum genome. A polyclonal antibody against the protein was prepared and purified, and its expression and localization in the parasite were detected using western blot (WB) and immunofluorescence assay (IFA). The ΔNcAP2XII-4 strain was constructed from the Nc1 strain using CRISPR/Cas9 to study its effect on the growth and development of N. caninum, and DAP-Seq and electrophoretic mobility shift assay (EMSA) were used to verify the transcriptional regulatory functions of the gene. Bioinformatic analysis showed that NcAP2XII-4 consists of 11,976 bp and encodes 3991 amino acids, with a predicted molecular mass of 410 kDa. The protein has two AP2 domains, 1207aa-1251aa and 3453aa-3500aa, and is predicted to be located in the nucleus. The results of PCR, WB, and IFA were in accordance with the bioinformatics analysis. ΔNcAP2XII-4 was successfully constructed, but the strain could not be released and ultimately succumbed within parasitophorous vacuoles (PVs). Plaque assays demonstrated that parasites lacking this gene could not form plaques. One motif was successfully identified using DAP-Seq technique. Two prokaryotic expression vectors containing the AP2 domain of NcAP2XII-4 were successfully constructed, and two prokaryotic expression proteins, AP2-D1 and AP2-D2, and ROP5 biotinylated probes were prepared. Using EMSA, NcAP2XII-4 was shown to regulate ROP5 transcription by binding to its promoter. NcAP2XII-4 is an essential gene in N. caninum. This study provides a foundation for further research on transcriptional regulation in N. caninum and identifies a new candidate factor for the development of vaccines against N. caninum.