Neospora caninum ROP16 play an important role in the pathogenicity by phosphorylating host cell STAT3

Neospora caninum is a common cause of abortions in cattle and nervous system dysfunctions in dogs. Our analysis shows that NcROP16 and TgROP16 have similar structures and may have similar functions. To our surprise, we found that similar to the T. gondii RH strain, the N. caninum Nc-1 strain could phosphorylate STAT3^Y705, but in contrast to T. gondii, N. caninum Nc-1 could not phosphorylate STAT6^Y641. We constructed a gene-knockout plasmid and screened ΔNcROP16 strains at the gene, protein and transcription levels. Plaque assays, invasion assays and intracellular proliferation tests indicated that the ΔNcROP16 strain phenotypes had changed, resulting in smaller plaques and slower intracellular growth. A virulence analysis showed that the cerebral loads of the parasite in mice infected with the ΔNcROP16 strain were significantly reduced compared to the loads in mice infected with the Nc-1 strain. In contrast, the overexpression of ROP16 led to the largest number of parasites observed in the mouse brains. Similarly, the overexpression of ROP16 caused the most powerful virulence in mice. In addition, NcROP16 takes part in the STAT3 signaling pathway in different host cells. This occurs by the secretion of NcROP16 into the host cell, where it phosphorylates STAT3, and phosphorylated STAT3 then migrates to the cell nucleus. NcROP16 can enter the host nucleus and continuously phosphorylate STAT3, resulting in the induction of host cell apoptosis. The parasites engineered to over express the NcROP16 induce the increased transcription of apoptotic-related genes, such as Fas, FasL and Bax and enhanced ANA1 cell apoptosis. The results show that NcROP16 is a key virulence factor in N. caninum, promoting the host cell apoptosis and enhancing the pathogenicity of the parasites for the host by phosphorylating STAT3.