The Molecular Mechanism Of Rhoptry Protein18in Virulence Difference Between Toxoplasma Gondii And Neospora Caninum

Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) are both obligate intracellular protozoan parasites of the phylum Apicomplexa, and they share many common morphological and biological features. Despite these similarities the two parasites differ dramatically in virulence, but the factors involved in virulence differences between the two parasites remain unknown. A secreted serine-threonine kinase called rhoptry protein18(TgROPI8) was identified to play a crucial role on virulence differences among different T. gondii clonal lineages. Intriguingly, we found that ROP18in Nc1strain of N. caninum (NcROP18) is a pseudogene due to several interrupting stop codons in the sequence. Thus, we assume that the difference of ROP18leads to virulence differences between T. gondii and N. caninum.In order to test this hypothesis, we firstly constructed the transfer vector pDMG-TgROP18-GFP carrying the pyrimethamine-resistant DHFR-TS gene, the TgROP18gene, and the reporter GFP gene. The tachyzoites of Ncl were transfected with pDMG-TgROP18-GFP by electroporation, and a clone of N. caninum stably expressing TgROP18was isolated by flow cytometry after10generations of selection by pyrimethamine. The results of PCR, qRT-PCR, and western blot demonstrated that the transgenic parasite was successfully constructed.Plaque assay indicated that at least one of the steps in the lytic cycles of the transgenic parasite changed compared to the Ncl wild-type strain and the expression of TgROP18in Nc1strain was most likely responsible for the change. The results of Transwell assay, Gliding motility assay, and cell invasion assay showed that the motility and invasion ability of the transgenic parasite did not change, but the result of intracellular replication assay showed that TgROP18expression in N. caninum led to a significant increase in parasite proliferation compared to the Nc1wild-type strain.Since the results above demonstrated that expression of TgROP18in N. caninum led to increased intracellular parasite proliferation, the mouse infection assay was then performed to assess the contribution of TgROP18to virulence of the transgenic parasite. The data showed that transfection of Ncl with TgROP18enhanced dramatically the virulence of Nc1compared to the parental Nc1wide-type. IRG phosphorylation assay showed the transgenic parasite can inactivate the IRGs due to the transgenic expression of TgROP18.Collectively, here is the first study to investigate the factors involved in virulence differences between T. gondii and N. caninum, and our findings identified ROP18as a key factor responsible for virulence difference between the two parasites, which laid the foundations for the further study of pathogenicity difference between T. gondii and N. caninum.