Compensatory Roles Of Rhoptry Neck Protein TgRON4L1 And TgRON4 At The Moving Junction Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasitic phylum of the apicomplexan protozoa that establishes infection in almost all nucleated cells of warm-blooded animals(birds and mammals),causing Toxoplasmosis and worldwide widely distributed and prevalent.In recent years,the incidence of Toxoplasmosis is on the rise with the increase of immunodeficiency patients.T.gondii invasion of host cells requires the formation of a moving junction(MJ)complex at the contact between the parasite and the host cell membrane to help the parasite anchor on the surface of the host cell and complete the invasion processes.The function of rhoptry neck protein(RON)2,4,5 has been reported.However,the interaction modes among these proteins are still unclear.TgRON4 is the key molecule during parasite invasion.The previous studies have shown that knockout of TgRON4 could affect the invasion rate but not fatal.In this study,we identified that TgRON4L1 is a homologous gene of TgRON4 through homology comparison.We would like to explore whether it plays a compensatory role in the invasion of parasite when TgRON4 is absent.Based on the sequence analysis of TgRON4Ll gene in ToxoDB(http://ToxoDB.org),the truncated sequences were amplified.Polyclonal antibodies against TgRON4Ll were prepared in mice inoculated by the recombinant protein.The IFAT results indicated that TgRON4L1 was located in moving junction complex during the parasite invasion.Then,the TgRON4 and its homologues TgRON4L1 and TgRON5 were expressed in eukaryotic cells in vitro.The direct interaction between TgRON4Ll/RON4 and TgRON5 was confirmed by co-immunoprecipitation assay.In order to further explore the function of TgRON4L1,we knocked out of the TgRON4Ll gene in tachyzoite stage of T.gondii by CRISPER/Cas9 technology,and monoclonal strains were screened out.The results reveal that the loss of TgRON4Ll did not affect the ability of the parasite to invade and escape from host cells and replicate in host cells,whereas the expression of TgRON4Ll was significantly increased in TgRON4-deleted strains.Overall,these results suggest that the rhoptry neck protein TgRON4Ll is a member of the moving junction complex.It maybe plays a compensatory role with TgRON4 in MJ during parasite invasion.