Background
Toxoplasma gondii is an opportunistic protozoan infecting almost one-third of the world's population. Toxoplasma gondii rhoptry protein 18 (TgROP18) is a key virulence factor determining the parasite's acute virulence and is secreted into host cells during infection. We previously identified the interaction of TgROP18 and host cell immune-related receptor protein IL20RB, and observed the activation of STAT3 in human keratinocytes (HaCaT) cells infected by the rop16 knockout RH strain, though TgROP16 is regarded as being responsible for host STAT3 activation during T. gondii invasion. Therefore, we hypothesize TgROP18 can activate host STAT3 through binding to IL20RB.
Conclusions
These findings indicate that TgROP18-IL20RB interaction upon T. gondii invasion was involved in STAT3 activation, which is associated with host cell defense.
Methods
CRISPR-CAS9 technology was used to generate the ROP16 and ROP18 double knockout RH strain, RH-∆rop16∆rop18. SDS-PAGE and western blot were used to detect STAT3 activation in different HaCaT cells with high endogenous IL20RB expression treated with T. gondii tachyzoites infection, recombinant ROP18, or IL-20. FRET and co-immunoprecipitation (Co-IP) was used to detect the protein-protein interaction.
Results
We observed that TgROP18 was involved in a synergic activation of the host JAK/STAT3 pathway together with TgROP16 in human HaCaT cells infected with T. gondii or treated with recombinant TgROP18 protein, stimulating host proinflammatory immune responses such as expression of TNF-α. The effect of recombinant ROP18 on STAT3 phosphorylation was presented in a dose-dependent manner. Additionally, TgROP18 was identified to target IL20RB on its extracellular domain. When we treated different cell lines with the recombinant ROP18, STAT3 phosphorylation could only be observed in the cells with endogenous IL20RB expression, such as HaCaT cells. Conclusions: These findings indicate that TgROP18-IL20RB interaction upon T. gondii invasion was involved in STAT3 activation, which is associated with host cell defense.