The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites
Figure 2
Mapping the interactions between TgRON2 and TgAMA1.
(A) Co-immunoprecipitation of TgAMA1 and TgRON2 after 0.6% SDS lysis and Triton X100 renaturation of tachyzoite extract: anti-RON2-1 allows the recovery of both proteins, while anti-RON2-2 only recovers TgRON2. (B) Transiently transfected BHK-21 cells express TgAMA1 at their surface in its native conformation, with the ectodomain exposed outside (revealed with antibody B3.90, “ -ecto”) and the cytosolic tail only detected after permeabilization (revealed with antibody CL22, “ -tail”). (C) TgAMA1-expressing BHK-21 cells were incubated with 0.1 µg/ml TgRON2-2 (a GST-fusion protein), washed and the binding of recombinant TgRON2 fragment to TgAMA1-expressing cells was revealed with anti-GST antibody. Untransfected BHK-21 cell on the left showed no TgRON2 binding. (D) Far Western blot analysis of TgRON2-2 binding to native TgAMA1. Extracts of tachyzoites expressing wild-type levels of AMA1 (WT) or depleted for this protein (KOiAMA1+Atc) were separated by SDS-PAGE and transferred on a nitrocellulose membrane, which was incubated with GST alone or TgRON2-2 and revealed with anti-GST. Incubation with TgRON2-2 revealed a band corresponding to the size of TgAMA1, as shown by specific antibodies and absent from the AMA1-depleted cell line extracts, while anti-GST antibody did not react with tachyzoites. Anti-SAG1 was used as a loading control. (E) TgRON2-2 binds specifically to surface-exposed TgAMA1. Fixed extracellular conditional KOi AMA1 tachyzoites induced (+Atc) to repress AMA1 expression, or non-induced (no Atc), were incubated with TgRON2-2, which was then revealed with anti-GST antibody. TgAMA1 was revealed with anti-ectodomain B3.90 antibody. (F) TgRON2-2 binds specifically to TgAMA1 in micronemes. Fixed intracellular induced or non-induced conditional KOi AMA1 tachyzoites were permeabilized and incubated with TgRON2-2, which labelled micronemes only when TgAMA1 was expressed. MIC3, an unrelated microneme marker, was used as a control.