Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1

Abstract

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.

Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA–RON2 pair

“…and spatial expression patterns appear to be inconsistent with formation of a functional complex (23,24), and thus the RhopH1/ Clag proteins were not included in the final figure. The RON2 ancestor underwent a duplication in the Eimeriorina into RON2 and RON2 L2 , consistent with both AMA1 and AMA2 binding RON2 and only AMA3 binding RON2 L2 (14,15). Interestingly, the putative partner of AMA4, RON2 L1 , was only recovered from lineages with AMA4 and underwent an Eimeria-specific duplication ( Fig.…”

“…One clade is subdivided into AMA1 of apicomplexan hemoparasites (Plasmodium, Babesia, and Theileria) and of Eimeriorina (Toxoplasma, Eimeria, and Neospora), with the latter having two duplications of the AMA1 ancestor resulting in AMA1, AMA2, and AMA3 paralogs (Fig. 1, Left) (14,15). In contrast, the second AMA clade consists of AMA4 from Toxoplasma and its orthologs in Eimeria and Neospora.…”

“…Intriguingly, recent studies have shown that the AMA1-RON2 complex is an attractive target for therapeutic intervention (9)(10)(11)(12). The importance of the AMA1-RON2 pairing is also reflected in the observation that many apicomplexan parasites encode functional paralogs that are generally expressed in a stage-specific manner (13)(14)(15). We recently showed that, in addition to AMA1 and RON2, T. gondii harbors three additional AMA paralogs and two additional RON2 paralogs (14,15): TgAMA2 forms a functional invasion complex with TgRON2 (15), TgAMA3 (also annotated as SporoAMA1) selectively coordinates TgRON2 L2 (14), and TgAMA4 binds TgRON2 L1 (15).…”

“…The importance of the AMA1-RON2 pairing is also reflected in the observation that many apicomplexan parasites encode functional paralogs that are generally expressed in a stage-specific manner (13)(14)(15). We recently showed that, in addition to AMA1 and RON2, T. gondii harbors three additional AMA paralogs and two additional RON2 paralogs (14,15): TgAMA2 forms a functional invasion complex with TgRON2 (15), TgAMA3 (also annotated as SporoAMA1) selectively coordinates TgRON2 L2 (14), and TgAMA4 binds TgRON2 L1 (15). Despite substantial sequence divergence, structural characterization of all AMA-RON2D3 complexes solved to date [TgAMA1-TgRON2D3 (16), PfAMA1-PfRON2D3 (17), and TgAMA3-TgRON2 L2 D3 (14)] reveal a largely conserved architecture and binding paradigm.…”

“…We recently showed that, in addition to AMA1 and RON2, T. gondii harbors three additional AMA paralogs and two additional RON2 paralogs (14,15): TgAMA2 forms a functional invasion complex with TgRON2 (15), TgAMA3 (also annotated as SporoAMA1) selectively coordinates TgRON2 L2 (14), and TgAMA4 binds TgRON2 L1 (15). Despite substantial sequence divergence, structural characterization of all AMA-RON2D3 complexes solved to date [TgAMA1-TgRON2D3 (16), PfAMA1-PfRON2D3 (17), and TgAMA3-TgRON2 L2 D3 (14)] reveal a largely conserved architecture and binding paradigm. Intriguingly, however, sequence analysis indicates that TgAMA4 and TgRON2 L1 are likely to adopt substantially divergent structures with an atypical assembly mechanism.…”