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.
Highlights
Host cell invasion by Apicomplexan parasites, including Toxoplasma gondii, has generally focused on the asexual stages
A single Rhoptry Neck protein 2 (RON2) homologue is present in the representative Plasmodium, Babesia and Theileria species examined and the clade that includes these latter RON2 sequences is distinct from both the generic RON2 and sporoRON2 clades seen with the three Eimeriorina (Toxoplasma, Eimeria and Neospora)
This suggests that the duplication that led to the two RON2 clades in the Eimeriorina occurred after its split from the Haemospororina
Summary
Host cell invasion by Apicomplexan parasites, including Toxoplasma gondii, has generally focused on the asexual stages. Invasion begins with a tight attachment, reorientation (or high-affinity apical attachment) and the onset of gliding motility to help the parasite propel its way into the host cell. This latter step involves the formation of an intimate ring of attachment between the plasma membranes of the host cell and parasite [10,11] that migrates down the length of the parasite as it invades. The intimate, high-affinity interaction of domain 3 of RON2 and the ectodomain of AMA1 is crucial for efficient invasion [13,18] and structural analyses of the association for both Toxoplasma and Plasmodium asexual stages has shown an extensive, buried region of interaction between the two proteins [13,20]
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