Abstract
Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Motility and invasion also require thrombospondin-related anonymous protein (TRAP) adhesins, which are secreted apically and translocated to the posterior end of the parasite before they are shed by the activity of a rhomboid protease. TRAP orthologs, including Toxoplasma gondii MIC2 (microneme protein 2), possess a short cytoplasmic tail, which is essential for motility. Previous studies have shown that aldolase forms a critical bridge between actin filaments and the cytoplasmic domains of MIC2 and TRAP. The cytoplasmic tails of TRAP family members harbor a conserved penultimate tryptophan, which is essential for aldolase binding, and clustered acidic residues. Herein, we determined the role of the conserved acidic residues by using alanine point mutants to investigate aldolase binding in vitro and to test functionality in the parasite. Our studies revealed two separate acidic residue clusters in the cytoplasmic domain of MIC2 that are essential for parasite survival. One region, located at the extreme C terminus, is required for the direct interaction with aldolase, whereas the second upstream acidic region is not necessary for aldolase binding but is nonetheless essential to parasite survival. Both acidic domains are conserved throughout TRAP orthologs, implicating a central role for these motifs in apicomplexan motility.
Highlights
Toxoplasma gondii is a member of the phylum Apicomplexa, which is unified by a complex cytoskeleton; the presence of an apical conoid structure in most members; and three sets of apical secretory organelles: the micronemes, rhoptries, and dense granules [1,2,3]
T. gondii MIC2 and other members of the thrombospondin-related anonymous protein (TRAP) family containing extracellular adhesive domains are anchored in the plasma membrane by a single transmembrane domain and contain short cytoplasmic tails
The short cytoplasmic tails of T. gondii MIC2 and P. falciparum TRAP mediate a specific interaction with the glycolytic enzyme aldolase [23]
Summary
Toxoplasma gondii is a member of the phylum Apicomplexa, which is unified by a complex cytoskeleton; the presence of an apical conoid structure in most members; and three sets of apical secretory organelles: the micronemes, rhoptries, and dense granules [1,2,3]. Productive invasion of the host cell requires a cleavage event, severing the microneme extracellular adhesive component from the parasite surface by an intramembrane rhomboid protease [13, 14]. MIC2 (microneme protein 2) and other TRAP family members are type I transmembrane proteins, the extracellular domains of which contain two motifs: an A-domain and one or more type 1 thrombospondin repeats [22]. Both proteins possess a short C-terminal cytoplasmic domain or tail. Deletion of the TRAP cytoplasmic tail (TRAPt) in Plasmodium berghei sporozoites results in non-motile parasites, despite the presentation of the ectodomain on the parasite surface [24]. The experimental results strongly suggest that the cytoplasmic tail has a conserved function in motility
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