Abstract
Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.
Highlights
Protozoan parasites are a significant cause of morbidity and mortality in humans worldwide
We introduced an HA-tag to the C-terminus of TgRON2 in RHDhxgprt parasites by using a vector to homologously recombine a cassette that contains the coding sequence of the HA tag and the hypoxanthinexanthine-guanine phosphoribosyltransferase (HXGPRT) gene flanked by targeting sequences from upstream and downstream of the 39 end of the TgRON2 gene
The results presented here demonstrate that TgRON2 localizes to the moving junction (MJ) of invading parasites and that at least two regions within the last,200 amino acids of TgRON2 independently and associate with the ectodomain of TgAMA1
Summary
Protozoan parasites are a significant cause of morbidity and mortality in humans worldwide. Apicomplexans are related by an anterior complex of specialized secretory organelles that secrete molecules necessary for active host cell invasion and subsequent development of the parasitophorous vacuole (PV) around the penetrating parasite [1]. In contrast to many intracellular pathogens that use conventional host-uptake pathways to enter a target cell, apicomplexans actively invade in a rapid, multi-step process that is dependent on the parasite actinomyosin machinery [2,3]. A distinctive feature of this process is the formation of a close apposition between the parasite and host plasma membranes that is reminiscent of a tight junction in mammalian cells [4,5]. Beginning with its apical end, the parasite moves through this ring-like structure which is referred to as the ‘moving junction’ (MJ) and which functions to generate the PV membrane from the invaginated host plasma membrane [6]. The MJ appears to act as a molecular sieve that somehow excludes certain host membrane proteins from the forming PV membrane [7]
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