Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii

Abstract

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually all nucleated cells in warm-blooded animals. The ability of Toxoplasma tachyzoites to infect and successfully manipulate its host is dependent on its ability to transport “GRA” proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside. GRAs have diverse roles in Toxoplasma’s intracellular lifecycle, including co-opting crucial host cell functions and proteins, such as the cell cycle, c-Myc and p38 MAP kinase. Some of these GRA proteins, such as GRA16 and GRA24, are secreted into the parasitophorous vacuole (PV) within which Toxoplasma replicates and are transported across the PV membrane (PVM) into the host cell, but the translocation process and its machinery are not well understood. We previously showed that TgMYR1, which is cleaved by TgASP5 into two fragments, localizes to the PVM and is essential for GRA transport into the host cell. To identify additional proteins necessary for effector transport, we screened Toxoplasma mutants defective in c-Myc up-regulation for their ability to export GRA16 and GRA24 to the host cell nucleus. Here we report that novel proteins MYR2 and MYR3 play a crucial role in translocation of a subset of GRAs into the host cell. MYR2 and MYR3 are secreted into the PV space and co-localize with PV membranes and MYR1. Consistent with their predicted transmembrane domains, all three proteins are membrane-associated, and MYR3, but not MYR2, stably associates with MYR1, whose N- and C-terminal fragments are disulfide-linked. We further show that fusing intrinsically disordered effectors to a structured DHFR domain blocks the transport of other effectors, consistent with a translocon-based model of effector transport. Overall, these results reveal a novel complex at the PVM that is essential for effector translocation into the host cell.

The secreted acid phosphatase domain-containing GRA44 fromToxoplasma gondiiis required for C-myc induction in infected cells

“…MYR1 was initially identified through a forward genetic 345 screen for Toxoplasma mutants unable to activate host cMYC and translocate effectors such 346 as GRA16 and GRA24 ( 22). Further screening for such mutants identified MYR2 and MYR3 347 as also required for effector translocation (27). Thus, MYR1/2/3 appear to be part of a putative translocon complex, although only MYR1 and MYR3 have been confirmed to directly interact 349 (27) these findings support the notion that GRA44 is a member of the MYR1/2/3 translocon 360 machinery.…”

“…Significantly, of the are likely secreted proteins (Table 1). Among these are eight known GRA proteins (GRA9, 16, 274 25, 33, 34, 45, 50, and 52), the parasitophorous vacuole membrane associated protein MAF1, 275 and MYR1 a known member of the effector translocation complex (26,27). 276…”

Toxoplasma gondiibradyzoites induce transcriptional changes to host cells and prevent IFNγ-mediated cell death

“…These include cell cycle regulator GRA16 17 , p38 MAPK activator GRA24 18 , Inhibitor of STAT1 Transcription (IST) 19,20 , GRA28 (effects on host are unknown) 21 , -catenin signalling modulator GRA18 is associated with anti-inflammatory responses 22 and, more recently, HCE1/ TEEGR, which has been shown to affect the cyclin proteins 23 and host immune regulation by the suppression of NF-κB-regulated cytokines 24 . Interestingly, all the exported dense granule proteins are predicted to be disordered, which could be essential to the translocation process 25 .…”

“…All known effector proteins traffic via Toxoplasma's secretory pathway 26 and are translocated across the PVM using parasite-derived machinery 25,27,28 . Upon trafficking through the parasite secretory pathway, some effectors are matured by a resident Golgi Aspartyl Protease, ASP5.…”

“…This has similarities with the export process of proteins in Plasmodium spp. [31][32][33][34] ; however, unlike Plasmodium spp., it appears that not all TEXEL-containing protein are exported 18,27,35 Recent evidence suggests a protein complex, consisting in part of MYR1, MYR2 and MYR3, is required for the translocation of proteins across the PVM 25,28 , but the exact mechanism it yet to be defined. Furthermore, MYR1-dependent transcriptional changes account for a large portion of the changes in host transcription induced by tachyzoite infection, consistently affecting immune response pathways 36 Despite its extensive ability to shut down defence mechanisms, Toxoplasma is tightly controlled by the innate and adaptive immune responses.…”