Co-immunoprecipitation with MYR1 identifies three additional proteins within theToxoplasmaparasitophorous vacuole required for translocation of dense granule effectors into host cells

Abstract

24Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively 25 modifies infected host cells through secreted effector proteins. Many such effectors 26 must be translocated across the parasitophorous vacuole (PV) in which the parasites 27 replicate, ultimately ending up in the host cytosol or nucleus. This translocation has 28 previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, 29 ROP17, and ASP5. We report here the identification of several MYR1-interacting and 30 novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 31 (dubbed MYR4), TGGT1_204340 (dubbed GRA54) and TGGT1_270320 (PPM3C). 32Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and 33 MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16, 34and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and 35 GRA45 contain ASP5-processing motifs, but like MYR1, processing at these sites 36 appears to be nonessential for their role in protein translocation. These results expand 37 our understanding of the mechanism of effector translocation in Toxoplasma and 38 indicate that the process is highly complex and dependent on at least eight discrete 39 proteins. 40 41 3 Importance 42 Toxoplasma is an extremely successful intracellular parasite and important 43 human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative 44 vacuole and translocates parasite effectors across this vacuole to function from the host 45 cytosol and nucleus. These effectors play a key role in parasite virulence. The work 46reported here newly identifies three parasite proteins that are necessary for protein 47 translocation into the host cell. These results significantly increase our knowledge of the 48 molecular players involved in protein translocation in Toxoplasma-infected cells, and 49 provide additional potential drug targets. 50 either rhoptry (ROP) or dense granule (GRA) proteins, which it introduces into the host 62 during or following invasion (2). In recent years, several Toxoplasma GRAs, including 63 GRA16, GRA24, IST, HCE1/TEEGR, GRA28, and GRA18, have been identified that are 64 translocated across the PVM into the host cell cytosol and/or nucleus, where they can 65 have profound effects on host processes (3-9). The machinery that is responsible for 66 the translocation of these effectors across the Toxoplasma PVM is incompletely 67 defined. A recent forward genetic screen identified several parasite proteins essential 68 for GRA protein translocation, including MYR1, MYR2, MYR3, (named for their effect on 69 host c-Myc regulation) and the rhoptry-derived protein kinase, ROP17 (10-12). 70Precisely how these proteins function to promote protein translocation across the PVM 71 is poorly understood. Of the four, the only protein with a known biochemical function is 72 5 ROP17, a serine/threonine protein kinase that phosphorylates host, and perhaps 73 parasite proteins at the PVM (6,13, 14). 74In addi...

Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular ParasiteToxoplasma gondii

“…Many GRAs remain in the PV lumen or PV membrane (PVM) (13–17), but others traverse the PVM to reach the host cytosol and often proceed to the host nucleus (18–24). The translocation of this latter class of GRAs across the PVM is dependent on a group of PVM proteins called the MYR complex, i.e., MYR1, MYR2, and MYR3, which are so-named because they are required for parasite-dependent host c- My c regulation (25–27). Host signaling pathways modulated by the PVM-embedded, MYR-independent GRAs (MIGs) include the nuclear factor kappa light chain enhancer of activated B cells (NF-kB) pathway.…”