Toxoplasma gondii is an obligate intracellular parasite that establishes a favorable environment in the host cells in which it replicates. We have previously reported that it uses MYR-dependent translocation of dense granule proteins to elicit a key set of host responses related to the cell cycle, specifically, E2F transcription factor targets, including cyclin E. We report here the identification of a novel Toxoplasma effector protein that is exported from the parasitophorous vacuole in a MYR1-dependent manner and localizes to the host’s nucleus. Parasites lacking this inducer of host cyclin E (HCE1) are unable to modulate E2F transcription factor target genes and exhibit a substantial growth defect. Immunoprecipitation of HCE1 from infected host cells showed that HCE1 efficiently binds elements of the cyclin E regulatory complex, namely, DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, or in uninfected human foreskin fibroblasts (HFFs), showed localization of the expressed protein to the host nuclei and strong cyclin E upregulation. Thus, HCE1 is a novel effector protein that is necessary and sufficient to impact the E2F axis of transcription, resulting in co-opting of host functions to the advantage of Toxoplasma. IMPORTANCE Like most Apicomplexan parasites, Toxoplasma gondii has the remarkable ability to invade and establish a replicative niche within another eukaryotic cell, in this case, any of a large number of cell types in almost any warm-blooded animals. Part of the process of establishing this niche is the export of effector proteins to co-opt host cell functions in favor of the parasite. Here we identify a novel effector protein, HCE1, that the parasites export into the nucleus of human cells, where it modulates the expression of multiple genes, including the gene encoding cyclin E, one of the most crucial proteins involved in controlling when and whether a human cell divides. We show that HCE1 works through binding to specific transcription factors, namely, E2F3, E2F4, and DP1, that normally carefully regulate these all-important pathways. This represents a new way in which these consummately efficient infectious agents co-opt the human cells that they so efficiently grow within.

Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the singlecelled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3 ′ ′ ′ ′ ′ -UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.

No abstract

Toxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation.

Parasites interact intimately with their hosts, and the interactions shape both parties. The common human parasite Toxoplasma gondii replicates exclusively in a vacuole in a host cell and alters its host cell’s environment through secreted proteins. One of these secreted proteins, MAF1b, acts to concentrate mitochondria around the parasite’s vacuole, and this relocalization alters the host immune response. Many other intracellular pathogens also recruit host mitochondria, but the identities of the partners that mediate this interaction have not previously been described in any infection. Here, we show that Toxoplasma MAF1b binds to the multifunctional MIB protein complex on the host mitochondria. Reducing the levels of the proteins in this mitochondrial complex reduces the close association of host cell mitochondria and the parasite’s vacuole. This work provides new insight into a key host-pathogen interaction and identifies possible targets for future therapeutic intervention as well as a more molecular understanding of important biology.

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelopes its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three novel parasite proteins (TGGT1_269950, TGGT1_215360, and TGGT1_217530) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface.

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...