Nonreplicating <i>Toxoplasma</i> uracil auxotrophs (NRTUAs) do not replicate in living host animals.

<p>Left panel: <i>Toxoplasma</i> NRTUAs invade host cells in vitro and replicate normally if the nutrient uracil is added to the culture medium. Center panel: <i>Toxoplasma</i> NRTUAs invade host cells in vitro but do not replicate in the absence of uracil supplementation. Right panel: Mammalian hosts have extremely low uracil concentrations because they do not express the uracil phosphoribosyltransferase enzyme, and therefore, pyrimidine salvage instead occurs through nucleoside kinases that salvage the nucleoside uridine into uridine 5′-monophosphate (UMP) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006523#ppat.1006523.ref017" target="_blank">17</a>]. In living hosts, <i>Toxoplasma</i> NRTUAs invade host cells but do not replicate because there is insufficient uracil to support replication.</p

Genotype of parasite strains developed in this study.

<p>Genotype of parasite strains developed in this study.</p

Secretion and active invasion is required for the antitumor response.

<p>ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS, or were vaccinated i.p. with 4BPB treated, mycalolide B treated, or untreated tachyzoites of uracil auxotrophs (OMP) using the standard three-dose treatment schedule. Data is representative of three independent experiments. ns was not significant, ****P<0.0001.</p

Secreted rhoptry proteins ROP35 and ROP38 and dense granule proteins GRA2, GRA12, and GRA24 are required for the antitumor response.

<p>(A) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS or were vaccinated i.p. with tachyzoites of uracil auxotrophs using the three-dose treatment schedule. PBS treated or vaccinated i.p. with OMP or uracil auxotrophs lacking rhoptry proteins ROP21, ROP35, or ROP38, or vaccinated with a ROP35 complemented strain. (B) Validation of apical rhoptry localization of expressed C-terminal HA-tagged ROP35 in the complemented OMPΔ<i>rop35</i>::<i>ROP35</i> strain. DAPI stains the nuclei of both parasites and the host HFF cells they invaded. Localization of the HA tag (revealed by green fluorescence) is associated with the apical rhoptry organelles. Vacuole locations in the host cell are shown by differential interference contrast (DIC) microscopy. (C) PVM localization of ROP35. Parasites were allowed to invaded HFF cells for 14 h and after fixation the cytosolic surface of the PVM was exposed using 0.002% digitonin to expose the cytosolic surface of the PVM and ROP35 was localized. Vacuole locations (PVM) in the host cell are shown by differential interference contrast (DIC) microscopy (red arrowheads). The ROP35 HA tag is associated with the PVM. (D) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS or were vaccinated i.p. with tachyzoites of uracil auxotrophs using the three-dose treatment schedule. PBS treated or vaccinated i.p. with OMP or uracil auxotrophs lacking dense granule proteins GRA2, GRA12, or GRA24. Data is representative of two independent experiments. ns was not significant, **p<0.01, ***P<0.001, ****P<0.0001.</p

CD8⁺ and CD4⁺ T cells are required for the antitumor response.

<p>(A) ID8DV ovarian tumors were established in wild-type or <i>CD8</i>^<i>-/-</i> C57BL/6 mice and groups of mice were treated with PBS or were vaccinated i.p. with tachyzoites of uracil auxotrophs (OMP) using the three-dose treatment schedule. (B-C) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS or were vaccinated i.p. with tachyzoites of uracil auxotrophs (OMP) on day 8 and day 20. Groups of mice were treated with isotype control antibody, or (B) αCD8 antibody, or (C) αCD4 antibody on days 7, 8, 11, 19, 20, 23 after tumor challenge. Data is representative of two independent experiments. ns was not significant, **p<0.01, ***P<0.001, ****p<0.0001.</p

Parasite extracts and heat killed uracil auxotrophs fail to stimulate antitumor responses or IFN-γ production in the tumor microenvironment.

<p>(A) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS, vaccinated i.p. with tachyzoites of uracil auxotrophs (OMP or CPS) or vaccinated i.p. with heat killed (HK) tachyzoites of uracil auxotrophs (OMP or CPS) using the three-dose treatment schedule. (B) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS, vaccinated i.p. with tachyzoites of uracil auxotrophs (CPS) or vaccinated i.p. with excreted/secreted protein (ESA) extracts, tachyzoite lysate antigen (TLA) extracts, or soluble tachyzoite antigen (STAg) extracts using the three-dose treatment schedule. (C-E) ID8DV tumors were established in groups of C57BL/6 mice for 25 days then tumor-bearing mice were treated with PBS, vaccinated with heat killed (HK) uracil auxotrophs or vaccinated with live uracil auxotrophs (experiments used the yellow fluorescent protein expressing CPS strain CPS-YFP [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006189#pgen.1006189.ref096" target="_blank">96</a>]) and peritoneal levels of (C) IL-12p40, (D) IL-12p70, (E) IFN-γ, and (F) the absolute number or percentage of YFP⁺CD11c⁺ cells present in the tumor microenvironment were measured 18 and 66 h after treatments. Data is representative of two independent experiments. ns was not significant, *p<0.05, **p<0.01, ****P<0.0001.</p

Rhoptry proteins ROP5, ROP17, and ROP18 are required for the antitumor response.

<p>(A) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS, or were vaccinated i.p. with tachyzoites of uracil auxotrophs (OMP), or were vaccinated i.p. with uracil auxotrophs (OMP) lacking rhoptry proteins ROP5, ROP17, or ROP18 using the three-dose treatment schedule. (B) Mouse embryonic fibroblasts were stimulated with IFN-γ and parasite survival (measured as PFU) was determined for uracil auxotrophs mutants lacking specific rhoptry or dense granule proteins. (C) Relative parasite invasion efficiency of uracil auxotrophs was measured in MEFs. The parasite to PFU ratios (invasion efficiency) was measured in at least 4 independent assays and compared to the invasion efficiency of the parental OMP strain. (D) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS, or were vaccinated i.p. with tachyzoites of type I uracil auxotrophs (OMP), or were vaccinated i.p. with type II OMP, or were vaccinated i.p. with type II strains lacking ROP5 or ROP18 using the three-dose treatment schedule. Data is representative of at least two independent experiments. ns was not significant, *p<0.05, **p<0.01, ****P<0.0001.</p

Immunity to <i>T</i>. <i>gondii</i> does not diminish the potency of the antitumor response stimulated by uracil auxotrophs.

<p>(A) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with phosphate buffered saline (PBS) or mice were vaccinated i.p. with tachyzoites (the acute replicative form of <i>T</i>. <i>gondii</i>) of uracil auxotrophs (OMP [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006189#pgen.1006189.ref013" target="_blank">13</a>] or CPS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006189#pgen.1006189.ref005" target="_blank">5</a>]) at 8, 20, and 32 d after tumor challenge (the three-dose treatment schedule). (B) ID8DV ovarian tumors were established in C57BL/6 mice and groups of mice were treated with PBS or were vaccinated once (8 d), twice (8, 20 d), three times (8, 20, 32 d), or five times (8, 20, 32, 44, 56 d) i.p. with tachyzoites of uracil auxotrophs. (C) Groups of C57BL/6 mice were vaccinated (VAC) with uracil auxotrophs to establish protective immunity or mice were treated with PBS (naive). Twelve months later ID8DV tumors were established in vaccinated or age matched naive mice and tumors were treated with PBS or were vaccinated with uracil auxotrophs using the three-dose treatment schedule. Data is representative of at least two independent experiments. ns was not significant, *p<0.05, **p<0.01, ***P<0.001, ****p<0.0001.</p

Targeted deletion of TgENO1 reduces cyst burden in the brain of chronically infected mice.

<p>A) The total number of cysts per brain of mice infected with 5×10² tachyzoites from the Pru<i>Δku80ΔTgeno1</i> mutant or parental Pru<i>Δku80</i> was counted after staining with FITC-labeled <i>dolichol biflorus</i> lectin. A group of nine mice was used for each experiment, and the experiment was repeated twice with similar results (n = 2, P<0.001). Cyst burden (total number of cysts per brain) of the Pru<i>Δku80ΔTgeno1</i> mutant was significantly lower than that of the parental Pru<i>Δku80</i> strain. B) Western blots of total SDS-extracted proteins from knockout Pru<i>Δku80ΔTgeno1</i> mutants (lane 1) and parental Pru<i>Δku80</i> tachyzoites (lane 2). Left panel was probed with the polyclonal anti-ENO2 antibodies while the right panel was stained with the monoclonal anti-actin antibodies. C) Western blots of mutants and wild type parasites. Lane 1, total SDS-protein extracts from wild type 76K tachyzoites. Lane 2, total SDS-extracted proteins from transgenic E1-5 tachyzoites. Lane 3, total SDS-extracted proteins from transgenic E2-4. Lane 4, total SDS-extracted proteins from transgenic E2-10. Lane 5, total SDS-extracted proteins from parental Pru<i>Δku80</i> tachyzoites. Lane 6, total SDS-extracted proteins from knock-out Pru<i>Δku80ΔTgeno1</i> mutants. Blots were stained with polyclonal antibodies specific to LDH1, LDH2, G6PI and monoclonal antibodies specific to actin. The numbers on the left indicate molecular markers in kilodaltons.</p

Nuclear Glycolytic Enzyme Enolase of <i>Toxoplasma gondii</i> Functions as a Transcriptional Regulator

<div><p>Apicomplexan parasites including <i>Toxoplasma gondii</i> have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that <i>T. gondii</i> enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin <i>in vivo</i>. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in <i>T. gondii</i>.</p></div