Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma Gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity

Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Secreted Effectors Modulating Immune Responses to Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that chronically infects a third of humans. It can cause life-threatening encephalitis in immune-compromised individuals. Congenital infection also results in blindness and intellectual disabilities. In the intracellular milieu, parasites encounter various immunological effectors that have been shaped to limit parasite infection. Parasites not only have to suppress these anti-parasitic inflammatory responses but also ensure the host organism’s survival until their subsequent transmission. Recent advancements in T. gondii research have revealed a plethora of parasite-secreted proteins that suppress as well as activate immune responses. This mini-review will comprehensively examine each secreted immunomodulatory effector based on the location of their actions. The first section is focused on secreted effectors that localize to the parasitophorous vacuole membrane, the interface between the parasites and the host cytoplasm. Murine hosts are equipped with potent IFNγ-induced immune-related GTPases, and various parasite effectors subvert these to prevent parasite elimination. The second section examines several cytoplasmic and ER effectors, including a recently described function for matrix antigen 1 (MAG1) as a secreted effector. The third section covers the repertoire of nuclear effectors that hijack transcription factors and epigenetic repressors that alter gene expression. The last section focuses on the translocation of dense-granule effectors and effectors in the setting of T. gondii tissue cysts (the bradyzoite parasitophorous vacuole)

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

The Toxoplasma Gondii Rhoptry Kinome is Essential for Chronic Infection

Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Delta rop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5, ROP17, ROP18, ROP35, or ROP38/29/19 (ROP38, ROP29, and ROP19) severely reduced cyst burdens. Delta rop5 and Delta rop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited \u3e100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Delta rop17 strain exhibited a \u3e12-fold defect in virulence; however, virulence was not affected in the Delta rop35 or Delta rop38/29/19 strain. Resistance to host IRGs was not affected in the Delta rop17, Delta rop35, or Delta rop38/29/19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii. IMPORTANCE Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections

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

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

Genotype of parasite strains developed in this study.

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