AIMing 2 Curtail Cancer

The dysregulation of the relationship between gut microbiota and innate immune homeostasis can lead to a range of complex diseases. In this issue, Man et al. reveal that the intracellular innate sensor AIM2 regulates microbial and stem cell homeostasis in the gut to protect against colorectal cancer

Type II Toxoplasma Gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection

Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8+ T cell epitopes that elicit corresponding antigen-specific CD8+ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission

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

Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen

Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation

Toxoplasma gondii Rhoptry Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control

The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection

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

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

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