GRA24-Based DNA Vaccine Prolongs Survival in Mice Challenged With a Virulent Toxoplasma gondii Strain.

Toxoplasma gondii causes infections in a wide range of intermediate hosts and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Dense granule protein 24 (GRA24) is a novel essential virulence factor that is transferred into the nucleus of host cells from the parasitophorous vacuole to regulate gene expression. In the present study, bioinformatic analysis showed that GRA24 had a high score for B-cell and T-cell epitopes compared with surface antigen 1 (SAG1), which has been studied as a promising vaccine candidate. As a DNA vaccine, pVAX1-GRA24 was injected intramuscularly into BALB/c mice and the induced immune response was evaluated. pVAX1-GRA24 induced high levels of a mixed Th1/Th2 cytokines at 6 weeks after immunization. Antibody determinations, cytokines [interferon gamma (IFN-γ), interleukin (IL)-12, IL-4, IL-10], antigen-specific lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, and cytotoxic T lymphocyte activity showed that mice immunized with pVAX1-GRA24 produced specific humoral and cellular immune responses. The expression levels of interferon regulatory factor 8 (IRF8), nuclear factor kappa B (NF-κB), and T-Box 21 (T-bet) were significantly higher in the pVAX1-GRA24 immunization group than in the control groups. Survival times were prolonged significantly (24.6 ± 5.5 days) in the mice immunized with pVAX1-GRA24 compared with the mice in the control groups, which died within 7 days of T. gondii challenge (p < 0.05). The results of the present study showed that pVAX1-GRA24 induced a T. gondii-specific immune response and thus represents a promising candidate vaccine to treat toxoplasmosis.

Neighbors Working Together: a Toxoplasma Rhoptry Protein That Facilitates Dense Granule Protein Translocation into the Host Cell.

The opportunistic pathogen Toxoplasma gondii is highly adept at manipulating host cell functions. While inside a host cell, Toxoplasma divides within a parasitophorous vacuole from which it secretes numerous effector proteins from its dense granules. Many of these so-called GRA proteins are translocated from the parsitophorous vacuole into the host cell where they directly disrupt host signaling pathways. The machinery that drives the translocation of GRA proteins across the parasitophorous vacuole membrane is being elucidated through both genetic and biochemical approaches. A new mSphere research article (M. W. Panas, A. Ferrel, A. Naor, E. Tenborg, et al., mSphere 4:e00276-19, 2019, https://doi.org/10.1128/mSphere.00276-19) describes how the kinase ROP17, which is secreted from the parasite's rhoptries into the host cell during invasion, regulates the translocation of GRA effectors.

Toxoplasma Does Not Secrete the GRA16 and GRA24 Effectors Beyond the Parasitophorous Vacuole Membrane of Tissue Cysts.

After invasion, Toxoplasma resides in a parasitophorous vacuole (PV) that is surrounded by the PV membrane (PVM). Once inside the PV, tachyzoites secrete dense granule proteins (GRAs) of which some, such as GRA16 and GRA24, are transported beyond the PVM likely via a putative translocon. However, once tachyzoites convert into bradyzoites within cysts, it is not known if secreted GRAs can traffic beyond the cyst wall membrane. We used the tetracycline inducible system to drive expression of HA epitope tagged GRA16 and GRA24 after inducing stage conversion and show that these proteins are not secreted beyond the cyst wall membrane. This suggests that secretion of GRA beyond the PVM is not important for the tissue cyst stage of Toxoplasma.

Structural Basis for the Subversion of MAP Kinase Signaling by an Intrinsically Disordered Parasite Secreted Agonist.

The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.