FIKK kinases are a novel family of kinases unique to the Apicomplexa. While most apicomplexans encode a single FIKK kinase, Plasmodium falciparum expresses 21 and piroplasms do not encode a FIKK kinase. FIKK kinases share a conserved C-terminal catalytic domain, but the N-terminal region is highly variable and contains no known functional domains. To date, FIKK kinases have been primarily studied in P. falciparum and Plasmodium berghei. Those that have been studied are exported from the parasite and associate with diverse locations in the infected erythrocyte cytosol or membrane. Deletion of individual P. falciparum FIKK kinases indicates that they may play a role in modification of the infected erythrocyte. The current study characterizes the single FIKK gene in Toxoplasma gondii to evaluate the importance of the FIKK kinase in an apicomplexan that has a single FIKK kinase. The TgFIKK gene encoded a protein of approximately 280 kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein (YFP) showed that the FIKK protein exclusively localized to the posterior end of tachyzoites. A YFP-tagged FIKK and a Ty-tagged FIKK both co-localized with T. gondii membrane occupation and recognition nexus protein (TgMORN1) to the basal complex and were localized apical to inner membrane complex protein-5 (IMC5) and Centrin2. Deletion of TgFIKK, surprisingly, had no detectable effect on the parasite’s lytic cycle in vitro in human fibroblast cells or in acute virulence in vivo. Thus, our results clearly show that while the FIKK kinase is expressed in tachyzoites, it is not essential for the lytic cycle of T. gondii.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependentinnate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the disease toxoplasmosis. Chronic infection is established through the formation of tissue cysts predominantly in cardiac and neurologic tissues. A defining characteristic of T. gondii is its ability to evade the host’s immune defenses; specifically, T. gondii can invade and persist within host phagocytes, using them to disseminate to the brain and central nervous system where cysts are then formed. This protocol is used to evaluate the ability of Toxoplasma gondii to survive and replicate within naive and activated murine bone marrow-derived macrophages at the level of single infected cells. In the following protocol macrophages are naive or activated with IFN-γ and LPS but different activation stimuli can be utilized as well as different host cell populations and diverse inhibitors. Parasite replication is determined by evaluating the number of parasites per vacuole over time using immunofluorescence staining for parasties and microscopic analysis. Kinetic determination of parasite number per vacuole accurately reflects parasite replication over time as vacuoles-containing parasites do not fuse with one another. Isolation of murine bone marrow-derived macrophages, preparation of conditioned L929 cells for collection of macrophage colony-stimulating factor, and staining for fluorescence microscopy included in the protocol has broad applicability. This protocol works well for pathogens like Toxoplasma gondii that reside in vacuoles that do not fuse with one another and that can be visualized by microscopy.