Calcium signaling through a transient receptor channel is important for Toxoplasma gondii growth.

Transient receptor potential (TRP) channels participate in calcium ion (Ca2+) influx and intracellular Ca2+ release. TRP channels have not been studied in Toxoplasma gondii or any other apicomplexan parasite. In this work, we characterize TgGT1_310560, a protein predicted to possess a TRP domain (TgTRPPL-2), and determined its role in Ca2+ signaling in T. gondii, the causative agent of toxoplasmosis. TgTRPPL-2 localizes to the plasma membrane and the endoplasmic reticulum (ER) of T. gondii. The ΔTgTRPPL-2 mutant was defective in growth and cytosolic Ca2+ influx from both extracellular and intracellular sources. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrates that TgTRPPL-2 is a non-selective cation channel that conducts Ca2+. Pharmacological blockers of TgTRPPL-2 inhibit Ca2+ influx and parasite growth. This is the first report of an apicomplexan ion channel that conducts Ca2+ and may initiate a Ca2+ signaling cascade that leads to the stimulation of motility, invasion, and egress. TgTRPPL-2 is a potential target for combating toxoplasmosis.

Characterization of Two EF-hand Domain-containing Proteins from Toxoplasma gondii.

The universal role of calcium (Ca2+ ) as a second messenger in cells depends on a large number of Ca2+ -binding proteins (CBP), which are able to bind Ca2+ through specific domains. Many CBPs share a type of Ca2+ -binding domain known as the EF-hand. The EF-hand motif has been well studied and consists of a helix-loop-helix structural domain with specific amino acids in the loop region that interact with Ca2+ . In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF-hand motif. The majority of these genes have not been characterized. We report the characterization of two EF-hand motif-containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca2+ measurements showed that Δtg216620 cells did not respond to extracellular Ca2+ as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca2+ influx while TgGT1_280480 may be playing a different role in the rhoptries.

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii.

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca2+) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca2+ signaling is essential for the cellular and developmental changes that support T. gondii parasitism. The characterization of the molecular players and pathways directly activated by Ca2+ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca2+ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca2+ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.

Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase.

Dihydroorotate dehydrogenase (DHODH) mediates the fourth step of de novo pyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. In Toxoplasma gondii, disruption of the first, fifth, or sixth step of de novo pyrimidine biosynthesis induced uracil auxotrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH of T. gondii (TgDHODH) failed. To further address the essentiality of TgDHODH, mutant gene alleles deficient in TgDHODH activity were designed to ablate the enzyme activity. Replacement of the endogenous DHODH gene with catalytically deficient DHODH gene alleles induced uracil auxotrophy. Catalytically deficient TgDHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show that TgDHODH is essential for the synthesis of pyrimidines and suggest that TgDHODH is required for a second essential function independent of its role in pyrimidine biosynthesis.

The Toxoplasma gondii Rhoptry Kinome Is Essential for Chronic Infection.

UNLABELLED: 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 Δrop16 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. Δrop5 and Δrop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δrop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δrop35 or Δrop38/29/19 strain. Resistance to host IRGs was not affected in the Δrop17, Δrop35, or Δrop38/29/19 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.

Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii.

The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60µM for l-dihydroorotate, and a K(m)=29µM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91µM, 96µM, and 60µM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs.

Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii.

A full-length dihydroorotase (DHOase) sequence was cloned from a Toxoplasma gondii tachyzoite cDNA library. The sequence had a calculated molecular mass of 44.2 kDa and a pI of 5.72, and was most similar to type IIa DHOases. A recombinant protein was expressed and purified with a yield of approximately 20 mg L(-1) of cell culture. Polyclonal antibodies raised against purified recombinant protein reacted with a band of the expected molecular mass in tachyzoite extracts. Specific activities of 18.3 micromol/min/mg in the biosynthetic direction and 18.4 micromol/min/mg in the degradative direction, with K(m, carbamyl aspartate) = 323 microM and K(m, dihydroorotate) = 64.3 microM, were measured for purified recombinant protein. Size exclusion chromatography/laser light scattering showed a single, monodisperse peak with a molecular mass of 45.6 kDa, suggesting that the native protein is a monomer.