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 aux- otrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH of T. gondii (Tg DHODH) failed. To further address the essentiality of Tg DHODH, mutant gene alleles deficient in Tg DHODH 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 Tg DHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show that Tg DHODH is essential for the synthesis of pyrimidines and suggest that Tg DHODH is required for a second essential function independent of its role in pyrimidine biosynthesis

Detección y localización de la enzima dihidroorotasa (DHOasa) de la síntesis de pirimidina en taquizoitos de Toxoplasma gondii

ilMagíster en MicrobiologíaMaestrí

Dihydroorotate dehydrogenase of Toxoplasma gondii - kinetic characterization and intracellular localization

The pyrimidine biosynthesis patway in the protozoan pathogen Toxoplosma gondii is essenial for parasite growth during infection. To investigate the properties of Dihydroorotate dehydrogenase (TgDHOD. TgDHOD exhibited a specific activity of 83.8 U/mg, a kcat of 89.2 sec-1 ± 1.5. a Km = 60.3 ±0.002 æM for L-dihydroorotate, and a Km = 28.9 æM ± 1.8 for decylubiquinone (QD). Quinones lacking or having short isoprenoid side chains yielded lower kcats and higher Kms than QD. As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The determined for Redoxal and A77-1726 were 253.3 æM ± 13.3 and 91.2 æM ±2.2, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. TgDHOD exhibits a 157 ? residue N-terminal extension, consistent with potential organellar targeting, but bioinformatic analysis failed to reveal a consensus subcellular destination, Preliminary inmunolocalization studies of TgDHOD performed in intra- and extracellular parasites treated with the polyclonal antibodies raised against purified recombinant TgDHOD exhibited fluorescence that appeared to colocalize with the apicoplast in fluorescence was observed in -10% of mitochondria stained with Mitotracker Red in extracellular parasites. These findings suggest that TgDHOD is associated with both the apicoplast and mitochondrion, making it a member of a growing list of a metabolic enzymes that are dual targeted in T. gondiiDoctor en Ciencias - BiologíaDoctorad

Toxoplasma Gondii - cien años de historia

Lo que ha hecho de Toxoplasma uno de los parásitos de mayor éxito y ubicuidad ha sido su capacidad de invadir cualquier tipo de célula nucleada en huéspedes de sangre caliente. Si tomamos como ejemplo las células del sistema inmune encargadas de la defensa, este parásito es capaz de utilizarlas como "caballos de troya" para moverse rápidamente a través del hospedero en la invasión, logrando llegar hasta órganos tan protegidos como el mismo cerebr

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