Toxoplasma gondii excreted/secreted proteases disrupt intercellular junction proteins in epithelial cell monolayers to facilitate tachyzoites paracellular migration.

Toxoplasma gondii shows high dissemination and migration properties across biological barriers infecting immunologically privileged organs. Toxoplasma uses different routes for dissemination; however, the mechanisms are not fully understood. Herein, we studied the effects of proteases present in excretion/secretion products (ESPs) of Toxoplasma on MDCK cell monolayers. Ultrastructural analysis showed that ESPs of Toxoplasma disrupt the intercellular junctions (IJ) of adjacent cells. The tight junction (TJ) proteins ZO-1, occludin, and claudin-1 suffered a progressive decrease in protein levels upon ESPs treatment. In addition, ESPs induced mislocalization of such TJ proteins, along with the adherent junction protein E-cadherin, and this was prevented by pre-treating the ESPs with protease inhibitors. Reorganisation of cytoskeleton proteins was also observed. Endocytosis inhibitors, Dyngo®-4a and Dynasore, impeded the modifications, suggesting that TJ proteins internalisation is triggered by the ESPs proteases hence contributing to the loss of IJ. The observed disruption in TJ proteins went in line with a decrease in the transepithelial electrical resistance of the monolayers, which was significantly blocked by pre-treating ESPs with metalloprotease and serine protease inhibitors. Moreover, exposure of cell monolayers to ESPs facilitated paracellular migration of tachyzoites. Our results demonstrate that Toxoplasma ESPs contain proteases that can disrupt the IJ of epithelial monolayers and this could facilitate the paracellular route for Toxoplasma tissue dissemination and migration.

In silico identification of immunotherapeutic and diagnostic targets in the glycosylphosphatidylinositol metabolism of the coccidian Sarcocystis aucheniae.

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host-pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI-anchored proteins (GPI-APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well-characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N-terminal signal peptide and a single C-terminal transmembrane region containing a GPI anchor signal. Twenty-four GPI-anchored peptides were identified, of which nine are likely S. aucheniae-specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI-anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra- and inter-specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI-APs as drug targets and/or as vaccine or diagnostic antigens.

Exploring protein myristoylation in Toxoplasma gondii.

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.

Characterization of metalloproteases and serine proteases of Toxoplasma gondii tachyzoites and their effect on epithelial cells.

Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatin-degrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.

A CTP Synthase Undergoing Stage-Specific Spatial Expression Is Essential for the Survival of the Intracellular Parasite Toxoplasma gondii.

Cytidine triphosphate synthase catalyzes the synthesis of cytidine 5'-triphosphate (CTP) from uridine 5'-triphosphate (UTP), the final step in the production of cytidine nucleotides. CTP synthases also form filamentous structures of different morphologies known as cytoophidia, whose functions in most organisms are unknown. Here, we identified and characterized a novel CTP synthase (TgCTPS) from Toxoplasma gondii. We show that TgCTPS is capable of substituting for its counterparts in the otherwise lethal double mutant (ura7Δ ura8Δ) of Saccharomyces cerevisiae. Equally, recombinant TgCTPS purified from Escherichia coli encodes for a functional protein in enzyme assays. The epitope-tagged TgCTPS under the control of its endogenous promoter displays a punctate cytosolic distribution, which undergoes spatial reorganization to form foci or filament-like structures when the parasite switches from a nutrient-replete (intracellular) to a nutrient-scarce (extracellular) condition. An analogous phenotype is observed upon nutrient stress or after treatment with a glutamine analog, 6-diazo-5-oxo-L-norleucine (DON). The exposure of parasites to DON disrupts the lytic cycle, and the TgCTPS is refractory to a genetic deletion, suggesting an essential requirement of this enzyme for T. gondii. Not least, this study, together with previous studies, supports that CTP synthase can serve as a potent drug target, because the parasite, unlike human host cells, cannot compensate for the lack of CTP synthase activity.

Molecular docking to Toxoplasma gondii thymidylate synthase-dihydrofolate reductase and efficacy of raltitrexed in infected mice.

Toxoplasmosis is a zoonosis of worldwide distribution. Currently, two drugs, pyrimethamine and sulfadiazine, are used as a reference in the treatment of toxoplasmosis, but the resistance of Toxoplasma gondii appears as a relevant public health problem. In order to identify new drugs to toxoplasmosis treatment, we performed a molecular docking of raltitrexed to T. gondii thymidylate synthase-dihydrofolate reductase (TS-DHFR) and also evaluated its efficacy in infected mice. Initially, raltitrexed was docked on the crystallographic structures of TS-DHFR from T. gondii and Mus musculus. Then, 48 h after infection with the T. gondii RH strain, different groups of mice received an oral dose of raltitrexed (0.15, 0.75, and 1.5 mg kg-1). Two days after treatments, raltitrexed was able to prevent mortality and reduce the number of tachyzoites in the peritoneal fluid and liver imprints from infected mice. The results showed that raltitrexed has important protective activities against the T. gondii RH strain. Molecular docking still suggests that the effects against the parasite may be dependent on the inhibition of T. gondii thymidylate synthase. This study opens new perspectives for the use of raltitrexed in patients infected with T. gondii, especially when conventional treatments do not exhibit the expected efficacy.

Antiparasitic Activity of Sulfur- and Fluorine-Containing Bisphosphonates against Trypanosomatids and Apicomplexan Parasites.

Based on crystallographic data of the complexes 2-alkyl(amino)ethyl-1,1-bisphosphonates-Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic activity, whereas the linear α-fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates were unexpectedly devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data turned out to be very important to establish a structural activity relationship.

In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC50) of 0.11 ± 0.02 µM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 µM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 µM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii.

Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans.

Role of the 52 KDa thioredoxin protein disulfide isomerase of Toxoplasma gondii during infection to human cells.

Toxoplasma protein disulfide isomerase (PDI) is a 52 KDa thioredoxin of interest because have a great immunogenicity for humans. We cloned and produced a recombinant protein (recTgPDI) used to test its effect during infection to different human cell lines (epithelial and retinal). We also determine if there were differences in gen expression during in vitro infection. Expression of the gen was lower after entry into the host cells. PDI's inhibitors bacitracin and nitroblue tetrazolium reduced the percent of infected cells and small amounts of recTgPDI proteins interfered with the invasion step. All these results support a role of Toxoplasma PDI during the first steps of infection (adhesion and invasion). Toxoplasma PDI is a protein linked to early steps of invasion, it would be of importance to identify the host proteins substrates during invasion steps.

Toxoplasma gondii Chitinase Induces Macrophage Activation.

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.

[Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii]. / Polimorfismos en los genes de dihidrofolato-reductasa ( dhfr ) y dihidropteroato-sintasa ( dhps ) y modelado estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii.

INTRODUCTION: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. OBJECTIVE: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . MATERIALS AND METHODS: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. RESULTS: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. CONCLUSIONS: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Polimorfismos en los genes de dihidrofolato-reductasa ( dhfr ) y dihidropteroato-sintasa ( dhps ) y modelado estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii / Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii

Introducción. No existen reportes sobre las variaciones en la secuencia de los genes blanco de los medicamentos anti- Toxoplasma en aislamientos provenientes de Suramérica. Objetivo. Clonar y secuenciar los genes de la dihidrofolato-reductasa ( dhfr ) y la dihidropteroato-sintetasa ( dhps ) de la cepa de referencia RH y de dos aislamientos colombianos de Toxoplasma gondii. Materiales y métodos. Se obtuvieron dos aislamientos de T. gondii en líquido céfalorraquídeo de pacientes colombianos positivos para HIV con toxoplasmosis cerebral. Se extrajo el ADN de los genes dhfr y dhps y se amplificaron mediante reacción en cadena de la polimerasa (PCR). Los productos fueron clonados en el vector pGEM-T y secuenciados. Resultados. Se encontró un cambio de adenina por guanina (A « G) en la posición 235 del exón 2 del gen dhps , dos cambios de guanina por citocina (G « C) en las posiciones 259 y 260 y un cambio de timina por guanina (T « G) en la posición 371 del exón 4 del gen dhps. Por análisis bioinformático, en este último exón se identificó un polimorfismo no sinónimo en la región codificante, que podría llevar al cambio de una Glu (CAA o CAG) por una His (codificada por los codones AAU o AAC). Se calculó el modelo estructural de la enzima dihidropteroato-sintetasa (DHPS) de T. gondii y se identificaron las modificaciones en la estructura secundaria ocasionadas por las mutaciones. Conclusiones. La metodología estandarizada puede servir como base para la búsqueda de polimorfismos en muestras de pacientes con diferentes manifestaciones clínicas de toxoplasmosis y para establecer su posible relación con los cambios en la sensibilidad a los antifolatos y la reacción al tratamiento.

Introduction: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Materials and methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. Results: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Complementary data on four methods for sampling free-living ticks in the Brazilian Pantanal / Dados complementares de quatro métodos para amostragem de carrapatos em vida livre no Pantanal brasileiro

In this study, four methods for sampling free-living ticks that are used in ecological and human tick-bite risk studies were evaluated. Cloth dragging, carbon dioxide traps and visual searches and inspection of plant litter on the ground were used in field and forest areas within the Brazilian Pantanal. Among the three tick species collected, Amblyomma sculptum predominated, followed by Amblyomma parvum and Amblyomma ovale. Dragging, a cheap and simple technique, yielded the highest numbers of ticks, particularly nymphs. The visual search detected a high number of adult ticks and provided information on tick questing height. Even though laborious, plant litter examination showed that large numbers of ticks may use this stratum. Carbon dioxide (CO2) traps are expensive and difficult to handle, but they are highly efficient for adult ticks, especially A. parvum. These data indicate that one method alone is incapable of providing a representative sample of the tick fauna in a particular area and that multiple techniques should be used for tick population studies.

Neste estudo, foram avaliados quatro métodos de amostragem de carrapatos em vida livre, usados em estudos ecológicos e avaliação do risco de picadas em humanos. Arraste de flanela, armadilhas de gás carbônico (CO2), busca visual e inspeção de serrapilheira foram aplicados em áreas campestres e florestais no Pantanal brasileiro. Dentre três espécies coletadas, a predominância foi de Amblyomma sculptum, seguida por Amblyomma parvum e Amblyomma ovale. O arraste, técnica simples e de baixo custo, resultou em maior número de carrapatos, particularmente de ninfas. A busca visual detectou alto número de carrapatos adultos e forneceu informações sobre altura de espera por hospedeiros. Apesar de trabalhoso, o exame da serrapilheira demonstrou que grande número de carrapatos pode utilizar esse estrato. Armadilhas de CO2 têm custo elevado e são difíceis de manusear, entretanto, são altamente eficientes para carrapatos adultos, em especial para A. parvum. Esses dados indicam que somente um método é incapaz de fornecer amostra representativa da ixodofauna em uma área particular e que, para estudos populacionais, técnicas múltiplas devem ser usadas.

New insights into molecular recognition of 1,1-bisphosphonic acids by farnesyl diphosphate synthase.

As part of our project pointed at the search of new antiparasitic agents against American trypanosomiasis (Chagas disease) and toxoplasmosis a series of 2-alkylaminoethyl-1-hydroxy-1,1-bisphosphonic acids has been designed, synthesized and biologically evaluated against the etiologic agents of these parasitic diseases, Trypanosoma cruzi and Toxoplasma gondii, respectively, and also towards their target enzymes, T. cruzi and T. gondii farnesyl pyrophosphate synthase (FPPS), respectively. Surprisingly, while most pharmacologically active bisphosphonates have a hydroxyl group at the C-1 position, the additional presence of an amino group at C-3 resulted in decreased activity towards either T. cruzi cells or TcFPPS. Density functional theory calculations justify this unexpected behavior. Although these compounds were devoid of activity against T. cruzi cells and TcFPPS, they were efficient growth inhibitors of tachyzoites of T. gondii. This activity was associated with a potent inhibition of the enzymatic activity of TgFPPS. Compound 28 arises as a main example of this family of compounds exhibiting an ED50 value of 4.7 µM against tachyzoites of T. gondii and an IC50 of 0.051 µM against TgFPPS.

Homology modeling of dihydrofolate reductase from T. gondii bonded to antagonists: molecular docking and molecular dynamics simulations.

The aim of this work was to solve the structure of the enzyme dihydrofolate reductase from Toxoplasma gondii (TgondiiDHFR) as a target for drug discovery on account of recent reports of parasite's growing resistance to pyrimethamine (CP6), which is the reference pharmaceutical used to treat toxoplasmosis and malaria. The tertiary structure of the protein bonded to NADP(+) and CP6 was solved by homology modeling. The best output model was subjected to conjugate gradient minimization and the comparison with templates shows important replacements at the inhibitor's binding site allowing selective drug design. CP6 redocking in TgondiiDHFR shows a ΔGbinding of -8.66 kcal mol(-1), higher than those found for templates Plasmodium vivax (-9.01) and P. falciparum (-8.99). Virtual screening of ligands similar to CP6 was performed using the ZINC database and docking procedures were carried out. The result indicates the substances ZINC14966516, ZINC13685962, ZINC13685929 and ZINC13686062 with a ΔGbinding of -10.57, -10.09, -9.87, and -9.76 kcal mol(-1), respectively, as the best choices. NPT molecular dynamics with the complexes indicates that they remained stable along the 10 ns simulation and they dock to TgondiiDHFR by salt bridges to the Asp 30 and to nine other residues in the contact region, which makes it more difficult for single mutations to acquire resistance. The contact frequency of protein residues with ligands suggests plausible explanations for site-directed mutagenesis studies regarding CP6 resistance described previously in the literature. All results indicate that the new ligands could be tested as pyrimethamine substitutes in the treatment of toxoplasmosis, in addition to other protozoonosis diseases.

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.

1-(Fluoroalkylidene)-1,1-bisphosphonic acids are potent and selective inhibitors of the enzymatic activity of Toxoplasma gondii farnesyl pyrophosphate synthase.

α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease, and against Toxoplasma gondii, the agent responsible for toxoplasmosis, and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T. gondii (TgFPPS). Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) proved to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range, exhibiting an IC(50) of 30 nM. This compound was two-fold more potent than risedronate (IC(50) = 74 nM) that was taken as a positive control. This enzymatic activity was associated with a strong cell growth inhibition against tachyzoites of T. gondii, with an IC(50) value of 2.7 µM.

Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase.

The effect of long-chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii was investigated. Particularly, compound 26 proved to be an extremely potent inhibitor against the intracellular form of T. cruzi, exhibiting IC(50) values at the nanomolar range. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 26 was an effective agent against T. cruzi (amastigotes) exhibiting an IC(50) value of 0.67 µM, while this compound showed an IC(50) value of 0.81 µM against the target enzyme TcFPPS. This drug was less effective against the enzymatic activity of T. cruzi solanesyl diphosphate synthase TcSPPS showing an IC(50) value of 3.2 µM. Interestingly, compound 26 was also very effective against T. gondii (tachyzoites) exhibiting IC(50) values of 6.23 µM. This cellular activity was also related to the inhibition of the enzymatic activity towards the target enzyme TgFPPS (IC(50)=0.093 µM) As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control different tropical diseases.

Toxoplasma gondii: molecular cloning and characterization of a nitric oxide synthase-like protein.

Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of l-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21 nmol/min/ml nitrites per microgram of protein and followed Michaelis-Menten kinetics, with a K(m) for L-arginine of 2.3mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from L-arginine as substrate.