Protective effect of luteoloside against Toxoplasma gondii-induced liver injury through inhibiting TLR4/NF-κB and P2X7R/NLRP3 and enhancing Nrf2/HO-1 signaling pathway.

Toxoplasma gondii (T. gondii) infection can cause liver injury by inducing inflammation and oxidative stress. The Chinese herbal extract luteoloside (Lut) has considerable anti-inflammatory and antioxidant properties, but its effects on the liver injury during T. gondii infection have not been reported. This study investigated the hepatoprotective effects of Lut by treating T. gondii-infected mice with 0-200 mg/kg doses of Lut and further examined the expression of key proteins in the inflammation and oxidative stress-related pathways in the liver to investigate the potential mechanism of the hepatoprotective effects of Lut. Results showed that Lut remarkably reduced serum ALT and AST levels, considerably decreased inflammatory factors TNF-α, IL-6, and IL-1ß, as well as oxidative products MDA, and greatly increased antioxidant enzymes SOD and GSH. The expression of key proteins TLR4, Myd88, TRAF6, p-NF-κB p65 in the TLR4/NF-κB pathway and P2X7R, NLRP3, caspase 1, IL-1ß, IL-18 in the P2X7R/NLRP3 pathway were significantly decreased in the liver. And the expression of key proteins Nrf2, HO-1, NQO-1, and GCLC in the Nrf2/HO-1 antioxidant-related pathway was significantly upregulated. In conclusion, Lut attenuated T. gondii-induced liver injury by inhibiting the inflammatory response and enhancing antioxidant capacity. The hepatoprotective mechanisms of Lut are involved in inhibiting TLR4/NF-κB and P2X7R/NLRP3 inflammatory signaling pathways, as well as enhancing the Nrf2/HO-1 antioxidant pathway. These findings not only provide some reference for further exploring the specific hepatoprotective mechanism of Lut during T. gondii infection, but also provide some theoretical basis for the future clinical application of Lut as a hepatoprotective drug in T. gondii infection.

Resveratrol inhibits Toxoplasma gondii-induced lung injury, inflammatory cascade and evidences of its mechanism of action.

BACKGROUND: Toxoplasma gondii is an opportunistic protozoan that can infect host to cause toxoplasmosis. We have previously reported that resveratrol (RSV) has protective effects against liver damage in T. gondii infected mice. However, the effect of RSV on lung injury caused by T. gondii infection and its mechanism of action remain unclear. PURPOSE: In this work, we studied the protective effects of RSV on lung injury caused by T. gondii infection and explored the underlying mechanism. METHODS: Molecular docking and localized surface plasmon resonance assay were used to detect the molecular interactions between RSV and target proteins. In vitro, the anti-T. gondii effects and potential anti-inflammatory mechanisms of RSV were investigated by quantitative competitive-PCR, RT-PCR, ELISA, Western blotting and immunofluorescence using RAW 264.7 cells infected with tachyzoites of T. gondii RH strain. In vivo, the effects of RSV on lung injury caused by T. gondii infection were assessed by observing pathological changes and the expression of inflammatory factors of lung. RESULTS: RSV inhibited T. gondii loads and T. gondii-derived heat shock protein 70 (T.g.HSP70) expression in RAW 264.7 cells and lung tissues. Moreover, RSV interacts with T.g.HSP70 and toll-like receptor 4 (TLR4), respectively, and interferes with the interaction between T.g.HSP70 and TLR4. It also inhibited the overproduction of inducible nitric oxide synthase, TNF-α and high mobility group protein 1 (HMGB1) by down-regulating TLR4/nuclear factor kappa B (NF-κB) signaling pathway, which is consistent with the effect of TLR4 inhibitor CLI-095. In vivo, RSV improved the pathological lung damage produced by T. gondii infection, as well as decreased the number of inflammatory cells in bronchoalveolar lavage fluid and the release of HMGB1 and TNF-α. CONCLUSION: These findings indicate that RSV can inhibit the proliferation of T. gondii and T.g.HSP70 expression both in vitro and in vivo. RSV can inhibit excessive inflammatory response by intervening T.g.HSP70 and HMGB1 mediated TLR4/NF-κB signaling pathway activation, thereby ameliorating lung injury caused by T. gondii infection. The present study provides new data that may be useful for the development of RSV as a new agent for the treatment of lung damage caused by T. gondii infection.

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma.

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9-assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

Susceptibility of human villous (BeWo) and extravillous (HTR-8/SVneo) trophoblast cells to Toxoplasma gondii infection is modulated by intracellular iron availability.

Congenital toxoplasmosis is a serious health problem that can lead to miscarriage. HTR-8/SVneo is a first trimester extravillous trophoblast, while BeWo is a choriocarcinoma with properties of villous trophoblast cells. In the placenta, iron is taken up from Fe-transferrin through the transferrin receptor being the ion an important nutrient during pregnancy and also for Toxoplasma gondii proliferation. The aim of this study was to evaluate the role of iron in T. gondii proliferation in BeWo and HTR-8/SVneo cells and in human chorionic villous explants. The cells were infected with T. gondii, iron supplemented or deprived by holo-transferrin or deferoxamine, respectively, and parasite proliferation and genes related to iron balance were analyzed. It was verified that the addition of holo-transferrin increased, and DFO decreased the parasite multiplication in both trophoblastic cells, however, in a more expressive manner in HTR-8/SVneo, indicating that the parasite depends on iron storage in trophoblastic cells for its growth. Also, tachyzoites pretread with DFO proliferate normally in trophoblastic cells demonstrating that DFO itself does not interfere with parasite proliferation. Additionally, T. gondii infection induced enhancement in transferrin receptor mRNA expression levels in trophoblastic cells, and the expression was higher in HTR-8/SVneo compared with BeWo. Finally, DFO-treatment was able to reduce the parasite replication in villous explants. Thus, the iron supplementation can be a double-edged sword; in one hand, it could improve the supplement of an essential ion to embryo/fetus development, and on the other hand, could improve the parasite proliferation enhancing the risk of congenital infection.

Comprehensive Characterization of Toxoplasma Acyl Coenzyme A-Binding Protein TgACBP2 and Its Critical Role in Parasite Cardiolipin Metabolism.

Acyl coenzyme A (CoA)-binding protein (ACBP) can bind acyl-CoAs with high specificity and affinity, thus playing multiple roles in cellular functions. Mitochondria of the apicomplexan parasite Toxoplasma gondii have emerged as key organelles for lipid metabolism and signaling transduction. However, the rationale for how this parasite utilizes acyl-CoA-binding protein to regulate mitochondrial lipid metabolism remains unclear. Here, we show that an ankyrin repeat-containing protein, TgACBP2, is localized to mitochondria and displays active acyl-CoA-binding activities. Dephosphorylation of TgACBP2 is associated with relocation from the plasma membrane to the mitochondria under conditions of regulation of environmental [K+]. Under high [K+] conditions, loss of ACBP2 induced mitochondrial dysfunction and apoptosis-like cell death. Disruption of ACBP2 caused growth and virulence defects in the type II strain but not in type I parasites. Interestingly, mitochondrial association factor-1 (MAF1)-mediated host mitochondrial association (HMA) restored the growth ability of ACBP2-deficient type II parasites. Lipidomics analysis indicated that ACBP2 plays key roles in the cardiolipin metabolism of type II parasites and that MAF1 expression complemented the lipid metabolism defects of ACBP2-deficient type II parasites. In addition, disruption of ACBP2 caused attenuated virulence of Prugniuad (Pru) parasites for mice. Taking the results collectively, these data indicate that ACBP2 is critical for the growth and virulence of type II parasites and for the growth of type I parasites under high [K+] conditions.IMPORTANCEToxoplasma gondii is one of the most successful human parasites, infecting nearly one-third of the total world population. T. gondii tachyzoites residing within parasitophorous vacuoles (PVs) can acquire fatty acids both via salvage from host cells and via de novo synthesis pathways for membrane biogenesis. However, although fatty acid fluxes are known to exist in this parasite, how fatty acids flow through Toxoplasma lipid metabolic organelles, especially mitochondria, remains unknown. In this study, we demonstrated that Toxoplasma expresses an active ankyrin repeat containing protein TgACBP2 to coordinate cardiolipin metabolism. Specifically, HMA acquisition resulting from heterologous functional expression of MAF1 rescued growth and lipid metabolism defects in ACBP2-deficient type II parasites, manifesting the complementary role of host mitochondria in parasite cardiolipin metabolism. This work highlights the importance of TgACBP2 in parasite cardiolipin metabolism and provides evidence for metabolic association of host mitochondria with T. gondii.

Novel Approaches To Kill Toxoplasma gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite.

Toxoplasma gondii, an obligate intracellular parasite replicating in mammalian cells within a parasitophorous vacuole (PV), is an avid scavenger of lipids retrieved from the host cell. Following lipid uptake, this parasite stores excess lipids in lipid droplets (LD). Here, we examined the lipid storage capacities of Toxoplasma upon supplementation of the culture medium with various fatty acids at physiological concentrations. Supplemental unsaturated fatty acids (oleate [OA], palmitoleate, linoleate) accumulate in large LD and impair parasite replication, whereas saturated fatty acids (palmitate, stearate) neither stimulate LD formation nor impact growth. Examination of parasite growth defects with 0.4 mM OA revealed massive lipid deposits outside LD, indicating enzymatic inadequacies for storing neutral lipids in LD in response to the copious salvage of OA. Toxoplasma exposure to 0.5 mM OA led to irreversible growth arrest and lipid-induced damage, confirming a major disconnect between fatty acid uptake and the parasite's cellular lipid requirements. The importance of neutral lipid synthesis and storage to avoid lipotoxicity was further highlighted by the selective vulnerability of Toxoplasma, both the proliferative and the encysted forms, to subtoxic concentrations of the acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) pharmacological inhibitor T863. T863-treated parasites did not form LD but instead built up large membranous structures within the cytoplasm, which suggests improper channeling and management of the excess lipid. Dual addition of OA and T863 to infected cells intensified the deterioration of the parasite. Overall, our data pinpoint Toxoplasma DGAT as a promising drug target for the treatment of toxoplasmosis that would not incur the risk of toxicity for mammalian cells.

The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells.

Phosphoinositide (PtdInsP) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes/late endosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological and enzyme-based assays dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized with Rab5-positive early endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

Design of inhibitory peptide targeting Toxoplasma gondii RON4-human ß-tubulin interactions by implementing structural bioinformatics methods.

Toxoplasma gondii an obligate intracellular parasite causes toxoplasmosis in homeothermic animals. Host invasion of this parasite is mediated by the formation of Moving Junction (MJ) complex which encompasses a network of microneme and Rhoptry Neck proteins (RONs) 2/4/5/8. Among these proteins, RON4 is the only cytosolic secretory protein that is considered as a crucial member, as it directly facilitates the motility of MJ complex by interacting with host tubulin. It is also prominently localized at the host-pathogen interface during the invasion, thus projecting it as a potential drug target. The structure of RON4 is yet to be crystallized. Hence, in this study, fold recognition and Free Energy Landscape sampling was performed to predict the plausible 3D structure of RON4. Further, its interacting pattern with the reported crystal structure of human tubulin was analyzed using molecular docking. Subsequently, a ß-tubulin based inhibitory peptides were derived based on its interacting interface observed in RON4-ß-tubulin docked complex. Following which, a stepwise validation of these peptides for various physico-chemical properties and its homology with antimicrobial peptides were also screened. The peptide (RT_pep) surpassing all these validation filters was modeled and its stability was analysed by Molecular Dynamics simulation. To validate further, the stable conformation of the RT_pep was docked to RON4. Finally, essential molecular dynamics simulation was conducted to determine the stability and atomic motions of native RON4 and also to decipher its association with ß-tubulin and RT_pep. All these analyses cumulatively suggest the therapeutic potential of RT_pep in targeting toxoplasmosis.

Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii.

Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochinlike quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases, including toxoplasmosis, malaria, and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of the cytochrome bc1 complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the specific mechanism of ELQ-316 remain unknown. Here, we describe the selection and genetic characterization of T. gondii clones resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interactions of Qi site inhibitors with the apicomplexan cytochrome bc1 complex.

Vaccination with Toxoplasma gondii calcium-dependent protein kinase 6 and rhoptry protein 18 encapsulated in poly(lactide-co-glycolide) microspheres induces long-term protective immunity in mice.

BACKGROUND: Toxoplasmosis is a worldwide zoonosis caused by the intracellular parasite Toxoplasma gondii. However, no effective vaccine is yet available. Poly(lactide-co-glycolide) polymers can reduce protein degradation and sustain the release of antigens over a long period, which could generate a long-lasting immune response in vivo. Using a mouse model of toxoplasmosis, we evaluated the protective efficacy of vaccination with two recombinant proteins, which are formulated in biodegradable polymers. METHODS: Two recombinant proteins, rCDPK6 and rROP18, were encapsulated in poly(D,L-lactide-co-glycolide) (PLG), and then injected subcutaneously into Kunming mice. The mice immune responses were evaluated in terms of lympho-proliferation, cytokine expression, and antibodies. The survival of infected mice and brain cyst formation were also evaluated at 6 weeks after challenge with T. gondii RH strain (genotype I) or PRU strain (genotype II). RESULTS: Both protein vaccines induced Th1-biased immune responses, with increased specific antibodies and T cells, high levels of interferon-γ and interleukin 2, and strong lymphocyte proliferative responses. The mice immunized with the various protein vaccines survived slightly longer time than the control groups (P > 0.05) after injection with T. gondii RH strain. There were fewer brain cysts in the mice in all the immunized groups than that in the control groups, and the brain cysts were significantly reduced in mice immunized with proteins + 206, rCDPK6 + PLG and rCDPK6 + rROP18 + PLG (P < 0.05) compared controls. Further comparison of the immune responses to the proteins adjuvanted with PLG or Montanide™ ISA 206 VG 6 weeks after the last immunization revealed that antigens encapsulated in PLG conferred greater protective immunity against challenge. CONCLUSIONS: These findings suggest that the two recombinant T. gondii proteins encapsulated in PLG conferred immunity to T. gondii for an extended period, providing the foundation for the further development of a commercial vaccine against toxoplasmosis.

The FIKK kinase of Toxoplasma gondii is not essential for the parasite's lytic cycle.

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 characterises 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 280kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein showed that the FIKK protein exclusively localised to the posterior end of tachyzoites. A Yellow Fluorescent Protein-tagged FIKK and a Ty-tagged FIKK both co-localised with T. gondii membrane occupation and recognition nexus protein to the basal complex and were localised apical to inner membrane complex protein-5 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.

Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii.

Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.

The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell.

Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In Toxoplasma gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here, we demonstrate that the ER-localized fatty acid elongation (ELO) complexes are essential for parasite growth. Conditional knockdown of the nonredundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. (13) C-glucose and (13) C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long-chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was bypassed by supplementing the media with specific fatty acids, indicating active but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii.

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite's lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.

Inhibition of p-aminobenzoate and folate syntheses in plants and apicomplexan parasites by natural product rubreserine.

Glutamine amidotransferase/aminodeoxychorismate synthase (GAT-ADCS) is a bifunctional enzyme involved in the synthesis of p-aminobenzoate, a central component part of folate cofactors. GAT-ADCS is found in eukaryotic organisms autonomous for folate biosynthesis, such as plants or parasites of the phylum Apicomplexa. Based on an automated screening to search for new inhibitors of folate biosynthesis, we found that rubreserine was able to inhibit the glutamine amidotransferase activity of the plant GAT-ADCS with an apparent IC(50) of about 8 µM. The growth rates of Arabidopsis thaliana, Toxoplasma gondii, and Plasmodium falciparum were inhibited by rubreserine with respective IC(50) values of 65, 20, and 1 µM. The correlation between folate biosynthesis and growth inhibition was studied with Arabidopsis and Toxoplasma. In both organisms, the folate content was decreased by 40-50% in the presence of rubreserine. In both organisms, the addition of p-aminobenzoate or 5-formyltetrahydrofolate in the external medium restored the growth for inhibitor concentrations up to the IC(50) value, indicating that, within this range of concentrations, rubreserine was specific for folate biosynthesis. Rubreserine appeared to be more efficient than sulfonamides, antifolate drugs known to inhibit the invasion and proliferation of T. gondii in human fibroblasts. Altogether, these results validate the use of the bifunctional GAT-ADCS as an efficient drug target in eukaryotic cells and indicate that the chemical structure of rubreserine presents interesting anti-parasitic (toxoplasmosis, malaria) potential.

Tenellones A and B from a Diaporthe sp.: two highly substituted benzophenone inhibitors of parasite cGMP-dependent protein kinase activity.

Parasite cGMP-dependent protein kinase (PKG) has been recently validated as a biochemical target for the treatment of coccidiosis. To discover new anticoccidial leads, we have screened our library of natural product extracts for inhibitors of parasite PKG. Bioassay-guided fractionation of the microbial extracts has led to the discovery of tenellones A (2) and B (3), two new highly substituted benzophenones. The isolation, structure, and activity of these compounds are described.

Anti-protozoal efficacy of high performance liquid chromatography fractions of Torilis japonica and Sophora flavescens extracts on Neospora caninum and Toxoplasma gondii.

We previously reported that alcoholic extracts of Sophora flavescens and Torilis japonica from South Korea demonstrated good efficacy in reducing replication of Toxoplasma gondii and Neospora caninum. To characterize the chemical component associated with anti-protozoal activity, specific fractions were isolated by high performance liquid chromatography (HPLC) and used for in vitro testing. These fractions were evaluated in vitro against T. gondii and N. caninum. Fractions of the herb extracts were serially diluted to final concentrations of 2.850 to 0.356 ng/ml in medium and added to wells containing replicating T. gondii and N. caninum. To determine the ability of each fraction to inhibit parasite proliferation, 3H-uracil incorporation was used to determine parasite replication. In cultures infected with T. gondii, a fraction of T. japonica (TJ2) inhibited T. gondii proliferation by 99.2, 94.4, 88.6 and 27.0% in the range from 2.850 to 0.356 ng/ml. Four fractions of S. flavescens (SF1-SF4) inhibited T. gondii proliferation by 99.6-60.6, 96.9-48.1, 92.3-68.2 and 95.4-52.9% in the range from 2.850 to 0.356 ng/ml. In cultures infected with N. caninum, a fraction of T. japonica (TJ2) inhibited N. caninum proliferation by 98.3, 95.5, 79.7 and 30.6% in the range from 2.850 to 0.356 ng/ml. Four fractions of S. flavescens (SF1-SF4) inhibited N. caninum proliferation by 97.1-25.9, 94.8-35.5, 95.9-33.7 and 95.4-49.4% in the range from 2.850 to 0.356 ng/ml. These fractions of T. japonica and S. flavescens extracts are currently undergoing in vivo evaluation in experimentally infected mice.

Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii.

A number of cysteine and serine protease inhibitors blocked the intracellular growth and replication of Toxoplasma gondii tachyzoites. Most of these inhibitors caused only minor alterations to parasite morphology irrespective of the effects on the host cells. However, three, cathepsin inhibitor III, TPCK and subtilisin inhibitor III, caused extensive swelling of the secretory pathway of the parasite (i.e. the ER, nuclear envelope, and Golgi complex), caused the breakdown of the parasite surface membrane, and disrupted rhoptry formation. The disruption of the secretory pathway is consistent with the post-translational processing of secretory proteins in Toxoplasma, and with the role of proteases in the maturation/activation of secreted proteins in general. Interestingly, while all parasites in an individual vacuole (the clonal progeny of a single invading parasite) were similarly affected, parasites in different vacuoles in the same host cell showed different responses to these inhibitors. Such observations imply that there are major differences in the biochemistry/physiology between tachyzoites within different vacuoles and argue that adverse effects on the host cell are not always responsible for changes in the parasite. Treatment of established parasites also leads to an accumulation of abnormal materials in the parasitophorous vacuole implying that materials deposited into the vacuole normally undergo proteolytic modification or degradation. Despite the often extensive morphological changes, nothing resembling lysosomal bodies was seen in any treated parasites, consistent with previous observations showing that mother cell organelles are not recycled by any form of autophagic-lysosomal degradation, although the question of how the parasite recycles these organelles remains unanswered.

Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii.

A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned. The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively. The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa. Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii genome.