Pyrimidine salvage in Toxoplasma gondii as a target for new treatment

Toxoplasmosis is a common protozoan infection that can have severe outcomes in the immunocompromised and during pregnancy, but treatment options are limited. Recently, nucleotide metabolism has received much attention as a target for new antiprotozoal agents and here we focus on pyrimidine salvage by Toxoplasma gondii as a drug target. Whereas uptake of [3H]-cytidine and particularly [3H]-thymidine was at most marginal, [3H]-uracil and [3H]-uridine were readily taken up. Kinetic analysis of uridine uptake was consistent with a single transporter with a Km of 3.3 ± 0.8 µM, which was inhibited by uracil with high affinity (Ki = 1.15 ± 0.07 µM) but not by thymidine or 5-methyluridine, showing that the 5-Me group is incompatible with uptake by T. gondii. Conversely, [3H]-uracil transport displayed a Km of 2.05 ± 0.40 µM, not significantly different from the uracil Ki on uridine transport, and was inhibited by uridine with a Ki of 2.44 ± 0.59 µM, also not significantly different from the experimental uridine Km. The reciprocal, complete inhibition, displaying Hill slopes of approximately -1, strongly suggest that uridine and uracil share a single transporter with similarly high affinity for both, and we designate it uridine/uracil transporter 1 (TgUUT1). While TgUUT1 excludes 5-methyl substitutions, the smaller 5F substitution was tolerated, as 5F-uracil inhibited uptake of [3H]-uracil with a Ki of 6.80 ± 2.12 µM (P > 0.05 compared to uracil Km). Indeed, we found that 5F-Uridine, 5F-uracil and 5F,2’-deoxyuridine were all potent antimetabolites against T. gondii with EC50 values well below that of the current first line treatment, sulfadiazine. In vivo evaluation also showed that 5F-uracil and 5F,2’-deoxyuridine were similarly effective as sulfadiazine against acute toxoplasmosis. Our preliminary conclusion is that TgUUT1 mediates potential new anti-toxoplasmosis drugs with activity superior to the current treatment

O papel da sumoilação de proteínas na biologia celular de giardia lamblia

Protein post-translational modification (PTM) increases the functional diversity of the proteome by the covalent addition of functional groups or proteins. PTMs include the bonding of a chemical group (acetyl, methyl), modifications of amino acids (deamination, elimination), addition of more complex molecules like sugars (glycosylation), lipids (isoprenylation) or another protein. In the latter case, ubiquitination (addition of an ubiquitin) and SUMOylation (addition of a SUMO) are well known PTMs occurring in eukaryotic cells. Modification of a protein by SUMO (Small Ubiquitin-like MOdifier) is known to play a role in many cellular processes such as nuclear-cytoplasmic transport, transcriptional regulation, progression through cell cycle, protein stability and protein cellular localization. In this thesis the biological role of protein SUMOylation was investigated in Giardia lamblia and the data herein show that Giardia genome contains a single SUMO gene, codifying for a SUMO protein (GlSUMO) with 42,7 % identity with the human SUMO-1. In trophozoites GlSUMO is localized mainly at the cell cytoplasm, but also in the nuclei, the perinuclear region and at the cell periphery where it co- localizes with VSPs, suggesting a possible role for SUMOylation in antigenic variation. Silencing of of GlSUMO by RNA interference resulted in trophozoites with abnormal morphologies, adhesion-deficient and slower growth rates. Compared to wild type parasites Flow cytometry and EdU assays suggest that cells with silenced expression of GlSUMO arrest in the G1/S transition. Moreover, the levels of transcripts for cyclin B and ?-giardin are reduced in trophozoites GlSUMO depleted. Ablation of GlSUMO also interfered in encystment, RNAi GlSUMO trophozoites produces less cyst, also the cysts with silenced expression of GlSUMO present deformed cyst walls. CMGC kinase, malate dehydrogenase, glycil t-RNA synthetase, uridine kinase, actin, poly-A polymerase, Lek 1, dnaJ chaperone, elongation factor 2 and arginine deiminase were identified as possible GlSUMO susbtrates by immunoprecipitation with anti-GlSUMO antibody and mass spectrometry. The present study provides strong evidence for a role for SUMO in the regulation of genes involved in cell cycle and cell architecture in Giardia lamblia and has revealed a potential mechanism for SUMO-mediated growth arrest.Modificações pós-traducionais de proteínas (PTMs) aumentam a diversidade funcional do proteoma pela ligação covalente de grupos funcionais ou proteínas. PTMs incluem a adição de grupos químicos (acetil, metil, nitrosil), modificações nos aminoácidos (deaminação, eliminação), a ligação de moléculas mais complexas como açúcares (glicosilação), lipídeos (isoprenilação) ou outras proteínas. No último caso, ubiquitinação (adição de ubiquitina) e SUMOilação (adição de SUMO) são PTMs que ocorrem exclusivamente em eucariotos e que desempenham um papel central em uma série de processos biológicos. Por exemplo, a modificação de proteínas por SUMO (Small Ubiquitin-like MOdifier) está relacionada com o transporte núcleo- citoplasma, regulação de transcrição gênica, progressão no ciclo celular, estabilidade e localização celular de proteínas. Nesta tese, investigou-se o papel biológico da SUMOilação de proteínas em Giardia lamblia e os dados obtidos mostram que o genoma do parasito possui um único gene que codifica para uma proteína SUMO (GlSUMO) com 42,7 % de identidade em aminoácidos com a SUMO-1 humana. Nos trofozoítos GlSUMO pode ser encontrada majoritariamente no citoplasma, nos núcleos, na região perinuclear e na periferia das células onde co-localiza-se com as VSPs, sugerindo uma possível função da SUMO na variação antigênica de Giardia lamblia. Trofozoítos silenciados para a expressão de GlSUMO (pela técnica de RNA de interferência) apresentam-se como células morfologicamente aberrantes, com menor capacidade de adesão e crescimento lento quando comparados aos parasitos selvagens. Análise por citometria de fluxo e ensaios com análogo de timina (EdU) sugerem que as células com expressão de GlSUMO silenciada acumulam nas fases G1/S do ciclo celular. Além disso, os níveis de mRNA de ciclina B e de ?-giardina são diminuídos nos trofozoítos depletados de GlSUMO. O silenciamente de GlSUMO também interferiu no encistamento, de modo que os trofozoítos RNAi GlSUMO encistam menos; além disso os cistos produzidos apresentam a parede cística deformada em comparação com os cistos WT. CMGC quinase, malato desidrogenase, glicil t-RNA sintetase, quinase de uridina, actina, poli-A polimerase, Lek 1, chaperona dnaJ, fator de enlongação 2 e arginina deiminase foram identificados como possíveis substratos da GlSUMO após imunoprecipitação com um anticorpo anti-GlSUMO e por análise de espectrometria de massas. O conjunto de resultados sugerem que a SUMOilação está envolvida na regulação de genes do ciclo celular e da arquitetura celular em Giardia lamblia.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction.

Many eukaryotic microbes have complex life cycles that include both sexual and asexual phases with strict species specificity. Whereas the asexual cycle of the protistan parasite Toxoplasma gondii can occur in any warm-blooded mammal, the sexual cycle is restricted to the feline intestine. The molecular determinants that identify cats as the definitive host for T. gondii are unknown. Here, we defined the mechanism of species specificity for T. gondii sexual development and break the species barrier to allow the sexual cycle to occur in mice. We determined that T. gondii sexual development occurs when cultured feline intestinal epithelial cells are supplemented with linoleic acid. Felines are the only mammals that lack delta-6-desaturase activity in their intestines, which is required for linoleic acid metabolism, resulting in systemic excess of linoleic acid. We found that inhibition of murine delta-6-desaturase and supplementation of their diet with linoleic acid allowed T. gondii sexual development in mice. This mechanism of species specificity is the first defined for a parasite sexual cycle. This work highlights how host diet and metabolism shape coevolution with microbes. The key to unlocking the species boundaries for other eukaryotic microbes may also rely on the lipid composition of their environments as we see increasing evidence for the importance of host lipid metabolism during parasitic lifecycles. Pregnant women are advised against handling cat litter, as maternal infection with T. gondii can be transmitted to the fetus with potentially lethal outcomes. Knowing the molecular components that create a conducive environment for T. gondii sexual reproduction will allow for development of therapeutics that prevent shedding of T. gondii parasites. Finally, given the current reliance on companion animals to study T. gondii sexual development, this work will allow the T. gondii field to use of alternative models in future studies

Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how T. gondii manipulates host metabolism to support its overall growth rate and non-essential metabolites. To investigate this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in expression of metabolic enzymes. T. gondii infection changed metabolite abundance in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, were mirrored by changes in parasite transcription, while changes in others, like the pentose phosphate pathway, were paired with changes in both the host and parasite transcriptomes. Further experiments led to the discovery of a T. gondii enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into the pentose phosphate pathway through an energetically driven dephosphorylation reaction. This additional route for ribose synthesis appears to resolve the conflict between the T. gondii tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in T. gondii central carbon metabolism that allows T. gondii to energetically-drive ribose synthesis without using NADP+

A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease.

Identification of regulators of Toxoplasma gondii bradyzoite development and cyst formation is the most direct way to address the importance of parasite development in long-term persistence and reactivation of this parasite. Here we show that a T. gondii gene (named Regulator of Cystogenesis 1; ROCY1) is sufficient for T. gondii bradyzoite formation in vitro and in vivo. ROCY1 encodes an RNA binding protein that has a preference for 3 regulatory regions of hundreds of T. gondii transcripts, and its RNA-binding domains are required to mediate bradyzoite development. Female mice infected with ΔROCY1 parasites have reduced (>90%) cyst burden. While viable parasites can be cultivated from brain tissue for up to 6 months post-infection, chronic brain-resident ΔROCY1 parasites have reduced oral infectivity compared to wild type. Despite clear defects in bradyzoite formation and oral infectivity, ΔROCY1 parasites were able to reactivate with similar timing and magnitude as wild type parasites for up to 5 months post-infection. Therefore while ROCY1 is a critical regulator of the bradyzoite developmental pathway, it is not required for parasite reactivation, raising new questions about the persisting life stage responsible for causing recrudescent disease

A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease

Abstract Identification of regulators of Toxoplasma gondii bradyzoite development and cyst formation is the most direct way to address the importance of parasite development in long-term persistence and reactivation of this parasite. Here we show that a T. gondii gene (named Regulator of Cystogenesis 1; ROCY1) is sufficient for T. gondii bradyzoite formation in vitro and in vivo. ROCY1 encodes an RNA binding protein that has a preference for 3’ regulatory regions of hundreds of T. gondii transcripts, and its RNA-binding domains are required to mediate bradyzoite development. Female mice infected with ΔROCY1 parasites have reduced (>90%) cyst burden. While viable parasites can be cultivated from brain tissue for up to 6 months post-infection, chronic brain-resident ΔROCY1 parasites have reduced oral infectivity compared to wild type. Despite clear defects in bradyzoite formation and oral infectivity, ΔROCY1 parasites were able to reactivate with similar timing and magnitude as wild type parasites for up to 5 months post-infection. Therefore while ROCY1 is a critical regulator of the bradyzoite developmental pathway, it is not required for parasite reactivation, raising new questions about the persisting life stage responsible for causing recrudescent disease

MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii

This work was supported by the National Institutes of Health (AI139201 and AI137767 to GEW, each including salary support; GM141743 to DMW, including salary support; F31AI145214 to RVS, including predoctoral fellowship stipend support; and T32AI055402 to GEW, including predoctoral fellowship stipend support for AKS). The work was also supported by the Canadian Institutes of Health Research (148596 to MJB), the Canada Research Chair program (to MJB, salary support) and the American Heart Association (20POST35220017 to RSK, including postdoctoral fellowship stipend support).Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.Publisher PDFPeer reviewe