Search

Radical cure of Plasmodium vivax malaria must include elimination of quiescent "hypnozoite" forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets, we screened the Repurposing, Focused Rescue, and Accelerated Medchem library against P. vivax liver stages and identified the DNA methyltransferase inhibitors hydralazine and cadralazine as active against hypnozoites. We then used bisulfite sequencing and immunostaining to identify cytosine modifications in the infectious stage (sporozoites) and liver stages, respectively. A subsequent screen of epigenetic inhibitors revealed hypnozoites are broadly sensitive to histone acetyltransferase and methyltransferase inhibitors, indicating that several epigenetic mechanisms are likely modulating hypnozoite persistence. Our data present an avenue for the discovery and development of improved radical cure antimalarials.

Introduction Mammalian hibernators, including Richardson's ground squirrels (Urocitellus richardsonii), undergo extreme behavioural and physiological changes to survive the winter months. In preparation for the switch between summer homeothermy and winter [...], Glycerol-3-phosphate dehydrogenase (G3PDH) bridges carbohydrate and lipid metabolism by interconverting glycerol3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). This reversible reaction converts G3P derived from triglyceride hydrolysis to DHAP that can then enter glycolysis or gluconeogenesis and, in the reverse reaction, makes G3P for use in triglyceride biosynthesis. Small hibernating mammals rely almost exclusively on triglyceride reserves as their fuel for energy production during torpor and the recovery of glycerol after lipolysis is an important source of carbohydrate over the nonfeeding winter months. G3PDH (~37 kDa) was purified from skeletal muscle of euthermic and hibernating Richardson's ground squirrels (Urocitellus richardsonii) using three column chromatography steps. Analysis of enzyme kinetic properties revealed that G3PDH from hibernator muscle had higher affinities for G3P and NAD at low (5 [degrees]C) assay temperature compared with high (21 or 37 [degrees]C) and a greater stability in the presence of denaturing agents (urea, guanidine hydrochloride) or high temperature (50 [degrees]C). Immunoblotting showed that hibernator muscle G3PDH had a higher phosphoserine content than the enzyme from euthermic controls and incubation studies showed that enzyme affinity for G3P changed significantly by stimulating endogenous protein kinases or phosphatases. Overall, this study suggests that the properties of ground squirrel muscle G3PDH are modulated by temperature and post-translational phosphorylation to alter enzyme function under euthermic versus hibernating states. Key words: glycerol-3-phosphate dehydrogenase, serine phosphorylation, muscle, Richardson's ground squirrel, hibernation. La glycerol-3-phosphate deshydrogenase (G3PDH) fait le pont entre le metabolisme des glucides et des lipides en catalysant l'interconversion du glycerol-3-phosphate (G3P) et de la dihydroxyacetone phosphate (DHAP). Cette reaction reversible convertit le G3P derive de l'hydrolyse des triglycerides en DHAP qui peut ainsi entrer dans les voies de la glycolyse ou de la gluconeogenese et, dans le cas de la reaction inverse, produit du G3P pour la biosynthese des triglycerides. Les petits mammiferes qui hibernent dependent presque exclusivement des reserves de triglycerides comme carburant pour produire de l'energie durant la periode de torpeur, et la recuperation de glycerol a la suite de la lipolyse constitue une source importante d'hydrates de carbone durant le jeune des mois d'hiver. La G3PDH (~37 kDa) a ete purifiee a partir du muscle squelettique de spermophile de Richardson (ecureuil terrestre de Richardson (Urocitellus richardsonii)) en euthermie et en hibernation, par trois etapes de chromatographie sur colonne. L'analyse des proprietes cinetiques de l'enzyme a revele que la G3PDH du muscle en hibernation presentait une affinite plus elevee envers le G3P et le NAD a faible temperature experimental (5 [degrees]C) comparativement a une temperature plus elevee (21 ou 37 [degrees]C), et une plus grande stabilite en presence d'agents denaturants (uree, chlorhydrate de guanidine) ou a haute temperature (50 [degrees]C). L'immunobuvardage montrait que la G3PDH du muscle en hibernation comportait un contenu en phosphoserine plus eleve que l'enzyme isolee du muscle controle en euthermie, et les etudes d'incubation ont montre que l'affinite de l'enzyme pour le G3P changeait de maniere significative a la suite de la stimulation des kinases et des phosphatases endogenes. Dans son ensemble, cette etude suggere que les proprietes de la G3PDH du muscle de l'ecureuil terrestre sont modulees par la temperature et la phosphorylation post-traductionnelle qui modifient la fonction de l'enzyme en fonction de l'euthermie ou de l'hibernation. [Traduit par la Redaction] Mots-cles: glycerol-3-phosphate deshydrogenase, serine phosphorylation, muscle, ecureuil terrestre de Richardson, hibernation.

The production of energy in the form of ATP by the mitochondrial ATP synthase must be tightly controlled. One well-conserved form of regulation is mediated via ATPase inhibitory factor 1 (IF1), which governs ATP synthase activity and gene expression patterns through a cytoprotective process known as mitohormesis. In apicomplexans, the processes regulating ATP synthase activity are not fully elucidated. Using the model apicomplexan Toxoplasma gondii , we found that knockout and overexpression of TgIF1, the structural homolog of IF1, significantly affected gene expression. Additionally, TgIF1 overexpression resulted in the formation of a stable TgIF1 oligomer and increased the presence of higher order ATP synthase oligomers. We also show that parasites lacking TgIF1 exhibit reduced mitochondrial cristae density, and that while TgIF1 levels do not affect growth in conventional culture conditions, they are crucial for parasite survival under hypoxia. Interestingly, TgIF1 overexpression enhances recovery from oxidative stress, suggesting a mitohormetic function. In summary, while TgIF1 does not appear to play a role in ATP synthase regulation under conventional growth conditions, our work uncovers its potential role in adapting to the stressors faced by T. gondii and other apicomplexans throughout their intricate life cycles.

The production of energy in the form of ATP by the mitochondrial ATP synthase must be tightly controlled. One well-conserved form of regulation is mediated via ATPase inhibitory factor 1 (IF1), which governs ATP synthase activity and gene expression patterns through a cytoprotective process known as mitohormesis. In apicomplexans, the processes regulating ATP synthase activity are not fully elucidated. Using the model apicomplexan Toxoplasma gondii , we found that knockout and overexpression of TgIF1, the structural homolog of IF1, significantly affected gene expression. Additionally, TgIF1 overexpression resulted in the formation of a stable TgIF1 oligomer that increased the presence of higher order ATP synthase oligomers. We also show that parasites lacking TgIF1 exhibit reduced mitochondrial cristae density, and that while TgIF1 levels do not affect growth in conventional culture conditions, they are crucial for parasite survival under hypoxia. Interestingly, TgIF1 overexpression enhances recovery from oxidative stress, suggesting a mitohormetic function. In summary, while TgIF1 does not appear to play a role in metabolic regulation under conventional growth conditions, our work highlights its importance for adapting to stressors faced by T. gondii and other apicomplexans throughout their intricate life cycles.