Determination of Chemical Inhibitor Efficiency against Intracellular &lt;em&gt;Toxoplasma Gondii&lt;/em&gt; Growth Using a Luciferase-Based Growth Assay

Key, Melanie; Bergmann, Amy; Micchelli, Chiara; Thornton, Justin A.; Millard, Sophie; Dou, Zhicheng

doi:10.3791/60985-v

Cited by 2 publications

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“…After allowing the parasites to invade the host cells for a 4-hour duration, the culture medium was replaced with fresh phenol-red free D10% to eliminate any non-invaded parasites. Bioluminescence signals were then recorded at 24-hr intervals over a total period of 96 hrs, following established protocols (10,38). The average signal readings at each time point were divided by the average readings at 4 hrs to calculate the fold change of intracellular growth for each strain.…”

Section: Luciferase-based Growth Assaymentioning

confidence: 99%

Toxoplasma gondiiharbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein

Key,

Baptista,

Bergmann

et al. 2023

Preprint

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Toxoplasma gondiiis an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. Duringin vivoinfection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements.Toxoplasmacande novobiosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog inToxoplasma. The mutantToxoplasmalacking CPOX displays significantly reduced growth, implying that TgCPDH potentially functions as an alternative enzyme to perform the same reaction as CPOX under low oxygen conditions. In this study, we demonstrated that TgCPDH exhibits coproporphyrinogen dehydrogenase activity by complementing it in a heme synthesis-deficientSalmonellamutant. Additionally, we observed an increase in TgCPDH expression inToxoplasmawhen it grew under hypoxic conditions. However, deletingTgCPDHin both wildtype and heme-deficient parasites did not alter their intracellular growth under both ambient and low oxygen conditions. This research marks the first report of a coproporphyrinogen dehydrogenase-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings suggest that it does not directly affect intracellular infection or the pathogenesis ofToxoplasmaparasites.IMPORTANCEToxoplasma gondiiis a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its lifecycle, these parasites must adapt to varying environmental conditions, including situations with low oxygen levels. Our research, in conjunction with studies conducted by other laboratories, has revealed thatToxoplasmaprimarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase, suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, coproporphyrinogen dehydrogenase has only been identified in some bacteria for heme biosynthesis. Our study discovered thatToxoplasmaharbors a functional enzyme displaying coproporphyrinogen dehydrogenase activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

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Section: Luciferase-based Growth Assaymentioning

confidence: 99%

Toxoplasma gondiiharbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein

Key,

Baptista,

Bergmann

et al. 2023

Preprint

Self Cite

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“…Instead of re ecting parasite viability, they only re ect parasite numbers [9]. Furthermore, radioactive isotopes have potential health hazards and safety issues [10].…”

Section: Introductionmentioning

confidence: 99%

Construction of luciferase-expressing Neospora caninum and drug screening

Wang,

Xue,

Pei

et al. 2023

Preprint

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Background: Neospora caninum is an apicomplexan parasite which is particularly responsible for abortions in cattle and neuromuscular disease in dogs. New therapeutics are urgently needed to control Neosporosis due to the limited effectiveness of currently available drugs. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. Methods: The CRISPR/Cas9 was used to construct the luciferase-expressing N. caninum (Nc1-Luc). After testing the luciferase expression and phenotype of Nc1-Luc strains, we determined the drug sensitivity of Nc1-Luc strains by treating them with known positive or negative drugs and half-maximal inhibitory concentration (IC50) calculation. Then the selective pan-RAF inhibitor TAK-632 was evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo pharmacodynamic studies. Results: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parent strains Nc1, and Nc1-Luc strains can be used to determine IC50 for anti-N. caninum drugs. Using Nc1-Luc strains, TAK-632 showed promising activities against N. caninum, with an IC50 of 0.6131 mM and a selectivity index (SI) of 62.53. In vitro studies showed that TAK-632 inhibited invasion, proliferation and divison of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burdens in the brain. Conclusions: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.

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Determination of Chemical Inhibitor Efficiency against Intracellular <em>Toxoplasma Gondii</em> Growth Using a Luciferase-Based Growth Assay

Cited by 2 publications

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Toxoplasma gondiiharbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein

Toxoplasma gondiiharbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein

Construction of luciferase-expressing Neospora caninum and drug screening

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