Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure

Kwan

et al. 2023

Malar J

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Background Spiroindolone and pyrazoleamide antimalarial compounds target Plasmodium falciparum P-type ATPase (PfATP4) and induce disruption of intracellular Na+ homeostasis. Recently, a PfATP4 mutation was discovered that confers resistance to a pyrazoleamide while increasing sensitivity to a spiroindolone. Transcriptomic and metabolic adaptations that underlie this seemingly contradictory response of P. falciparum to sublethal concentrations of each compound were examined to understand the different cellular accommodation to PfATP4 disruptions. Methods A genetically engineered P. falciparum Dd2 strain (Dd2A211V) carrying an Ala211Val (A211V) mutation in PfATP4 was used to identify metabolic adaptations associated with the mutation that results in decreased sensitivity to PA21A092 (a pyrazoleamide) and increased sensitivity to KAE609 (a spiroindolone). First, sublethal doses of PA21A092 and KAE609 causing substantial reduction (30–70%) in Dd2A211V parasite replication were identified. Then, at this sublethal dose of PA21A092 (or KAE609), metabolomic and transcriptomic data were collected during the first intraerythrocytic developmental cycle. Finally, the time-resolved data were integrated with a whole-genome metabolic network model of P. falciparum to characterize antimalarial-induced physiological adaptations. Results Sublethal treatment with PA21A092 caused significant (p < 0.001) alterations in the abundances of 91 Plasmodium gene transcripts, whereas only 21 transcripts were significantly altered due to sublethal treatment with KAE609. In the metabolomic data, a substantial alteration (≥ fourfold) in the abundances of carbohydrate metabolites in the presence of either compound was found. The estimated rates of macromolecule syntheses between the two antimalarial-treated conditions were also comparable, except for the rate of lipid synthesis. A closer examination of parasite metabolism in the presence of either compound indicated statistically significant differences in enzymatic activities associated with synthesis of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. Conclusion The results of this study suggest that malaria parasites activate protein kinases via phospholipid-dependent signalling in response to the ionic perturbation induced by the Na+ homeostasis disruptor PA21A092. Therefore, targeted disruption of phospholipid signalling in PA21A092-resistant parasites could be a means to block the emergence of resistance to PA21A092.

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Metabolic responses in blood-stage malaria parasites associated with increased and decreased sensitivity to PfATP4 inhibitors

Tewari

Kwan

et al. 2023

Malar J

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“…Parasite growth was monitored using an Attune Nxt Flow Cytometer (Thermo Fisher Scientific, MA, USA) as previously described (Swift et al, 2020; Tewari et al, 2022). For determining the growth dependence on mevalonate, cultures were seeded in the presence or absence of 50 µM mevalonate at 0.5% parasitemia and 2% hematocrit in a total volume of 250 µL, in quadruplicate for each condition.…”

Section: Methodsmentioning

confidence: 99%

ThePlasmodium falciparumapicoplast cysteine desulfurase provides sulfur for both iron sulfur cluster assembly and tRNA modification

Prigge

Swift

et al. 2022

Preprint

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Iron sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite Plasmodium falciparum. Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. By complementing the activity of the parasite MnmA and SufS with a bacterial MnmA and its cognate cysteine desulfurase, we showed that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.

“…To determine the half-maximal effective concentration (EC 50 ) of MMV030734, sorbitol-synchronized ring parasites were seeded at 3% parasitemia and 2% hematocrit in a 96-well flat bottom plate and grown in the presence of inhibitor at 37°C for 72 h. A concentration series for MMV030734 (17 pM to 3 μM) and chloroquine (487 pM to 250 nM) was tested. After 72 h, parasite growth was quantified using SYBR Green I (Invitrogen) to stain DNA and an Attune NxT Flow Cytometer as described previously (48, 49). Parasitemia from the MMV030734 and chloroquine samples were normalized to the 0.1% DMSO control.…”

Section: Methodsmentioning

confidence: 99%

“…A concentration series for MMV030734 (17 pM to 3 µM) and chloroquine (487 pM to 250 nM) was tested. After 72 h, parasite growth was quantified using SYBR Green I (Invitrogen) to stain DNA and an Attune NxT Flow Cytometer as described previously (48,49). Parasitemia from the MMV030734 and chloroquine samples were normalized to the 0.1% DMSO control.…”

Section: Testing Pathogen Box Compounds On Plasmodium Falciparum Asex...mentioning

confidence: 99%

Screening the Pathogen Box Compounds for Activity Against Plasmodium falciparum Sporozoite Motility

Kanatani

Kanchanabhogin

et al. 2022

Preprint

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As the malaria parasite becomes resistant to every drug that we develop, identification and development of novel drug candidates is essential. Many studies have screened compounds designed to target the clinically important blood stages. However, if we are to shrink the malaria map, new drugs that block transmission of the parasite are needed. Sporozoites are the infective stage of the malaria parasite, transmitted to the mammalian host as mosquitoes probe for blood. Sporozoite motility is critical to their ability to exit the inoculation site and establish infection and drug-like compounds targeting motility are effective in blocking infection in the rodent malaria model. In this study, we established a moderate throughput motility assay for sporozoites of the human malaria parasite Plasmodium falciparum, enabling us to screen the 400 drug-like compounds from the Pathogen box provided by Medicines for Malaria Venture for their activity. Compounds exhibiting inhibitory effects on P. falciparum sporozoite motility were further assessed against transmission-blocking activity and asexual stage growth. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility at 1 μM concentration and four of these compounds also showed significant inhibition on transmission of P. falciparum gametocytes to the mosquito and of these four, three had previously been shown to have inhibitory activity on asexual blood stage parasites. Our findings provide new antimalarial drug candidates that have multi-stage activity.