Suitability of methods for Plasmodium falciparum cultivation in atmospheric air

Crispim, Marcell; Verdaguer, Ignasi Bofill; Silva, Sofia Ferreira; Katzin, Alejandro M.

doi:10.1590/0074-02760210331

Cited by 3 publications

References 32 publications

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Beyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites

Katzin

Crispim

Verdaguer

et al. 2023

Preprint

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A promising treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation, by competition. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional gene knockouts were created to investigate the biological importance of the FOH/GGOH salvage pathway. The knockout parasites were viable but more susceptible to fosmidomycin, and their sensitivity to MEP inhibitors could not be rescued by the addition of prenols. Moreover, the knockout parasites lost their ability to use prenols for protein prenylation. These results demonstrate that FOH/GGOH salvage is an additional source of isoprenoids by malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme whose inhibition may potentiate the antimalarial efficacy of drugs that affect isoprenoid metabolism.

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Beyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites

Katzin

Crispim

Verdaguer

et al. 2023

Preprint

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Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites

Crispim,

Verdaguer,

Hernández

et al. 2024

PLoS Pathog

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A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.

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Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum

Basco

2023

Pathogens

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Successfully developed in 1976, the continuous in vitro culture of Plasmodium falciparum has many applications in the field of malaria research. It has become an important experimental model that directly uses a human pathogen responsible for a high prevalence of morbidity and mortality in many parts of the world and is a major source of biological material for immunological, biochemical, molecular, and pharmacological studies. Until present, the basic techniques described by Trager and Jensen and Haynes et al. remain unchanged in many malaria research laboratories. Nonetheless, different factors, including culture media, buffers, serum substitutes and supplements, sources of erythrocytes, and conditions of incubation (especially oxygen concentration), have been modified by different investigators to adapt the original technique in their laboratories or enhance the in vitro growth of the parasites. The possible effects and benefits of these modifications for the continuous cultivation of asexual intraerythrocytic stages of P. falciparum, as well as future challenges in developing a serum-free cultivation system and axenic cultures, are discussed.

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Suitability of methods for Plasmodium falciparum cultivation in atmospheric air

Cited by 3 publications

References 32 publications

Beyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites

Beyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites

Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites

Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum

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