Malaria parasites utilize pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage
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Evaluation Summary:
This work will be of interest to researchers seeking new ways to target malaria parasites. The work provides insight into the energy requirements of parasites during the first day of their two-day life cycle, a period during which they are significantly resistant to a wide variety of antimalarial drugs, and identifies an essential enzyme that could be targeted in early-stage parasites. The study shows this protein is necessary for normal development and growth of parasites in red blood cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
The malaria parasite relies on anaerobic glycolysis for energy supply when growing inside RBCs as its mitochondrion does not produce ATP. The ring stage lasts ∼ 22 hours and is traditionally thought to be metabolically quiescent. However, recent studies show that the ring stage is active for several energy-costly processes including gene transcription/translation, protein export, and movement inside the RBC. It has remained unclear if a low glycolytic flux can meet the energy demand of the ring stage. Here we show that the metabolic by-product, pyrophosphate, is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to pump protons across the parasite plasma membrane to maintain the membrane potential and cytosolic pH. Conditional deletion of PfVP1 leads to delayed ring stage development and a complete blockage of the ring-to-trophozoite transition, which can be partially rescued by Arabidopsis thaliana vacuolar pyrophosphatase 1, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae . Proton-pumping pyrophosphatases are absent in humans, which highlights the possibility of developing highly selective VP1 inhibitors against the malaria parasite.
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Author Response
Reviewer #1 (Public Review):
Proton pumps are necessary to set up gradients necessary for myriad biological processes. The malaria-causing parasite Plasmodium falciparum, uses two main pathways to achieve this, the vacuolar ATPase (V-type ATPase) and a more ancient vacuolar pyrophosphatase (PfPV1). The proton motive force set up across the parasite plasma membrane holds particular significance since it is necessary for transport of nutrients and waste products into and out of the cell. Motivated by the observation that the V-type ATPase is no expressed until several hours after the parasite has entered host cells, the present study examines the function of PfVP1. The authors demonstrate PfVP1 depletion blocks the early development of Plasmodium-specifically the transition from the ring to the trophozoite stage-and …
Author Response
Reviewer #1 (Public Review):
Proton pumps are necessary to set up gradients necessary for myriad biological processes. The malaria-causing parasite Plasmodium falciparum, uses two main pathways to achieve this, the vacuolar ATPase (V-type ATPase) and a more ancient vacuolar pyrophosphatase (PfPV1). The proton motive force set up across the parasite plasma membrane holds particular significance since it is necessary for transport of nutrients and waste products into and out of the cell. Motivated by the observation that the V-type ATPase is no expressed until several hours after the parasite has entered host cells, the present study examines the function of PfVP1. The authors demonstrate PfVP1 depletion blocks the early development of Plasmodium-specifically the transition from the ring to the trophozoite stage-and this is associated with changes to cellular pH and pyrophosphate levels, consistent with predicted functions. Complementation of the conditional knockdown suggests that pyrophosphatase activity alone is not sufficient to overcome the loss of PfVP1. Overall, data supporting a critical role for PfVP1 in parasite energetics is compelling. However, the lack of several key controls somewhat weakens the conclusions of the paper when it comes to complementation of the mutants and description of which activities are needed for parasite survival. Because the proximal activities of the enzyme ATP generation and the proton motive force are incompletely examined, some of the major conclusions from the study remain speculative.
We thank the reviewer for these constructive comments. We are grateful to the reviewer for his/her recognition of the significance of our study. The major discovery of this manuscript is to uncover PfVP1’s essential role in the early-stage development of the 48h asexual lifecycle in P. falciparum. Our data suggest PPi is an energy source when ATP level is likely low in the ring stage malaria parasite and its transition to the trophozoite stage. We have performed additional experiments and tried the best to address each comment from the reviewer.
Reviewer #2 (Public Review):
In this work, the authors characterize a proton pump from the parasite Plasmodium falciparum that uses pyrophosphate as an energy source (PfVP1).
They looked at the expression and localization of the pump in different stages of the parasite and determined that it localizes to the plasma membrane and it is highly expressed in the ring stage. They studied the biochemical function by expressing the gene in Saccharomyces followed by isolation of vesicles and measurements of proton transport and PPi hydrolysis. They also characterized the biological role of PfVP1 in the parasites by creating conditional mutants that express PfVP1 when cultured in the presence of anhydrotetracycline (ATC). Upon removal of ATC the expression of PfVP1 is downregulated, which impacted growth and transition to the trophozoite stage. Mutant parasites struggled to progress through the ring state and failed to become trophozoites in the second intraerythrocytic cycle. They complemented the mutants with the yeast inorganic pyrophosphatase gene and the Arabidopsis vacuolar pyrophosphatase.
We thank the reviewer for positive and constructive comments. We have seriously worked on every comment raised by the reviewer. We have tried the best to perform additional experiments.
Reviewer #3 (Public Review):
Solebo and coworkers investigated the energy requirements of blood-stage malaria parasites (the stage of infection that causes symptoms). Traditionally, parasites were thought to be somewhat quiescent during the first half of their life cycle in red blood cells and become metabolically active as they prepare for replication. Consequently, antimalarial drugs are more active against parasites during the second half of their life cycle. In this report, the authors show that the metabolic by-product pyrophosphate is an essential energy source for the development of early-stage malaria parasites and that it is consumed by a vacuolar pyrophosphatase (PfVP1). Knock down studies showed that PfVP1 is required for the development of early-stage parasites and localization studies established that it is located in the parasite plasma membrane. Characterization of PfVP1 heterologously expressed in yeast confirmed that it is a pyrophosphate hydrolyzing proton pump. Consequently, loss of PfVP1 in early-stage parasites results in reduced pyrophosphate consumption and a reduction in pH (accumulation of protons). The authors further show that a similar vacuolar pyrophosphatase from Arabidopsis thaliana can complement the loss of the parasite ortholog, but a general pyrophosphatase enzyme cannot. Consistent with this result, mutations designed to inactivate either the pyrophosphatase activity or the proton-pumping activity demonstrated that both activities are essential for the development and survival of early-stage parasites.
The conclusions of this paper are firmly supported by data, often from more than one type of experimental approach. The conclusions provide fundamental information about the stage of parasite development that has been hard to target with antimalarial drugs. The most energy-consuming process in a cell is the maintenance of membrane potential and in malaria parasites, it is known that proton pumps (rather than sodium pumps) are responsible for this process. Although PfVP1 was previously reported to be located internally in an organelle of the parasite, the data presented in this report clearly define its location on the plasma membrane and its essential role in maintaining the membrane potential. PfVP1 inhibitors could preferentially target early stage malaria parasites and the current results support efforts to find these inhibitors. Perhaps the most exciting aspect of this work is the potential to act synergistically and enhance the effect of current antimalarial drugs on early stage parasites. In this vein, the authors tested four antimalarial compounds in conjunction with knockdown of PfVP1 to determine whether there was enhanced activity. These experiments were not conducted in a systematic way and this is perhaps the only weakness of the paper.
We thank the reviewer for positive, constructive, and encouraging comments. We really appreciate that. We are also very excited about our discovery that a non-ATP driven proton pump plays essential roles in the early-stage development of the asexual lifecycle. Our data suggest PPi is an energy source in the malaria parasite P. falciparum.
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Evaluation Summary:
This work will be of interest to researchers seeking new ways to target malaria parasites. The work provides insight into the energy requirements of parasites during the first day of their two-day life cycle, a period during which they are significantly resistant to a wide variety of antimalarial drugs, and identifies an essential enzyme that could be targeted in early-stage parasites. The study shows this protein is necessary for normal development and growth of parasites in red blood cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Proton pumps are necessary to set up gradients necessary for myriad biological processes. The malaria-causing parasite Plasmodium falciparum, uses two main pathways to achieve this, the vacuolar ATPase (V-type ATPase) and a more ancient vacuolar pyrophosphatase (PfPV1). The proton motive force set up across the parasite plasma membrane holds particular significance since it is necessary for transport of nutrients and waste products into and out of the cell. Motivated by the observation that the V-type ATPase is no expressed until several hours after the parasite has entered host cells, the present study examines the function of PfPV1. The authors demonstrate PfPV1 depletion blocks the early development of Plasmodium-specifically the transition from the ring to the trophozoite stage-and this is associated …
Reviewer #1 (Public Review):
Proton pumps are necessary to set up gradients necessary for myriad biological processes. The malaria-causing parasite Plasmodium falciparum, uses two main pathways to achieve this, the vacuolar ATPase (V-type ATPase) and a more ancient vacuolar pyrophosphatase (PfPV1). The proton motive force set up across the parasite plasma membrane holds particular significance since it is necessary for transport of nutrients and waste products into and out of the cell. Motivated by the observation that the V-type ATPase is no expressed until several hours after the parasite has entered host cells, the present study examines the function of PfPV1. The authors demonstrate PfPV1 depletion blocks the early development of Plasmodium-specifically the transition from the ring to the trophozoite stage-and this is associated with changes to cellular pH and pyrophosphate levels, consistent with predicted functions. Complementation of the conditional knockdown suggests that pyrophosphatase activity alone is not sufficient to overcome the loss of PfPV. Overall, data supporting a critical role for PfPV1 in parasite energetics is compelling. However, the lack of several key controls somewhat weakens the conclusions of the paper when it comes to complementation of the mutants and description of which activities are needed for parasite survival. Because the proximal activities of the enzyme ATP generation and the proton motive force are incompletely examined, some of the major conclusions from the study remain speculative.
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Reviewer #2 (Public Review):
In this work, the authors characterize a proton pump from the parasite Plasmodium falciparum that uses pyrophosphate as an energy source (PfVP1).
They looked at the expression and localization of the pump in different stages of the parasite and determined that it localizes to the plasma membrane and it is highly expressed in the ring stage.
They studied the biochemical function by expressing the gene in Saccharomyces followed by isolation of vesicles and measurements of proton transport and PPi hydrolysis.
They also characterized the biological role of PfVP1 in the parasites by creating conditional mutants that express PfVP1 when cultured in the presence of anhydrotetracycline (ATC). Upon removal of ATC the expression of PfVP1 is downregulated, which impacted growth and transition to the trophozoite stage. …
Reviewer #2 (Public Review):
In this work, the authors characterize a proton pump from the parasite Plasmodium falciparum that uses pyrophosphate as an energy source (PfVP1).
They looked at the expression and localization of the pump in different stages of the parasite and determined that it localizes to the plasma membrane and it is highly expressed in the ring stage.
They studied the biochemical function by expressing the gene in Saccharomyces followed by isolation of vesicles and measurements of proton transport and PPi hydrolysis.
They also characterized the biological role of PfVP1 in the parasites by creating conditional mutants that express PfVP1 when cultured in the presence of anhydrotetracycline (ATC). Upon removal of ATC the expression of PfVP1 is downregulated, which impacted growth and transition to the trophozoite stage. Mutant parasites struggled to progress through the ring state and failed to become trophozoites in the second intraerythrocytic cycle. They complemented the mutants with the yeast inorganic pyrophosphatase gene and the arabidopsis vacuolar pyrophosphatase.
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Reviewer #3 (Public Review):
Solebo and coworkers investigated the energy requirements of blood-stage malaria parasites (the stage of infection that causes symptoms). Traditionally, parasites were thought to be somewhat quiescent during the first half of their life cycle in red blood cells and become metabolically active as they prepare for replication. Consequently, antimalarial drugs are more active against parasites during the second half of their life cycle. In this report, the authors show that the metabolic by-product pyrophosphate is an essential energy source for the development of early-stage malaria parasites and that it is consumed by a vacuolar pyrophosphatase (PfVP1). Knock down studies showed that PfVP1 is required for the development of early-stage parasites and localization studies established that it is located in the …
Reviewer #3 (Public Review):
Solebo and coworkers investigated the energy requirements of blood-stage malaria parasites (the stage of infection that causes symptoms). Traditionally, parasites were thought to be somewhat quiescent during the first half of their life cycle in red blood cells and become metabolically active as they prepare for replication. Consequently, antimalarial drugs are more active against parasites during the second half of their life cycle. In this report, the authors show that the metabolic by-product pyrophosphate is an essential energy source for the development of early-stage malaria parasites and that it is consumed by a vacuolar pyrophosphatase (PfVP1). Knock down studies showed that PfVP1 is required for the development of early-stage parasites and localization studies established that it is located in the parasite plasma membrane. Characterization of PfVP1 heterologously expressed in yeast confirmed that it is a pyrophosphate hydrolyzing proton pump. Consequently, loss of PfVP1 in early-stage parasites results in reduced pyrophosphate consumption and a reduction in pH (accumulation of protons). The authors further show that a similar vacuolar pyrophosphatase from Arabidopsis thaliana can complement the loss of the parasite ortholog, but a general pyrophosphatase enzyme cannot. Consistent with this result, mutations designed to inactivate either the pyrophosphatase activity or the proton-pumping activity demonstrated that both activities are essential for the development and survival of early-stage parasites.
The conclusions of this paper are firmly supported by data, often from more than one type of experimental approach. The conclusions provide fundamental information about the stage of parasite development that has been hard to target with antimalarial drugs. The most energy-consuming process in a cell is the maintenance of membrane potential and in malaria parasites, it is known that proton pumps (rather than sodium pumps) are responsible for this process. Although PfVP1 was previously reported to be located internally in an organelle of the parasite, the data presented in this report clearly define its location on the plasma membrane and its essential role in maintaining the membrane potential. PfVP1 inhibitors could preferentially target early stage malaria parasites and the current results support efforts to find these inhibitors. Perhaps the most exciting aspect of this work is the potential to act synergistically and enhance the effect of current antimalarial drugs on early stage parasites. In this vein, the authors tested four antimalarial compounds in conjunction with knockdown of PfVP1 to determine whether there was enhanced activity. These experiments were not conducted in a systematic way and this is perhaps the only weakness of the paper.
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