Thesis
The biochemical functionality and cellular importance of the vacuolar H⁺ translocating pyrophosphatase 1, PfVP1, in Plasmodium falciparum
Master of Science (M.S.), Drexel University
Aug 2021
DOI:
https://doi.org/10.17918/00000858
Abstract
Plasmodium falciparum, the causative agent behind the most severe cases of malaria, has been found to encode vacuolar H⁺ translocating pyrophosphatases, or H⁺ -PPases. These evolutionarily ancient proton pumps have been found within prokaryotes and two of the four kingdoms of Eukarya, with metazoa and fungi being the exceptions. The potassium-dependent H⁺ -PPase of P. falciparum, known as PfVP1, has recently been predicted to be essential and this combined with the absence of any homologs in animals have highlighted it as a possible drug target. Here, we have characterized several important aspects of PfVP1 including its subcellular localization, time of action, and mechanism of function using comprehensive genetic and biochemical approaches. We show that PfVP1 is highly expressed and primarily localized to the parasite plasma membrane, with some additional intracellular localization that does not coincide with the food vacuole as previously thought. Importantly, we discovered that PfVP1 is essential for the ring stage development and the PfVP1 knockdown parasite was completely arrested at the ring stage to trophozoite stage transition. We expressed and purified PfVP1 from the heterologous expression system in Saccharomyces cerevisiae and confirmed its pyrophosphate hydrolysis and proton pumping activities in vitro. To further verify PfVP1's mechanism of function in vivo, we complemented the knockdown parasite with pyrophosphatases that have either a sole function of PPi hydrolysis (S. cerevisiae soluble pyrophosphatase) or dual functionality of PPi hydrolysis and proton pumping, AVP1 (Arabidopsis thaliana VP1). These genetic studies revealed the dual functionality PfVP1 is required. Together, our data suggest PfVP1 is the major proton pump to maintain the plasma membrane potential during the ring stage development. Such a bioenergetically favorable proton pump is critical for the ring stage likely because this young malaria parasite needs to utilize alternative energy sources during the time it takes for nutrient acquisition routes to become fully established across the host cell membrane.
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Details
- Title
- The biochemical functionality and cellular importance of the vacuolar H⁺ translocating pyrophosphatase 1, PfVP1, in Plasmodium falciparum
- Creators
- Omobukola R. Solebo
- Contributors
- Hangjun Ke (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 102 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991015411988804721