Thesis
Characterisation of vacuolar type ATPase in the malaria parasite Plasmodium falciparum
Master of Science (M.S.), Drexel University
Dec 2021
DOI:
https://doi.org/10.17918/00000682
Abstract
V-type ATPases are highly conserved multi-subunit proton pumps found in all eukaryotic organisms. They are present in the membranes of various intracellular organelles such as lysosomes, endosomes and secretory vesicles. They use ATP hydrolysis to pump protons to acidify intracellular compartments and are thus crucial for various cellular processes. A unique feature of this machinery is the reversible assembly/disassembly of its domains (V1 and Vo), regulating the pump's function in response to extracellular conditions. This machinery is evolutionarily conserved in malaria parasites; however, its biological significance has been under-appreciated. To understand the localisation, mechanism and regulation of this proton pumping machinery in Plasmodium falciparum, we utilised CRISPR/Cas9 mediated gene editing tools to tag the subunit A of V-Type ATPase (V1A) with a triple hemagglutinin (3HA) epitope. We also employed a conditional knockdown system via TetR-DOZI-aptamers to regulate expression of the tagged subunit A with a small non-toxic molecule, anhydrotetracycline (aTc). By immunofluorescence assays, we identified that V-type ATPase is expressed throughout the asexual developmental cycle in RBCs and is mainly localised on the parasite's food vacuole and plasma membrane. Using BN-PAGE and Western blot, we detected a large complex (~ 1.2 M Da) corresponding to the molecular weights of V1 and Vo domains. Moreover, we also observed many intermediate complexes with unknown identities. Upon knockdown of V1A, we detected that the parasite Food Vacuole loses its integrity and does not digest haemoglobin efficiently. Overall, in this study we have revealed the localisation and essentiality of the V-type ATPase in the asexual developmental stage of Plasmodium falciparum.
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Details
- Title
- Characterisation of vacuolar type ATPase in the malaria parasite Plasmodium falciparum
- Creators
- Neeta Vijay Shadija
- Contributors
- Hangjun Ke (Advisor)Akhil B. Vaidya (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 103 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991016054028504721