Title page for ETD etd-05162012-185144

Type of Document Dissertation
Author Denloye, Titilola Ifeoma
Author's Email Address titid@vt.edu
URN etd-05162012-185144
Title Characterization of a glycerophosphodiester phosphodiesterase in the human malaria parasite Plasmodium falciparum
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Klemba, Michael W. Committee Chair
Dean, Dennis R. Committee Member
Helm, Richard Frederick Committee Member
Hernick, Marcy Committee Member
Larson, Timothy J. Committee Member
  • choline
  • lipid metabolism
  • fatty acid
  • malaria
  • glycerophosphodiester phosphodiesterase
  • Plasmodium falciparum
Date of Defense 2012-04-25
Availability unrestricted
Active lipid metabolism is a key process required for the intra-erythrocytic development of the malaria parasite, Plasmodium falciparum. Enzymes that hydrolyze host-derived lipids play key roles in parasite growth, virulence, differentiation, cell-signaling and hemozoin formation. Therefore, investigating enzymes involved in lipid degradation could uncover novel drug targets. We have identified in P. falciparum, a glycerophosphodiester phosphodiesterase (PfGDPD), involved in the downstream pathway of phosphatidylcholine degradation. PfGDPD hydrolyzes deacylated phospholipids, glycerophosphodiesters to glycerol-3-phosphate and choline. In this study, we have characterized PfGDPD using bioinformatics, biochemical and genetic approaches. Knockout experiments showed a requirement for PfGDPD for parasite survival. Sequence analysis revealed PfGDPD possesses the unique GDPD insertion domain sharing a cluster of conserved residues present in other GDPD homologues. We generated yellow fluorescent fusion proteins that revealed a complex distribution of PfGDPD within the parasite cytosol, parasitophorous vacuole and food vacuole. To gain insight into the role of PfGDPD, sub-cellular localization was modulated and resulted in a shift in protein distribution, which elicited no growth phenotype. Kinetic analyses suggest PfGDPD activity is Mg2+ dependent and catalytically efficient at the neutral pH environment of the parasitophorous vacuole. Next, our aim was to determine the upstream pathway that provides deacylated glycerophosphodiesters as substrate for PfGDPD. We identified via bioinformatics, a P. falciparum lysophospholipase (PfLPL1) that directly generates the substrate. Knockout clones were generated and genotyped by Southern and PCR analysis. The effects of PfLPL1 knockouts on parasite fitness were studied, and the results showed that PfLPL1was not required for parasite survival and proliferation.
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