The pathogenic protist Entamoeba histolytica is estimated to be responsible for 50,000 to 100,000 deaths each year and is a significant source of morbidity and mortality due to parasitic disease in humans, particularly in developing countries. This protist is also listed by NIAID as a category B priority pathogen with bioterrorism potential due to its low infectious dose and potential for dissemination through compromised food and water supplies. E. histolytica has a simple life cycle, existing in either an infectious cyst form or as motile trophozoites which can reside in the anaerobic confines of the human colon. This microbe is a scavenger that lacks ATP- producing mitochondria and relies on glycolysis as its primary pathway for generation of ATP. The PPi- dependent glycolytic pathway of Entamoeba has an increased net generation of ATP, which would be beneficial for organisms that lack a functional citric acid cycle and oxidative phosphorylation and must rely solely on substrate level phosphorylation to generate ATP from glucose breakdown. The proposed research examines the role of acetate fermentation in E. histolytica energy metabolism and physiology. Acetate is produced during growth in axenic culture, and the two enzymes of interest, acetate kinase (Ack) and ADP- forming acetyl-CoA synthetase (Acd), are both proposed to play a role in acetate production at the end of glycolysis. Acd is proposed to provide for generation of an additional ATP at the end of glycolysis. Recombinant E. histolytica Acd (EhAcd) has been shown to function well in both directions of the reaction, suggesting that this enzyme can operate in either direction in vivo, depending on the cell's metabolic needs. Recombinant E. histolytica Ack (EhAck) has been shown to have two unusual properties: production of PPi rather than ATP, and a strong preference for the acetate-forming direction of the reaction. Ack may thus provide a mechanism for supplying additional PPi for glycolysis under certain growth conditions that require a higher flux through this pathway. The two specific aims of this proposal are (1) to investigate the enzymology of EhAcd and EhAck using a structure-function approach; and (2) to determine the physiological roles of Acd and Ack in E. histolytica through transcriptome profiling and metabolic analysis of ACK and ACD RNAi and overexpression strains. The results from this research will provide a better understanding of a key metabolic pathway for ATP generation in this important eukaryotic pathogen.

Public Health Relevance

As Entamoeba histolytica is a leading cause of death due to parasitic disease, a thorough understanding of the physiology and biochemistry of this pathogen is necessary to fully combat it. To this end, this research examines the roles of two enzymes proposed to play a role in a key pathway for generation of ATP, the primary energy molecule used in all cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory Grants (P20)
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Clemson University
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Gordhan, Heeren M; Milanes, Jillian E; Qiu, Yijian et al. (2017) A targeted delivery strategy for the development of potent trypanocides. Chem Commun (Camb) 53:8735-8738
Welter, Brenda H; Sehorn, Michael G; Temesvari, Lesly A (2017) Flow cytometric characterization of encystation in Entamoeba invadens. Mol Biochem Parasitol 218:23-27
Altamirano, Sophie; Chandrasekaran, Srikripa; Kozubowski, Lukasz (2017) Mechanisms of Cytokinesis in Basidiomycetous Yeasts. Fungal Biol Rev 31:73-87
Bauer, Sarah T; McQueeney, Kelley E; Patel, Terral et al. (2017) Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum. J Eukaryot Microbiol 64:97-105
Gordhan, Heeren M; Patrick, Stephen L; Swasy, Maria I et al. (2017) Evaluation of substituted ebselen derivatives as potential trypanocidal agents. Bioorg Med Chem Lett 27:537-541
Hendrick, Holland M; Welter, Brenda H; Hapstack, Matthew A et al. (2016) Phosphorylation of Eukaryotic Initiation Factor-2? during Stress and Encystation in Entamoeba Species. PLoS Pathog 12:e1006085
Davis, Mindy I; Patrick, Stephen L; Blanding, Walker M et al. (2016) Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity. Antimicrob Agents Chemother 60:6023-33
Kelso, Andrew A; Goodson, Steven D; Chavan, Suchitra et al. (2016) Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase. Mol Biochem Parasitol 210:71-84