Date of Award
Doctor of Philosophy (PhD)
Genetics and Biochemistry
Cheryl Ingram-Smith, Committee Chair
James C. Morris
Kerry S. Smith
Entamoeba histolytica is a protozoan parasite that causes amoebic colitis and liver abscess in approximately 90 million people each year, resulting in 50,000-100,000 fatalities. Even though Entamoeba poses a significant public health problem worldwide, research dedicated to understanding the biology of this unique protozoan has been limited. This amitochondriate parasite lacks many essential biosynthesis pathways including the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. As a result, substrate level phosphorylation plays a necessary role in ATP production. Unlike the standard glycolytic pathway, E. histolytica glycolysis requires pyrophosphate (PPi) by replacing ATP - dependent phosphofructokinase and pyruvate kinase with PPi - dependent phosphofructokinase and phosphate pyruvate dikinase. E. histolytica infects and colonizes the human colon where glucose is limited and short chain fatty acids (acetate, propionate, and butyrate) are plentiful. Acetate is also a major end product that is excreted when E. histolytica is grown axenically on glucose. Acetate has been demonstrated to act as carbon and energy source for cellular growth in other organisms, acetogenesis can regenerate NAD+, recycle coenzyme A, and produce ATP when the TCA cycle or oxidative phosphorylation does not operate or when the carbon flux into the cell exceed its capacity. In E. histolytica, acetate can be generated by acetate kinase (ACK) and ADP - forming acetyl - CoA synthetase (ACD). ACK converts acetyl phosphate + orthophosphate (Pi) to acetate + PPi. Previous biochemical and kinetic characterization of recombinant ACK showed that it strongly prefers the acetate/PPi-forming direction. We hypothesized that ACK may function to supply PPi for the PPi oriented glycolytic pathway in E. histolytica. Recombinant ACD displayed high activity in both directions of the reaction to convert acetyl-CoA + orthophosphate + ADP to acetate + ATP + CoA. ACD may function to extend the glycolytic pathway to increase ATP production by 40% per molecule of glucose, or in the alternative direction to convert acetate to acetyl-CoA to meet the cell's metabolic needs. Using reverse genetics, an Ehacd silenced strain displayed a growth defect in normal high glucose media, while Ehack silenced cells showed enhance growth in medium without added tryptone and glucose. The presence of acetate and butyrate showed no effect on E. histolytica growth in the absence of glucose regardless of ACK or ACD activity. The presence of propionate, however, improved E. histolytica growth and impaired growth of the Ehacd silenced strain implicated ACD as the cause of this improvement. Our data suggest ACD plays a role in increasing ATP production during growth on glucose and utilization of propionate as a growth substrate. Our data do not support the previously hypothesized role for ACK but instead suggest it possesses a novel function. The basis for E. histolytica ACK's divergence from all other ACKs in phosphoryl substrate utilization was also explored. Currently, E. histolytica ACK is the only known ACK that uses pyrophosphate (PPi) or inorganic phosphate (Pi) as the phosphoryl donor or acceptor. All other known ACKs utilize ATP or ADP. In silico structural comparison and modeling of E. histolytica ACK against other ACKs identified structural differences that could affect substrate binding and selection. ACK variants were generated to test these predictions. Inhibition and structural activity relationship studies revealed an occlusion in the ADENOSINE motif of E. histolytica ACK reduced ATP and ADP binding affinity. However, alterations to alleviate the constriction did not confer activity with ATP or ADP. Our results suggest controlling access of the adenosine pocket influences phosphoryl substrate binding but is not the sole determinant of enzyme activity.
Dang, Thanh, "Acetate Metabolism: The physiological role of ADP-forming acetyl-CoA synthetase and acetate kinase in Entamoeba histolytica" (2017). All Dissertations. 1976.