Date of Award
Doctor of Philosophy (PhD)
Genetics and Biochemistry
Entamoeba histolytica is an amoebic parasite that infects an estimated 90 million people worldwide and causes approximately 100,000 deaths per year. As the causative agent of amoebic dysentery, this food- and water-borne pathogen represents a significant public health burden worldwide, particularly in areas with poor sanitation. While treatments for amoebiasis exist, they are often limited in their effectiveness. Thus, efforts to better understand the biology and physiology of this organism are vital to the development of novel treatments for this disease.
E. histolytica lacks the enzymes for many common metabolic pathways such as the citric acid cycle and oxidative phosphorylation and relies primarily on a pyrophosphate (PPi)-dependent glycolytic pathway for ATP production. This microaerophilic organism produces ethanol and acetate as the primary products of glucose metabolism. These metabolites are produced in an extended glycolytic pathway in which pyruvate is converted to acetyl-CoA, which is then converted to ethanol and acetate by the bifunctional alcohol/aldehyde dehydrogenase (ADHE) and ADP-forming acetyl-CoA synthetase (ACD), respectively.
The acetate kinase (ACK) of E. histolytica catalyzes the conversion of acetyl phosphate into acetate and acts preferentially in the acetate-forming direction. Uniquely, this enzyme produces PPi instead of ATP, raising the possibility that it may make a substantial contribution to acetate and PPi levels. However, the source of its acetyl phosphate substrate is unknown.
Ethanol production by ADHE consumes more NADH than is produced by glycolysis, creating an imbalance. The polyol pathway has been investigated in humans for its role as an alternative glucose metabolism pathway that results in the production of fructose and NADH. This pathway consists of two reactions. In the first reaction, aldose reductase (AR) catalyzes the NADPH-dependent conversion of a sugar into its corresponding sugar alcohol. In the second reaction, sorbitol dehydrogenase (SDH) catalyzes the NAD-dependent conversion of sorbitol into fructose, generating NADH. The presence of such a pathway in E. histolytica would potentially balance the NAD+/NADH requirement and produce fructose, which would re-enter glycolysis.
We investigated aldose reductase for its role in a potential polyol pathway in E. histolytica. We biochemically characterized two recombinant AR isozymes, termed EhAR1 and EhAR3, and found them to act primarily in the sugar alcohol-forming reaction direction. These isozymes demonstrate activity with a broad range of sugar and sugar alcohol substrates, with glyceraldehyde being the primary substrate. EhAR1 and EhAR3 display different oligomeric states as a monomer and a dimer, respectively, and an aldose reductase knockdown had no effect on cells grown in various concentrations of glucose. We also attempted to detect sorbitol dehydrogenase activity in cell extracts, but the inability to do so suggests that E. histolytica lacks a functional polyol pathway. This indicates that aldose reductase serves a different metabolic role as a glyceraldehyde reductase.
To investigate the contribution that acetate kinase makes towards acetate and PPi production, we measured concentrations of several extracellular and intracellular metabolites in an ACK knockdown strain. Acetate and ethanol levels were not affected; similarly, the ACK knockdown did not affect cell growth in various glucose concentrations. However, acetyl-CoA levels and the NAD+/NADH ratio were significantly elevated. Furthermore, we demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the ACK-dependent conversion of acetaldehyde to acetyl phosphate, revealing a source of substrate for acetate kinase.
Here, we have investigated two pathways for their potential roles in glucose metabolism and maintenance of the NAD+/NADH balance needed for completion of glycolysis. We have demonstrated that ACK fulfills this role by partitioning acetyl-CoA between ethanol and acetate production in the extended glycolytic pathway. AR, on the other hand, appears not to be involved in the production of NADH but rather in the reduction of aldehydes, which may provide a protective mechanism for E. histolytica against these reactive species found in the human intestinal tract.
Angel, Matthew B., "An Investigation into the Roles of Aldose Reductase and Acetate Kinase in the Metabolism of Entamoeba histolytica" (2022). All Dissertations. 2986.