Adaptation of metabolic enzyme activities of Trypanosoma brucei promastigotes to growth rate and carbon regimen

Abstract

The insect stage of Trypanosoma brucei adapted the activities of 16 metabolic enzymes to growth rate and carbon source. Cells were grown in chemostats with glucose, rate limiting or in excess, or high concentrations of proline as carbon and energy sources. At each steady state, samples were collected for measurements of substrate and end product concentrations, cellular parameters, and enzyme activities. Correlation coefficients were calculated for all parameters and used to analyze the data set. Rates of substrate consumption and end product formation increased with increasing growth rate. Acetate and succinate were the major nonvolatile end products, but measurable quantities of alanine were also produced. More acetate than succinate was formed during growth on glucose, but growth on proline yielded an equimolar ratio. Growth rate barely affected the relative amounts of end products formed. The end products accounted for the glucose consumed during glucose-limited growth and growth at high rates on excess glucose. A discrepancy, indicating production of CO 2 , occurred during slow growth on excess glucose and, even more pronounced, in cells growing on proline. The activities of the metabolic enzymes varied by factors of 2 to 40. There was no single enzyme that correlated with consumption of substrate and/or end product formation in all cases. A group of enzymes whose activities rigorously covaried could also not be identified. These findings indicate that T. brucei adapted the activities of each of the metabolic enzymes studied separately. The results of this complex manner of adaptation were more or less constant ratios of the end products and a very efficient energy metabolism.The carbohydrate metabolism of Trypanosoma brucei, causative agent of African sleeping sickness and of nagana in cattle, has been explored in great detail in order to identify targets for chemotherapy. In the mammalian host, it relies solely on glycolysis for ATP production, converting glucose to pyruvate (17). The procyclic form, corresponding to the insect stage, metabolizes glucose to CO 2 , acetate, succinate, and alanine (7,17). Seven enzymes of the glycolytic pathway are located in the glycosome, an organelle that is unique to the Kinetoplastida (17, 18). In the bloodstream form, the mitochondrion is involved only in maintaining the redox balance. The procyclic form has a more developed mitochondrion containing an active electron transport chain (16,33). Despite this, the metabolism remains primarily fermentative even in the presence of oxygen.The procyclic form grows even better on amino acids such as proline or glutamic acid than on glucose (8, 9). These carbon sources are abundantly available in the midgut of the tsetse fly. In most organisms, proline is metabolized in mitochondria, where it enters the tricarboxylic acid cycle as ␣-ketoglutarate after sequential conversion to pyrroline-5-carboxylate, glutamate ␥-semialdehyde, and glutamate (24). This is assumed also to be the pathway in T. brucei (7); thus, CO 2 can be expecte...

Glucose is toxic to glycosome-deficient trypanosomes

“…A decrease in glycolysis is unlikely to kill procyclic forms, as these parasites easily survive in the absence of glucose. Although the abundances of the glycolytic enzymes are much reduced in procyclic forms, the glycolytic capacity of procyclic forms remains relatively high (44,45). Glucose metabolism could be toxic in procyclic forms in the absence of the regulation imposed by glycosomal compartmentation.…”

The Adenosine Analog Tubercidin Inhibits Glycolysis in Trypanosoma brucei as Revealed by an RNA Interference Library

“…Based on the finding that procyclic trypanosomes can shift their metabolism from a state dependent upon glycolysis (in high glucose medium) to one dependent upon amino acids (in low glucose medium) (14), we propose that procyclic parasites survive without glycolysis only if they are given time to adapt to a glucose-free environment, a process possibly involving up-regulation of amino acid metabolism or transport. RNAi silencing of the glucose transporter or hexokinase probably occurs over several days, giving time for this adaptation.…”

“…Therefore, we conducted an experiment based on the finding that procyclic trypanosomes can suppress glycolysis and rely instead on amino acids as a carbon and energy source (14). Parental cells were adapted to amino acid metabolism by growth on low glucose medium for 7 days followed by growth in minimal glucose medium.…”

Intracellular Positioning of Isoforms Explains an Unusually Large Adenylate Kinase Gene Family in the Parasite Trypanosoma brucei

“…brucei is transmitted between mammalian hosts by its insect vector (Glossina sp., tsetse fly). Procyclic trypanosomes (found in the tsetse fly midgut) have an aerobic metabolism and use a variety of carbon sources (12)(13)(14), and alternative oxidase and cytochrome c oxidase constitute the two terminal enzymes of an essential branched respiratory chain (14,15). Although oxidative phosphorylation is an important facet of procyclic metabolism, pyruvate kinase and an acetate:succinate CoAtransferase/succinyl-CoA synthetase cycle catalyze important substrate phosphorylations.…”

“…Although oxidative phosphorylation is an important facet of procyclic metabolism, pyruvate kinase and an acetate:succinate CoAtransferase/succinyl-CoA synthetase cycle catalyze important substrate phosphorylations. The cytoplasmic phosphotransfer reaction catalyzed by the former enzyme appears to be essential, except perhaps under conditions where increased substrate availability can result in increased mitochondrial amino acid (proline) metabolism (12,13,16). Acetate:succinate CoA transferase is restricted in its evolutionary distribution to trypanosomatid mitochondria, anaerobic helminth mitochondria, and protist hydrogenosomes; it catalyzes formation of acetate from acetyl-CoA.…”