Uncovering the Role of 5'-Nucleotidases in Pyrimidine Metabolism in Trypanosoma brucei

The protozoal parasite Trypanosoma brucei causes the disease African trypanosomiasis in humans and nagana in cattle. Fifty-five million people in subtropical countries are at risk for contracting T. brucei through the tsetse fly vector, and infection is typically fatal if untreated. While recent interventions have reduced disease prevalence, current therapies are toxic or fail to target the substantial animal reservoir. A better understanding of parasite metabolism is necessary to address these issues in treatment and vector control. T. brucei generates nucleic acids and nucleotide sugars through two pathways: pyrimidine salvage or de novo synthesis using glutamine. However, T. brucei lacks several key pyrimidine transporters and enzymes found in mammals, creating an area of parasite vulnerability, especially in the ability to maintain balance between nucleotide pools. Previous work in our lab discovered that thymidine kinase (TK) is necessary to balance the activity of 5'-nucleotidases. I have characterized three proteins with 5'-nucleotidase activity in T. brucei belonging to the histidine-aspartate (HD) and haloacid dehalogenase (HAD) protein families. I found that HD 5'-nucleotidase (5'-HDNT) preferentially converts pyrimidine mononucleotides to nucleosides and was essential for parasite growth, while neither of the HAD enzymes was essential. Parasite viability was restored by expression of the human enzyme dCMP deaminase, which provides an alternate conversion path between cytosine and uracil pools. Metabolomics analysis revealed depletion of de novo pathway intermediates, including glutamine, UDP-sugars, and CDP-phospholipids, contributing to parasite death. Isotope uptake assays showed deficiency in glutamine uptake, and proteomics analysis identified two downregulated amino acid transporters. Finally, I used a PCR-based assay to study DNA strand breaks in cells lacking 5'-HDNT, and DNA damage was visualized using a comet assay. These results support the critical role of 5'-HDNT in facilitating interconversion between pyrimidine nucleotides and nucleosides, especially C and U. Disruption of this cycle leads to dNTP imbalance and DNA damage, which signals the downregulation of glutamine uptake. Through the study of 5'-HDNT, I have unmasked a potential cell death pathway that goes beyond de novo and salvage pathways to implicate cellular metabolism in T. brucei.

Author dc:creator

• Deng, Yue Wen

Contributors dc:contributor

• Tu, Benjamin
• Liszczak, Glen
• Wetzel, Dawn
• Phillips, Margaret A.

• Pyrimidines
• Trypanosoma brucei brucei
• Trypanosomiasis, African
• Nucleotidases

Language dc:language

en

Identifier

1535537211

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/10706