WITHDRAWN: Knockout of HD domain 5’-nucleotidase inT. bruceitriggers cell death linked to DNA damage and downregulation of glutamine uptake

Abstract

African trypanosomiasis is a fatal vector-borne disease caused by the protozoal parasite Trypanosoma brucei. This parasite encodes both de novo and salvage pathways for pyrimidine nucleotide biosynthesis, but in contrast to other eukaryotes, it lacks a dedicated enzyme to interconvert between cytidine (C) and uridine (U) nucleotide pools. Instead, we previously reported that nucleotide interconversion was mediated by thymidine kinase (TK) and an unknown 5’-nucleotidase enzyme. T. brucei encodes multiple potential 5’-nucelotidases, including one from the histidine-aspartate (HD) family (5’-HDNT) and two from the haloacid dehalogenase (HAD1 and HAD2) superfamily of metalloenzymes, but their relative roles and function in nucleotide metabolism remained unexplored. Herein we report that enzymes from both families catalyze dephosphorylation of pyrimidine monophosphate nucleotides, but only 5’-HDNT exhibited high activity on dCMP and was essential for T. brucei growth, while HAD1 and HAD2 were dispensable. Growth arrest upon loss of 5’-HDNT expression was rescued by ectopic expression of human dCMP deaminase (HsDCTD), which creates an alternate route for conversion between C and U pools. Knockout of 5’-HDNT led to depletion of pyrimidine pathway metabolites, altered ratios of pyrimidine to purine nucleotides, and DNA damage. Surprisingly, intracellular glutamine, the precursor of de novo pyrimidine biosynthesis, was also significantly reduced, and isotope tracing assays identified a deficiency in glutamine uptake, suggesting that the glutamine transporter was downregulated in response to loss of 5’-HDNT. This response would likely exacerbate perturbation of pyrimidine pathway homeostasis, suggesting we have uncovered a potential regulated cell death pathway in T. brucei.